[Advances in Enzymology - and Related Areas of Molecular Biology] Advances in Enzymology and Related...

THE P-REPLACEMENT-SPECIFIC PYRIDOXAL-P-DEPENDENT LYASES

By ALEXANDER E. BRAUNSTEIN and ELIZABETH V. GORYACHENKOVA, Institute of Molecular Biology, USSR Academy of Sciences, Moscow B-334, USSR

C O N T E N T S

I. Introduction A. B.

Principles of Formal and Rational Enzyme Classification Theory and Systematics of Pyridoxal-P-Dependent Chemical and Enzymic Reactions 1. General Concepts: Subclassification of PLP-Dependent

Enzymes 2. Pyridoxal-P-Dependent Lyases Catalyzing Elimination and

Replacement Reactions: Suggested Chemical Mechanisms and Subtypes

11. General Physical and Chemical Characterization of Individual Enzymes A. The P-Replacement-Specific Lyases

1. Cysteine Lyase (EC 4.4.1 .lo) 2. Serine Sulfhydrase (allelozymes of EC 4.2.1.22) 3. Cystathionine P-Synthase (allelozymes of EC 4.2.1.22) 4. P-Cyanoalanine Synthase (EC 4.4.1.9) Recent Studies Relating to Lyases of Other Subgroups I , Purification and Properties of Alliinase (EC 4.4.1.4) 2. Observations Concerning y-Cystathionase (EC 4.4.1. I ) and

Some Other Eliminating or Multifunctional Lyases

B.

111. Comparative Survey of Physicochemical and Catalytic Features of P-Replacement-Specific and Some Other PLP-Dependent Lyases A.

B.

Size and Stability Parameters: Quaternary Structure; Oligomer - Subunit and Holo - Apoenzyme Equilibria; Cation Effects Comparison of Relative Reaction Rates and Substrate Affinities

2 2

3

3

7

13 13 14 18 18 27 34 34

38

42

42 46

Ediror’sfoornote: We are pleased to publish this chapter. We hope it will facilitate new significant interactions between Soviet scientists and others interested in this area of vitamin Ba enzymology, and that it will offer useful correlations with other studies, such as those recently reviewed by L. Davis and D. E. Metzler (The Enzymes, 3rd ed. Vol. VII, pp. 33-74, 1972), E. E. Snell [Advances in Enzymology, 42, 287- 333 (1974)], and E. Miles [Advances in Enzymology, 49, 127-186 (197911.

1

Advances in Enzymology and Related Areas of Molecular Biology, Volume 56 Edited by Alton Meister

Copyright © 1984 by John Wiley & Sons, Inc.

2 ALEXANDER E. BRAUNSTEIN AND ELIZABETH V. GORYACHENKOVA

C.

D. E.

Spectral Features of the Pyridoxal-P-Dependent Lyases in the Visible Range lsotopic Exchange of Hydrogen Atoms and P-Substituents Interactions with Active-Site Directed Ligands and Group- Modifying Reagents 1. Coenzyme Analogs 2. Quasisubstrates and Nonspecific Carbonyl Reagents 3. Sodium Borohydride 4. Sulfhydryl Reagents 5. Cycloserine Enantiomers and Related Compounds 6. Mercapto-Amino Acids and Aminothiols 7. Michael Addition and Related Reactions in the Active

Center Reaction Types Indicated by Steady State Kinetics F.

1V. Discussion of the Reaction Mechanisms and General Conclusions Acknowledgments References

49 50

54 54 57 59 60 61 63

67 70 76 a2 a2

Abbreviations and Symbols

PLP or pyridoxalP, pyridoxal-5’-phosphate; PMP or pyridoxamine-P, pyridoxamine-5‘-phosphate; Lys(Pxy) N6-(pyridoxylidene)-lysine; HS-EtSOH, 2-mercaptoethanol; HS.Et.NH2, cysteamine; Val(3HS), penicillamine; Hcy, homocysteine; Ala(CN), p- cyanoalanine; Ala(Cl), p-chloroalanine; Ala(SCN), p-thiocyanoalanine = S-cyanocysteine; Mal > NaEt, N-ethylmaleimide; Gly(2Allyl), allylglycine; cSer, cycloserine.

I. Introduction

A. PRINCIPLES OF FORMAL AND RATIONAL ENZYME CLASSIFICATION

The general system of enzyme classification adopted by the En- zyme Commission of IUB, and retained in the latest revised ZWB Recommendations (1978) on Enzyme Nomenclature (1) is based on a formal principle-the overall equations of enzyme-catalyzed reactions. Elucidation of the structure and actual catalytic functions of active sites eventually provides more rational chemical criteria for “local” classification of some enzymes, for example, the nature of catalytically important functional groups, in the case of protei- nases (1). Development and verification of subclassifications based on catalytic-site chemistry is a helpful approach for confirming sug-

FREPLACEMENT-SPECIFIC PLP-DEPENDENT LYASES 3

gested molecular mechanisms of enzymic catalysis and detecting new ones.

In 1972, Braunstein (3) pointed out that currently available information relating to the structural and functional features of a large family of crucially important biocatalysts-the pyridoxal-P-dependent enzymes-was sufficient for elaboration, in rough outline, of their rational subclassification based on presumable molecular reaction mechanisms. Some theoretical and experimental criteria, developed to clarify such mechanisms (3,4), were verified by applying them in a series of recent studies, surveyed in this chapter.

Currently, the Enzyme Nomenclature (1) lists more than 90 pyridoxal-P-requiring enzymes (and the total is steadily growing). These are mainly in classes EC 2 ..., 4 ..., and 5 ..., with quite a few in the other classes of this system. In all living beings, pyridoxal-P-proteins catalyze key steps and many specialized reactions in the assimilation and metabolic transformations of nitrogen- and sulfur-containing compounds. These include a broad variety of elimination, exchange, and condensation reactions (e.g., transamination, racemization, decarboxylation reactions, cleavage or elongation of carbon chains, elimination or replacement of substituents), particularly at the a, p, and y C-atoms in amino acids, and in other NH2-containing compounds.

Progress in the study of structure, catalytic activities, biological functions, and regulation of PLP-dependent enzymes and their specialized subgroups has been surveyed many times in monographs (21,23), in chapters published in treatises and serial publications, such as The Enzymes, Methods in Enzymology, Annual Reviews of Biochemistry, Advances in Enzymology, Vitamins and Hormones, in the Proceedings of several Symposia on Pyridoxal Enzymes (Rome, 1962; New York, 1964; Moscow, 1966; Nagoya, 1967; Len- ingrad, 1974; Toronto, 1979; Athens, 1983; cf. refs. 3-1 1,20,78, and 103), and in the references at the end of this chapter.

B. THEORY AND SYSTEMATICS OF PYRIDOXAL-P-DEPENDENT CHEMICAL AND ENZYMIC REACTIONS

1 . General Concepts: Subclassification of PLP-Dependent Enzymes

In 1952-1954, Braunstein (5-8), Snell (9-12) and their associates developed similar interpretations of the general chemical mechanism responsible for a broad variety of transformations induced in amino


acids by interactions with pyridoxal or its phosphate ester (or analogous carbonyl compounds), and catalyzed in organisms by pyridoxal-P-dependent enzymes [cf. refs. 3,4,12, and 23 and an early formulation of the underlying basic concept (7)]. According to this well-known theory, the common cause of all such reactions is the greatly lowered electron density of the NH2-linked carbon atom (usually Ca) in imines (Schiff bases) readily formed from amino acids and electron-withdrawing carbonyl compounds-especially in PLP- aldimines and in the tautomeric PMP-ketimines. In such imines (in particular with imino-N rendered electropositive by hydrogen bonding, metal chelation, or protonation) the bonds between the a-carbon atom and all its substituents are strongly polarized and labilized. Eventual release of one of the substituents produces an aldimine carbanion with delocalized negative charge (3,4, and 23); see for- mulas 1 and 2 on scheme I. Secondary transformations of the imines,

-0OC R \ /

CH,OPOF -* -0OC R \c/

1 HN* H *'\/

CH,OPOr

-O& + H 2 -

#I H+ .+ H 3a - 3 - 2a -

Scheme I . Interconversion of PLP-aldimines and PMP-ketimines (1-3) and stereochemistry of their reduction with Na-borotritide (34. (See ref. 3.)

p-REPLACEMENT-SPECIFIC PLP-DEPENDENT LYASES 5

depending on the structure of the amino acid and on experimental conditions, result in diverse reaction types involving breaking and making bonds at the C atoms a, p, or y.

Early subclassifications of pyridoxal-P-dependent enzymes based on reaction type were somewhat dissimilar. Braunstein grouped the enzymes, according to the final result of reactions, into seven main types (Fig. 1) with different patterns of bonding and breaking ( 3 3 , and 6). Snell et al. (11,12) divided the enzymes into three main reaction types based on the nature of the p-substituent believed to be the primary leaving group, namely, the a-H atom, the a-carboxyl, or the R group (side-chain).

In nonenzymic model systems, transformations of pyridoxylidene-imino acids often proceed concomitantly in several direction.

H Y H

_____~

Type Bonds disrupted Reactions

1 C"-H 1 + 2 C"-H; C"-N

4 C"-H; CB-Y 3 C"-COOH

5 CB-H; Cy-X

6 C R - C p (and P-HI

7 CP-CY

_ _ ~ ~ ~

Racemization Transamination a-Decarboxylation Elimination or replacement of a-H and p-

Elimination or replacement of P-H and y-

a , P-Cleavage (and condensation) of carbon

p, y-Cleavage of carbon chain

substituent

substituent

chains

Figure 1. acids (8).

Scheme and list of main types of PLP-dependent transformations of amino

6 ALEXANDER E. BRAUNSTEIN AND ELIZABETH v. GORYACHENKOVA

Both Braunstein (5,6, and 8) and Snell (1 1) have emphasized that the superior efficiency and reaction specificity of catalysis by PLP- containing enzymes resulted from the nature of the individual enzyme proteins or apoenzymes. It was later recognized that these qualities result from contributions to substrate binding and to catalytic transformation by appropriately located functional groups of the protein (see, for example, refs. 4,11, and 24).

Dunathan (4,13,25) defined the special geometry of intermediate pyridoxylidene imines (Fig. 2) that controls the release of a particular leaving group from C" and the formation of the corresponding plan- ary transient carbanion (e.g., as in Scheme I, 2).

Stability of the carbanion is enhanced owing both to a gain in resonance energy of the extended n-system and to other factors. Dunathan pointed out (13) that, for quantum-chemical reasons, maximum cr-T overlap between the ring-imine cr,n system and the a-C bond to be broken occurs when the latter a-bond is in a plane orthogonal to the plane of the cofactor-imine system (Fig. 2a).

In model pyridoxylidene-imino acids, rotational freedom around the C"-N bond allows parallel occurrence of reactions initiated by the release of any one of the C" substituents. In PLP-enzyme- substrate aldimines (ES intermediates), conformation about this bond must be under control, since only one single group'in position CY is to be selectively labilized. This implies that in the active center the anchoring sites for groups R and COO- of substrate amino acids must have a specific dissymmetric relationship to the plane of the cofactor ring. Figure 2 shows three distinct PLP-aldimine-enzyme complexes, each with a different C" bond in the active perpendicular position. Experimental verification has thus far confirmed every pre- diction based on this model. Hence, subdivision of PLP-enzymes into three main types according to the primarily weakened C" bond now rests on a rational mechanistic basis, which is common to the "local" systematics of PLP-enzymes currently adopted by our school (3) and by other enzymologists (4,12, and 14).

Reliable criteria for correctly assigning a PLP-enzyme to one of the main types (Fig. 2a, b, or c) are obtained by isotopic studies revealing the stereochemistry of enzyme-catalyzed replacement of C"-linked groups by labeled hydrogen or other substituents. Evi- dence thus obtained has demonstrated that allocation of a PLP-enzyme to one of the three types should be based on the geometry of


Figure 2. Conformations of PL-amino-acid aldimines in the active site, favoring disruption of (a) C"-H bond ( e g transaminases), ( b ) C"-@ bond (threonine al- dolase), and (c) C"-COO- bond (amino acid a-decarboxylases). (Scheme drawn by E. Severin after Dunathan; cf. refs. 4 and 13.)

the C"-substituent primarily released, rather than on its chemical nature (H, COO-, or R) (see refs. 3,4,25, and 100).

2 . Pyridoxal-P-Dependent Lyases Catalyzing Elimination and Replacement Reactions: Suggested Chemical Mechanisms and

Subtypes

In the biosynthesis and metabolism of most p- or y-substituted amino acids, a group of PLP-enzymes classified as lyases (EC, class 4) play a pivotal role. They catalyze, more or less selectively, the


elimination and/or replacement (exchange) of electron-accepting groups (X) in positions Q or y (see eqs. 1-4).

cr,p-Elimination:

X'CHR--C"HN+H,-COO- + H20 * XH

+ NH$ + R'CH2-C"O-COO- (1)

P-Replacement :

X@CHR-C"HN+H3-COO- + YH S XH

+ Y'CHR-C"HN+ H3-COO- (2)

P,y-Elimination:

XYCHR-C'H2-CnHN+H3-C0O- + H20 + XH

+ NH$ + RTH~-CPH2-C"O-COO- (3)

y-Replacement :

X'CHR-C'H2-CnHN+H3-COO- + YH S XH

+ YYCHR-CBH2-C"HN+Hs-COO- (4)

In organisms of all classes, such reactions are key links in the catalysis and control of the synthesis of many protein-constituent and specialized amino acids, such as hydroxylated , sulfur-containing and aromatic amino acids, P-cyanoalanine, tryptophan, and other heterocyclic amino acids. The PLP-dependent lyases often display relative rather than absolute substrate specificities, and several are multifunctional, since they catalyze more than one reaction type (see examples in Table I).

The reaction mechanisms of types I-IV were interpreted in a rather similarly manner by Braunstein and Shemyakin (5,6, and 8) and Snell et al. (9,11,12, and 14). Both schools assumed, and isotopic studies of hydrogen exchange have later confirmed, that the primary step in all such reactions was dissociation of the a-H atom in enzyme-bound PLP-substrate aldimines. According to Snell (1 1,12) and others (13,14, and 25), this step is followed by desmotropic conversion of the PLP-aldimine carbanion [see Fig. 3, (2)] to a PMP-


ketimine intermediate (or a tautomeric quinonoid species). We agree with regard to reactions I11 and IV (elimination and replacement in position y), and also accept the mechanism suggested for eliminating equation 1 and ambivalent (eqs. 1 and 2) p-lyases. This mechanism comprises (as shown in Fig. 3),* transitory formation by eliminating a-H and the p-substituent (X) of ap-unsaturated intermediates [ P- pyridoxylidene-iminoacrylates, Fig. 3 (3)] bound at the active site of the specific protein. These AaP-intermediates are either decomposed by twofold hydrolysis to a-keto acid, ammonia, and free PLP- enzyme [a,p-elimination, Fig. 3 (3) + (5) + (611 or undergo Michael addition of a molecule of replacing agent (the cosubstrate YH) to the double bond, and hydrolysis to PLP-enzyme and the new amino acid YCHR-CHN’ H3-COO- [p-replacement, Fig. 3 (3) + (4)] (see ref. 14). PMP-ketimine and unsaturated Schiff-base intermediates are known to be essential in the case of the y-specific lyases (3-14,79). On the basis of theoretical considerations and experimental evidence presented below, we question the formation of intermediate enzyme-bound PMP-ketimines for the exclusively p-replacing lyases although this step seems to occur in the case of a$- eliminating enzymes. For reactions I and I1 Braunstein and She- myakin in 1953 [(6); cf. (3,8)], taking into account the strong elec- trophilic inductive effect of the p-X group (and external induction by the replacing agent YH), outlined reaction schemes requiring no PMP-ketimine intermediate (see ref. 8, Schemes J, K, and L).

In the second alternative scheme for p-replacement, shown in Fig. 4, no ap-unsaturated Schiff bases occur in the sequence of reaction intermediates. This view is supported by our subsequent findings concerning enzymic p-replacement (type I1 reactions). t S

* A more elaborate version of this mechanism, incorporating the recently demonstrated reversal of a$-elimination, that is, reaction I catalyzed by tryptophanase (15) or tyrosine phenol-lyase (16), is featured in Snell’s scheme for bacterial tryptophanase (IS). Using different p-substituents, this scheme could apply to other reactions of types I or I and 11.

t These enzymic Xp-replacement reactions proceed with retention of configuration (see Sections 1II.D and 1V); hence their actual mechanism must differ from that suggested in Figure 4, which represents a reaction of the Sr.12 type, involving con- figurational inversion at the p-C atom. (Cf. refs. 18,20,27,78,92,99, and 100.)

$ More recent publications (see refs. 127-130) have shown that several replacing agents of the “suicide inactivator” type may act by a novel type of crotonate condensation reaction, rather than by Michael addition. See Section III.E.7 for a detailed discussion.

Tab

le I

T

he P

yrid

oxal

-P-D

epen

dent

Lya

ses

Stud

ied'

Enz

ymes

" T

ypes

of

[Cla

ssif

icat

ion

(3).

nam

e, b

iolo

gica

l re

actio

ns

Pri

mar

y su

bstr

ates

R

epla

cing

age

nts

sour

ce]

cata

lyze

d ($

-sub

stitu

ted

a-am

ino

acid

s)

(cos

ubst

rate

s)

+

A 2, b. $

-Rep

laci

ng

Cys

tein

e ly

ase

(chi

cken

-em

bryo

I1

cy

s H

SO

F, A

lkSH

, C

ys,

HTS

yo

lk s

ac)

Seri

ne s

umyd

rase

(chi

cken

live

r;

Cys

tath

ioni

ne p

-syn

thas

e (mam- 'II

as f

or 2

as

for 2

; Ala

(3-S

eH)

mal

ian

liver

, yea

st;

aIIe

Iozy

me

of

Ser

, Cys

, Se

r(O

Acy

l), C

ys(S

Alk

) H

cy,

Alk

SH,

HzS

,NH

~(C

HZ

)~S

H,

bake

r's y

east

) A

la(C

I), A

la(C

N)

HO

(CH

2)zS

H

+

lyas

e 22

) p-

Cya

noal

anin

e sy

ntha

se (

lupi

ne

II C

ys, A

la(C

I), A

la(S

CN

) H

CN

, H

2S, A

IkSH

, HO

(CH

2)zS

H

seed

lings

)

+

A,2

,a. a

,f3-E

limin

atin

g 5

. A

lliin

ase

(gar

lic)

I A

lliin

(an

d an

alog

s)

6. S

erin

e de

hydr

atas

e (r

at l

iver

) 7

S

er, T

hr, e

ryth

ro-P

-HO

-Asp

A.2

.c. a

,P-E

limin

atin

g an

d f3-

-2

3- a

nd 5

-Alk

-Ind

; HzO

, H2S

, Alk

SH

Rep

laci

ng(a

mbi

func

tiona

l) ? --

7.

Try

ptop

hana

se (

E. c

oli)

1,

II

Trp

, S

er, C

ys, C

ys(S

A1k

)

A,l

,d. f

3,y-

and

a,p

-Spe

c$c

A

(mul

tifun

ctio

nal)

+

-

8,

y-C

ysta

thio

nase

' (r

at l

iver

) I,

II(?

) C

ysta

thio

nine

, H

Se,

Hcy

-,

Cys

, C

ysC

ys,

HSe

(OA

cyl)

, 11

1, IV

Ala

(3,S

eH),

Sel

ena-

cyst

athi

onin

e

From

ref

eren

ce 8

a, 2

0, and 78.

Enz

ymes

1-5

and

8 w

ere

puri

fied

to

95-1

00%

ho

mog

enei

ty b

y te

chni

ques

dev

elop

ed in

our

labo

rato

ry.

Evi

denc

e co

ncer

ning

rea

ctio

ns o

f ty

pes

I1 a

nd I

V i

s sp

arse

; se

e re

fere

nces

14,

79,

and

116

.


containing snryme

Y I H + Y n

‘a+ R-CH+-COO- I NH:

- p-replacement

Figure 3. Scheme of the mechanisms of a,p-elimination and P-replacement reactions catalyzed by specific PLP-dependent lyases (14).

During the last 15 years, we and our associates* have focused attention on the isolation and characterization of several high-purity lyases, of the P-replacement-specific type, that act on cysteine, serine, and some related a-amino acids. The main chemical and physical features and catalytic properties of these enzymes were studied in comparison with those of a few lyases in other subgroups [a$-eliminating, ambi- or multifunctional (3,18, and 78)]. The features investigated comprised: the scope of catalytic activities; specificities for cofactor, substrates, and various inhibitors (including analogs of the replacing agent, coenzyme, and substrate); spectral

* Main participants of the studies on pyridoxal-P-dependent lyases: T. N. Akopyan, S. V. Amontov, A. E. Braunstein, N. Dinh-Lac, V. L. Florentiev, S. M. Galoyan, E. V. Goryachenkova, R. A. Kazaryan, L. V. Kozlov, T. G. Leonova, E. 1. Loupou, R. N. Maslova, L. A. Polyakova, A. G. Rabinkov, A. S. Tikhonenko, E. A. Tolosa, I. H. Willhardt, L. L. Yefremova et al.

B-REPLACEMENTSPECIFIC PLP-DEPENDENT LY ASES 13

H cH,<-COO- + Pyridoxal- P-containing enzyme

y' I NH:

Figure 4. Suggested mechanism for p-replacement catalyzed by specific PLP-dependent lyases (Scheme L, from ref. 3).

properties; and kinetic characteristics of overall reactions and of the lyase-catalyzed isotopic exchange of H-atoms and P-substituent groups.

Substantial differences in reaction mechanisms between lyases with exclusive P-replacement function and the mono- or multifunctional lyases active in reactions of types I, I + 11, 111, IV, were revealed by the evidence now available (see refs. 18,78 and Section 111). The data presented support the claim that @replacing lyases constitute a separate subgroup in the rational classification of PLP- dependent enzymes, as outlined by Braunstein (3,18).

Table I lists the main objects studied (3) according to reaction specificities and indicates the known primary (amino acid) substrates and cosubstrates.

11. General Physical and Chemical Characterization of Individual Enzymes

A. THE p-REPLACEMENT-SPECIFIC LYASES

Four pyridoxal-P-dependent lyases listed in Table I (subgroup A,2,b) have been investigated by our team in considerable detail,


and were conclusively shown to catalyze only reactions of p-replacement. These are (1) cysteine lyase (EC 4.4.1.10) from chicken- embryo yolk-sac (32-34a); (2) serine sulfhydrase (EC 4.2.1.22) from chicken liver (40-44), or from baker’s yeast (45-49,94); (3) cystathionine p-synthase, from mammalian liver (62-64,78) [enzymes (2) and (3) are similar species-specific forms of one enzyme protein, or genetically determined allelozymes (2,26,50-56)]; and (4) p-cyanoalanine synthase (EC 4.4.1.9) from lupine seedlings (28,65-69,77,93).

An overview of the major physical, chemical, and biological characteristics of these four enzymes is given in Sections 1I.A. 1-4. For comparison, improved purification procedures were developed for a few lyases in other subgroups (e.g., alliinase, y-cystathionase, and some properties of these) and other lyases were reexamined (see Sections 1I.B. 1-2 and 1II.A-D).

The p-replacement-specific subgroup may include in some plants and microorganisms) further PLP-dependent lyases that catalyze the condensation between specific open-chain or heterocyclic metabo- lites and cysteine or serine (their S-alkyl or 0-acyl derivatives, respectively) to p-substituted a-aminopropionic acids (considered as “secondary assimilates”) acting as N (or S) reserve (e.g., by K. Mothes, L. Fowden, and others; see refs. 28a and 30) detoxication products (21), or repellents. Some plausible examples are p-pyra- zolyl-L-alanine synthase (EC 4.2.1.50) (29) and hypothetic lyases synthesizing alliins, willardiin, mimosin, lathyrin, and so on (30).

I . Cysteine Lyase (EC 4.4.1.10)

In 1957, an enzyme-catalyzing formation of cysteic acid (and H2S) from L-cysteine and sulfite ions (eq. 5) was discovered in chicken embryo yolk-sac, partially purified (about 22-fold), and named cysteine lyase by Chapeville and Fromageot (31); it has not been detected in objects other than the yolk-sac of avian (and chelonian (35) embryos. Evidence was presented indicating that cysteine lyase is a PLR-enzyme. In the absence of sulfite, crude preparations formed some lanthionine by a p-replacement reaction between 2 mole of cysteine and small amounts of HzS, NHZ and pyruvate (i.e., products of a,p-elimination). The semipurified enzyme catalyzed isotopic exchange of the a-hydrogen atom in cysteine at a rate ex- ceeding that of the overall p-replacement reaction with sulfite ( 3 3 ,


and also effected a slow substitution of labeled HS from [35S] sulfide for the HS-group of L-cysteine.

Thus, the catalytic mechanism presumably might involve the elimination of a-H and P-SH from enzyme-bound PLP-cysteine aldimine to form a transitory AmP-substrate-PLP aldimine, followed by its condensation with sulfite (eq. 5 ) or with a second molecule of cysteine (lanthionine formation; eq. 6: R = CH2-CHNC H3-cOO-) that is, a nucleophilic Michael addition reaction. This is essentially the mechanism shown in Figure 3 (2) + (3) --+ (4).

Tolosa et al. extensively purified the enzyme from yolk-sacs of 18-day-old chicken embryos, first 400-fold (32) and later approximately 800-fold, to virtual homogeneity, by an improved procedure (33) which includes delipidation of the tissue homogenate with di- ethyl ether, ammonium sulfate fractionation, adsorption on Ca3(P04)2 gel, desalting the eluted enzyme on Sephadex G-25 and, isoelectric focusing. The active fractions were filtered through Sephadex G-25 to remove low-molecular solutes. For larger-scale preparation, the final electrofocusing and molecular-sieving steps were efficiently replaced by fractionation on DE-52 cellulose with a gradient of pH 8.2 buffer, and filtration through Sephadex G-100 (33).

Activity assays were based either on determination of the release H2S or on spectrophotometry of the P-replacement product (YCHR-CHN + H3-cOO-) eluted from paper chromatograms (26,33); 95% purity and catalytic activities of 20-26 pmol.h-'.mg- I

were achieved in the best preparations of the enzyme. Even moderately purified (400-fold) cysteine lyase (32) was completely devoid of a$-elimination activity, which was evidently caused in the cruder preparations by such contaminating PLP-enzymes as cystathionase.

Cysteine lyase has an estimated M,. of 120,000; it is apparently a dimeric protein. During purification, the lyase is partially resolved to apo- and coenzyme; for activity assay it requires the addition of PLP in excess. Therefore, because of the nonproductive binding of PLP outside the catalytic site, titration of the apoenzyme with PLP fails to provide an exact estimate of the stoichiometcic content of PLP in fully saturated holoenzyme. (Tolosa observed restitution of most of the activity on preincubation of the apoenzyme overnight with approximately two equivalents of PLP). The enzyme has a fairly broad activity optimum about pH 8.7-8.8; its isoelectric point, PI, as determined by ampholine electrofocusing, is at 4.98. The pur-


ified lyase is very labile, can be stored at lower temperatures under ammonium sulfate solution with added PLP and a thiol (e.g., 2- mercaptoethanol). In neutral or weakly acidic solution, the pure enzyme rapidly undergoes denaturation.

Tolosa et al. (34,34a) noted that the rate of release of H2S from cysteine by this enzyme is markedly enhanced in the presence of certain aliphatic mercaptans, such as 2-mercaptoethanol, cysteamine, methane-, or ethanethiol. This clue led to the discovery that pure cysteine lyase utilizes these and some other mercaptans [including cysteine itself (34a) but not homocysteine (18,32), as cosubstrates in p-replacement reactions with L-cysteine (according to eq. 6), and produces the relevant cysteine thioether (e.g., S-hydroxyethyl-L-cysteine, thialysine, and ]anthionhe)* (see Table I1 and Fig. 5) . In contrast to serine sulfhydrase, which is able to effect such reactions in reverse (see Section II.A.21, the action of cysteine lyase in equations 5 and 6 is not reversible, but unidirectional under all experimental conditions tested. The exclusive amino substrate specificity for L-cysteine was verified using either sulfite (31) or mercapto compounds (33) as the cosubstrate in experiments with a broad variety of cysteine derivatives or analogs. None of these compounds displayed activity as a substitute for cysteine or as a competitive inhibitor (except L-serine, see below).

This indicates that the enzyme displays a practically absolute re- quirement for all functional groups of the substrate molecule, namely, p-SH, a-H, a-NHz, and 1-COOH.

Accordingly, cysteine lyase was redefined by us (3,18,27) as an exclusively p-replacing lyase [subgroup A,2, b (3)] with strict specificity for the primary substrate, L-cysteine, and relative specificity for several sulfur-containing cosubstrates (eqs. 5,6).

+ + HSCH2-CHNH3-COO- + HSOC + H2S + -0S02-CH2-CHNH3-COO- ( 5 )

+ + HSCH2-CHNHy-COO- + RSH + H2S + RSCH2-CHNH3-COO- (6)

+ (R = HJC; H5C2; HOaEt; NHZ-Et; -OOC-CH(NH3)-CH2; etc.)

Though maximum reaction rates with 2-mercaptoethanol are much higher than with sulfite (Table 11; cf. ref. 18), the only phys-

* Lanthionine formation is suppressed in the presence of adequate replacing agents-sulfite (Table 111) or a suitable thiol (see chromatoelectrophoregrams on Fig. 5).

TABLE I1 Relative Activities of Cysteine Lyase

Presence of some Thiols or Sulfite as Cosubstrate"

(H2S Liberation from M Cysteine) in the

Cosubstrate Activity (%)

C ysteine l00b M Mercaptoethanol 200

lo-* M Thioglycolate 210 M Thioglycolate 210

M Mercaptoethanol 550

M Dithiothreitol 200 M Dithiothreitol 300

250" 3 x lo-' M Sulfite

a From reference 33 and 34. Nonvolatile reaction product is mainly lan-

Reaction products: H2S + cysteic acid. thionine.

A t

I

pH V.0

Figure 5. Products formed by cysteine lyase from L-cysteine alone and in the presence of P-mercaptoethanol (A) or cysteamine (B) (34a). (Scheme of electrophore- tograms. Incubation period, 3 h.) Control samples: Heat-denatured enzyme with either P-mercaptoethanol (2) or cysteamine (5). Test samples: Active enzyme with either cysteine (31, cysteine and P-mercaptoethanol (4), or cysteine and cystearnine (6). Standard solutions of reference compounds ( I ) . Spots: (a) S-hydroxyethylcysteine, (b) lanthionine, (c) cysteine, (d) cysteamine, and (e) thialysine. (All spots were positive to ninhydrin and to iodoplatinate reagent).

17


iologically abundant replacing agent in yolk-sac is sulfite. The natural function of cysteine lyase is to produce, by way of reaction 5 , cysteic acid-the precursor of the fetal metabolite taurine (3 1-34).

As opposed to Chapeville et al., we found that a few cysteine analogs and thioethers can, at high concentrations, bind specifically at the enzyme's active site. It is noteworthy that L-serine acts as a competitive inhibitor with Ki = 8 mM, that is, with affhity to the binding site practically equal to the K,,, of cysteine (see refs. 18 and 33 and Table XV. Moreover, L-serine, in spite of its incapacity to undergo the complete P-replacement reaction with adequate cosubstrates (18,22-34a), (as well as S-hydroxyethyl-L-cysteine, which display low affhity for the enzyme), will exchange the H-atom in position a for 3H from tritiated solvent water (17,22) in the presence of appropriate cosubstrates (see Section 1II.D). It appears that these substrate analogs bind in the active site as coenzyme aldimines (18,27,33), as proven for L-serine by the presence of N-pyridoxyl- L-serine among the degradation products of the modified enzyme obtained by NaBH4-reduction of enzyme-serine-mercaptoethanol solutions (Tolosa et al., 1977; cited in ref. 78; cf. Sections III.E.2- 5) .

Cysteine lyase has a pH-independent spectral absorption peak with Am, 497 nm, which is not significantly altered by the addition of substrate amino acids, thiol cosubstrates, or their analogs (homocysteine and penicillamine). Borohydride reduction of the lyase considerably diminishes the band, with concomitant appearance of a band with Amax near 332 nm (34) (see Sections 1II.C and III.E.3).

Cysteine lyase is inactivated by modification of its HS groups, and its activity is impaired by the usual agents inhibitory to pyridoxal-P-dependent enzymes-carbonyl reagents, coenzyme derivatives, and certain substrate analogs. Like the other P-replacement- specific lyases, this one is also refractory to inhibition by L- and D-

cycloserine and by aminothiols. Interactions of the enzyme with these and other inhibitors and modifying reagents (see refs. 3,18,27,33, and 78) are discussed in Section 1II.E.

2. Serine Sulfhydrase (allelozymes of EC 4.2.1.22)

3 . Cystathionine P-Synthase (allelozymes of EC 4.2.1.22) The presence in yeast cells (and cell-free extracts) of an enzyme

catalyzing the reversible reaction of [ "S]-~-cysteine synthesis from L-serine and [35Slsulfide (eq. 7) was first reported in 1957 by Schloss-

P-REPLACEMENT-SPECIFIC PLP-DEPENDENT LYASES 19

TABLE 111 Cysteine Lyase; Reaction Products (kmol.h+ "mg- I)"

A Cysteic Samples A H S A Lanthioine acid A NH3b A Pyruvate

Controls (0')

Without Na2S03 1 .O mg of enzyme 0 0 0 0 0

0.5 mg of enzyme 1.44 I .20 0 0 0 I .O mg of enzyme 1.98 2.19 0 0.08 0 1.5 mg of enzyme 2.74 2.88 0 0.14 0

0.5 mg of enzyme + traces 6.3 0 0 1.0 mg of enzyme + + traces 10.5 0.10 0 1.5 mg of enzyme + + + traces 13.4 0.19 0

With Na2S03

~~~

" Conditions of experiment were: pH 8.5, temperature of 3YC, incubation time of 1 h in lo-' M Cys.

Enzyme preparation was the source of traces of NH3.

mann and Lynen (37) who named the enzyme serine suljhydrase. It was later detected in a variety of microorganisms, plants, and animal tissues (38,39,45-53), including avian and human hepatocytes and fibroblast cultures; the highest content was noted in chicken and human liver. With moderately (50- to 100-fold) purified preparations of the chicken liver or yeast enzyme (38,46-49), the equilibrium of equation 7

HOCH2-CHN+H3-COO- + H2S H20

+ HSCH2-CHNfH3-COO- (7)

strongly favors cysteine formation, while the rate of the back-reaction (7) is quite low.

In experiments with extracts from chicken yolk-sac, and fetal and adult chicken liver, Fromageot and associates (35,44a) used substrates labeled with isotopes: [33Slsulfide, Hll*O, 'HzO, [ 1 4 C ] - ~ ~ - serine, and [31P]phosphoserine. They observed slow reversible exchange of HI8* and H35S- with the P-substituent groups in cysteine, serine, and phosphoserine, and 3H-incorporation into serine at a rate amounting to 10-20% of the rate of overall cysteine synthesis. These isotope-exchange reactions were attributed to two enzymes, serine hydro-lyase and phosphoserine phospho-lyase. The


former enzyme, shown to be PLP-dependent, was supposedly identical to serine sulfhydrase. Phosphoserine phospho-lyase has never been adequately characterized.

Our colleague Dinh-Lac (40) purified chicken-liver serine sulfhydrase about 350-fold, eliminating contaminant PLP-enzyme activities, Yefremova and Goryachenkova (41,42) further improved his procedure and achieved 700-fold purification (Table IV). Their electrophoretically homogenous preparations catalyzed several p-replacement reactions of serine and some related amino-acid substrates with a number of cosubstrates, mainly thiols (see eq. 8). Specific activities up to or above 35 pmol-h-’/l mg protein were observed in the reaction with 2-mercaptoethanol.

Our studies of the general and catalytic properties of hepatic serine sulfhydrase (26,40-44) revealed that the rates of cysteine desulfhydration by this enzyme-like the liberation of H2S from cysteine catalyzed by cysteine lyase (Section 1I.A. 1 1)-were accelerated ma- nyfold in the presence of certain mercapto compounds (e.g., HOeEteSH, NHyEtSSH, and L-homocysteine; see Table V and Fig. 6). The formation of H2S and cysteine thioethers by p-replacement reactions between cysteine and the thiols was confirmed by the chro- matographic identification of corresponding thioethers, for example, S-hydroxyethyl-L-cysteine, S-aminoethyl-L-cysteine (“thialysine”), and cystathionine (43,44,48). The same thioethers (and HzO) are formed by the lyase at comparable rates from serine and the cor-

TABLE IV Purification of Serine Sulfhydrase (2) from 500 g Chicken Livef

Activity Total

protein Total Specific Recovery Enzyme fraction following- (mg) (p-mo1.h-I) (pmol.h-’/mg) (%)

(a) Homogeneization 37,860 1893 0.05 100 (b) Fractionation with NHc 1,802 1800 1 .00 100

(c) Adsorption on Cap(PO& 742 1710 2.3 90 sulfate

(d) Chromatography on DEAE- 68 954 14.0 52

(e) Fractionation on Sephadex 20.5 720 35.0 38 cellulose

(3-200

From reference 42. Activities expressed as pmo1.h- I H2S released in the reaction of L-CYS with 2-mercaptoethanol.

TABLE V Relative Specific Activities of Hepatic Serine Sulfhydrase, 2, (Chicken) and

Cystathionine p-Synthase, 3, (Rat) Toward Primary Substrates (Serine and its Analogs, Cysteine, and Cysteine Thioethers)"

Relative activities (%)

Primary substrate (1-2 M * I O - ' )

LSerine 3-Chloro-~~-danine DL-Serine O-sulfate 3-Cyano-~~-alanine S-Carboxy methyl-L-c ysteine S-Methyl-L-c y steine S-H ydroxyethyl-L-c ysteine Nu-Acet yl-L-thial y sine

~

Serine sulfhydrase Cystationine P-synthase (enzyme 2) (enzyme 3)

100 52 20 23 18 17 9 9

100 33 20 6

not tested 20 +

not tested

' From references 18 and 26. 2-Mercaptoethanol was used as cosubstrate with serine sulfhydrase and oL-homocysteine with cystathione p-synthase. No attempt was made to ensure complete saturation of the enzyme with the reactants.

A R L

+ 1 2 3 4 5 I I I I I

Figure 6. Products of serine sulfhydrase-catalyzed reactions of L-serine and L-cysteine with thiols: P-mercaptoethanol (A), oL-homocysteine (B), or cysteamine (C). (Scheme of chromatoelectrophoretic separation from ref. 43. Incubation, 2 h. at 37".) Samples: Serine ( I ) or PLP (2) with heat-inactivated enzyme and respective thiol (controls); (3) like ( I ) , and (4) like (2), but with active enzyme. Standard reference solutions: s-hydroxyethyl-L-cysteine (5A), cystathionine (5B), and "thial ysine" (SC). Spots: A-(a) serine and (b) S-hydroxyethyl-L-cysteine; B-(a) serine and (b) cystathionine; C-(a) serine, (b) thialysine, and (c) cysteamine. (Open spots-positive to ninhydrin; hatched spots-positive to ninhydrin and to iodoplatinate reagent).

21


responding mercapto compounds (Fig. 6). The S-alkylcysteines thus produced (and some other p-substituted a-aminopropionic acids) can act as amino-acid substrates in p-replacement reactions (eq. 8 = eq. 2) with various thiols as cosubstrates. This demonstrates conclusively that the action of serine sulfhydrase is reversible (see also refs. 48 and 49).

XCH2*CHN+HjCOO- + YH XH + YCH2CHN+H3*COO- (8)

(X = OH; SH; OAcyl; SAlkyl; C1; CN)

(Y = SH; SAL; S*Et*OH;'S.Et*NH2; S * C H K H N + H3*COO-)

Most such reactions with mercapto compounds are considerably more rapid than the serine $ cysteine interconversion (eq. 7). The rates are highest, and nearly equal, with serine and with cysteine when homocysteine is the replacing agent [(41,42); cf. Section 1II.B and Tables V and XIV]. Hence, the main physiological function of serine sulfhydrase in animals is evidently the synthesis of cystathionine from serine and homocysteine. That this lyase and cystathionine P-synthase are identical or closely related enzymes (26,18) has subsequently been demonstrated for all biological systems studied.

High-purity serine sulfhydrase from chicken liver (41,42) has M , = 90,OOO -+ 3000. It contains 2 g-mol of firmly bound pyridoxal-P. On denaturation in 6 M urea, the dimeric protein dissociates to two identical subunits; amino acid analysis has indicated a subunit M, of approximately 52,000. Apoenzyme, prepared by resolution of ox- imes of the sulfhydrase, does not undergo spontaneous dissociation to monomeric protein in weakly alkaline solution. Its complete reactivation with PLP (Kc,, = 1 x M) requires adjustment of the solution to pH - 7.0. The pure holoenzyme is rapidly inactivated at pH 9 7.0; it is also very labile to lyophilization or storage at 0-5°C and pH - 8, unless stabilized by a high-protein concentration in the solution and the addition of 2-mercaptoethanol (41,42); cf. Section 1II.C.

The most potent inhibitors of the enzyme are hydroxylamine and aminooxyacetic acid (Ki about 2 x M). It is refractory to inhibition by mercaptoamino acids and aminoisoxazolidones [(18,26); see Section III.E.5 and 61.

Using a procedure similar to that developed for lyase 2 from chicken liver (Table IV), Willhardt et al. (46,47) efficiently purified

B-REPLACEMENT-SPECIFIC PLP-DEPENDENT LYASES 23

serine sulfhydrase from baker's yeast (37) and further characterized it (48,94). This lyase, which we designate lyase 2a, accounts for the activity of "S-methylcysteine synthetase," first observed in yeast by Wolff et al. (45); (EC 4.2.1.22 in ref. 1). The best preparations of lyase 2a (at that time believed to contain 90% active protein) had specific activities of 25 pmol.h-'/mg in the cysteine + mercaptoethanol assay and 10 pmol.h-'/mg in the L-serine + N"-acetyl-cysteamine system (34), which has been used for preparative enzymic synthesis of Nu-acetylated and free ~-thialysine (49).

The yeast enzyme differs from chicken liver lyase 2 in relative catalytic efficiencies with various amino- and cosubstrates; however, the substrate specificities are qualitatively the same (cf. Tables V and XIV).

Some other minor differences in molecular features were noted between lyases 2a and 2. The molecular weight (M, ) of the yeast enzyme was estimated as 60,000 + 2000 and its PI as 4.8. The optimum range for activity was pH 7.7-8.2 (with phosphate buffer). Apoenzyme was prepared by resolution of lyase 2a at pH 6.8 with 5 x M hydroxylamine (cf. ref. 29); 100% reactivation was achieved on prompt, relatively brief contact of the apoenzyme with

Like hepatic serine sulfhydrase (2), the yeast enzyme (2a) displays high sensitivity to inhibition by carbonyl reagents (for aminooxyacetate Ki is -2 x lop6 M). It is refractory to inhibition by D and L enantiomers of cycloserine (up to lo-* M) and by P-mercapto-a- amino acids (18,26,78). (For further details concerning values of affinity for substrates, cosubstrates, coenzyme analogs and other ligands, stationary kinetics (94), etc., see Section 1II.A-F.)

Yamagata et al. reported (36a) the isolation, from strains of baker's yeast, of at least two serine sulfhydro-lyases with perplexing differences in the ranges of substrate specificities in replacement reactions, as indicated by the names and attributions proposed by these authors-0-acetylserine,O-acetylhomoserine sulfhydro-lyase, EC 4.2.99.10 (? Braunstein), and serine,O-acetylserine sulfhydro- lyase, EC 4.2.99.8 (?). Based on comparative studies of wild and mutant yeast strains, the latter enzyme is considered as the lyase responsible for cysteine biosynthesis. The authors do not state whether their enzymes possessed eliminating activities. Therefore, these lyases cannot be assigned umambiguously to definite subgroups in our classification of PLP-dependent lyases

s x 1 0 - ~ M P L P .


(3,18,20,78). Neither our studies nor those of Willhardt and Hermann (46-49,94) are mentioned in their report (36a).

Our team has camed out detailed comparative studies of lyases 2 and 2a (test reaction: cysteine + mercaptoethanol) with cystathionine P-synthase from rat liver (lyase 3) (standard test: L-serine + homocysteine); (see references 8a,20,26,36,41,57,58,78,92,94).

The PLP-dependent synthesis of cystathionine from serine and homocysteine in animal tissues was recognized in the early 1950s by Goryachenkova (5738) and Binkley et al. (59) as the first step in cysteine biosynthesis by “transsulfuration” [see (21)l. The second step is p,y-elimination catalyzed by y-cystathionase (EC 4.4.1 .I) (see Section II.B.2). In procaryotes and plants, the preferred immediate precursors of cysteine in its synthesis via transsulfuration are 0-acyl (acetyl, succinyl) derivatives of serine (22,79,80). But in the synthesis of homocysteine by “reverse transsulfuration,” green plants prefer 0-phosphohomoserine as a very active HS-acceptor (22).

In his early attempts at purification of cystathionine f3-synthase (3) from rat liver, Greenberg (60) mistakenly claimed that this enzyme (EC 4.2.1.22) copurifies with serine deaminase activity (EC 4.2.1.13), and supposed that these two enzymes were identical. However, use of improved preparative and analytic procedures elim- inated contamination with serine deaminase and led to isolation of essentially pure cystathionine p-synthase (61-64)-a dimeric protein of M, 112,000 with 2 gram-equivalents of rather firmly bound PLP (63).

Less pure preparations of the enzyme tend to form a tetrameric aggregate, with apparent M, - 250,000, and larger ones. Fairly detailed descriptions of physical-chemical features of the enzyme are available (14,41,61-64).

The smallest dimeric holoenzyme 3 (Mr - 94,000) with the highest reported values of specific activity-160 U (Fmol.h-’)/l mg protein (as cystathionine P-synthase) and 28. I U/1 mg (as sulfhydrase-forming cysteine from serine + sulfide)-was prepared and characterized by Kraus et al. (52) from human liver tissue. This homogeneous lyase 3 preparation was purified about 3000-fold. It had PI = 5.2, a broad activity optimum in thq pH range from 8.4 to 9.0, and un- usually high values of substrate aff‘inity (K(manP) = 1.15 mM for L-

serine and 0.59 mil4 for L-homocysteine); see Table XV.

P-REPLACEMENT-SPECIFIC PLP-DEPENDENT LY ASES 25

For comparative studies, we used (18,26,27,36) lyase 3 from rat liver, partially purified essentially according to Kimura et al. (63), but replacing the last two steps of their procedure with fractionation on a column of Biogel-200 (41). The preparations had specific activities of 12-15 p,mol-h-'/mg, measured as cystathionine produced from L-serine + homocysteine in the standard assay procedure (26,64). Serine sulfhydrase isolated from chicken liver (41) was as- sayed by measuring initial @replacement rates in the assay system with L-cysteine + 2-mercaptoethanol (26,64).

The experimental results clearly demonstrate the close functional homology of serine sulfhydrase from chicken liver (2) or yeast (2a) with cystathionine P-synthase (3) from rat (63) and human (52) hepatic tissue as species-specific allelozymes (2), as indicated by us in 1971 (26). These lyases have qualitatively coincident specificities for primary (amino acid) substrates, cosubstrates (replacing agents), and inhibitors (Tables I, V, XII, and Section 1II.E).

The difference in biological origin accounts for significant di- vergencies between these lyases in relative reaction rates with individual substrates and cosubstrates and in K,,, values (Michaelis constants); see Tables XII, XIV, XV, and Section 1II.B. There are also substantial differences in M , values and isoelectric points. Thus, PI is 4.8 for yeast lyase 2a (Willhardt); for lyase 3 in hepatocytes it was estimated as 5.2 (human), 5.5 (rat), and 6.0 (chicken) (41,42).

Dissimilarities in physical-chemical and catalytic features among lyases from various biological sources, designated as serine sulfhydrase (or sulfo-lyase), cysteine (or alkylcysteine) synthetase, and cystathionine p-synthase, obviously reflect species-specific differences in the structure and properties of multiple allelic forms [allelozymes (2)] of one enzyme protein, namely, EC 4.2.1.22.

Functional homology and the presumed common genetic deter- minism of serine sulfhydrase and cystathionine P-synthase is evident from the following findings (20,26). Fractionation of crude or partially purified enzyme extracts from chicken or rat livers by preparative electrofocusing (Fig. 7) or gel-filtration (18) revealed the presence of a single homogeneous protein fraction that differed in each case in molecular weight and charge but showed overlapping peak activities for serine sulfhydrase and for cystathionine P-synthase. An enzyme displaying the same activities is present in human liver tissue (52) and fibroblast cultures (51 33). Its deficient biosyn-

26 ALEXANDER E. BRAUNSTEIN AND ELIZABETH v . GORYACHENKOVA

0.8

0.4

0

Wnm

Figure 7. Fractionation by ampholine electrophoresis of “crude” preparations of (A) serine sulfhydrase (from chicken liver) and ( B ) cystathionine synthase (from rat liver) (from ref. 26). Amount applied on column: 80-120 mg protein; U, pH values; M, protein (A280 ,,,,,); O--O, A cystathionine ( A ~ s s ,,,,,I; A-A, AHzS.

thesis is the cause of the human hereditary metabolic error known as homocystinuria (see refs. 53a-56).

The allelozymic common derivation of serine sulfhydrase and cystathionine P-synthase was further confirmed by Pieniazek et al. (51). They reported a correlated impairment of the activities in mutant strains of Aspergillus niduluns with allelic defects in cysteine biosynthesis and similar parallel lyase deficiencies were found in other organisms (see, e.g., refs. 53a-56).

Soda, Tanaka et al. (1 16) have recently shown that an essential component of several enzyme proteins, selenacysteine (more correctly, 3-selenoalanine or 2-amino-3-hydroselenopropionic acid), can be


synthesized enzymically via the selena-analog of cystathionine, by the joint action of hepatic cystathionine P-synthase (lyase 3) and y- cystathionase (lyase 8, see Section III.B.2). This reaction sequence duplicates transsulfuration, but starts with the selena-analog of methionine (known likewise to occur in some proteins).*

The same authors (1 17) discovered a strictly specific PLP-dependent hepatic lyase, selenocysteine lyase, which catalyzes the reductive cleavage of 3-selenoalanine to HzSe + L-Ala, using dithiothreitol or a second molecule of selenoalanine as the reductant.

The reaction mechanism is still under investigation. It resembles either that of aspartate P-decarboxylase or that of a peculiar p-replacing lyase reacting with selenoalanine as the primary substrate. L-Cysteine (Ki = 1 mM) competes with selenoalanine ( K , = 0.83) as an inhibitor, but does not react as an amino substrate.

4 . P-Cyanoalanine Synthase (EC 4.4.1.9)

P-Cyanoalanine [Ala(CN)] was isolated by Fowden (29,71) and others (30,30a) from several species of Leguminosae, certain Ce- reals, for example, Sorghum sp. (72), and other plants; it has also been detected in some bacteria and fungi (29). Ala(CN), the 4-nitrile of L-aspartic acid, is a precursor and toxic component of neurola- thyrogenic agents such as y-glutamyl-P-cyanoalanine and related compounds (30). It acts as an intermediate in a major pathway of L-asparagine biosynthesis in a broad variety of plants, including species never thought to be cyanogenic (e.g., barley, wheat, asparagus, and spinach) (29-30a,73-75).

The immediate precursors of Ala(CN) are L-cysteine and cyanide. The enzyme catalyzing synthesis of Ala(CN) from these compounds by a P-replacement reaction was detected in seedlings of blue lupine and moderately purified by Hendrickson and Conn (72). O-acetylserine also reacted slowly as an amino-acid substrate with semipurified Ala(CN) synthase, but not with high-purity preparations of the enzyme (28,66). 3-Chloroalanine is an actively utilizable quas- isubstrate (see Table VIII).

Akopyan et al. (28,65) obtained preparations of Ala(CN) synthase (lyase 4 in Table I) from blue lupine seedlings by operations ensuring

* Lyase 3 forms the selena-analog of cystathionine from serine and a-amino-y- hydroselenobutyrate at 69% of the rate of cystathionine synthesis. Its degradation by lyase 8 (P,y-elimination to 3-selenoalanine, a-oxobutyrate, and NH3) proceeds at a rate about three times more rapid than that of cystathionine ( 1 16).

28 ALEXANDER E. BRAWNSTEIN AND ELIZABETH V. GORYACHENKOVA

3500-fold purification and an overall activity recovery of -17.2% (Table VI) in homogeneous form, as indicated by disc-electrophoresis and electrofocusing. The lyase has M, - 52,000 (based on the results of gel filtration through Sephadex G-200 and amino-acid analysis). The holoenzyme is apparently monomeric; it contains 1 equivalent of firmly bound pyridoxal-P (65,66). Fully reactivatable apoprotein was obtained by gel-filtration of the holoenzyme oxime (77).

Activity assays are based on the rates of formation of H2S [determined upon conversion to methylene blue or as colloidal PbS solution (32,33,46)] or of [14C]cyanoalanine (28,66).

The optimum activity range of Ala(CN) synthase (lyase 4) is from pH 8.8 (77) to 9.5 (28,66), indicating that both the primary substrate and cosubstrate react in anionic form; the enzyme's PI is 4.7. Pure Ala(CN) synthase is stable when stored at -20°C.

Cosubstrate specificity studies (Table VII) showed that 2-mercaptoethanol can be utilized ( K , - M) instead of KCN ( K , = 55 mM). Formation of S-hydroxyethylcysteine was demonstrated with either cysteine or chloroalanine as the amino-acid substrate (28,66). Some small thiols (e.g., methanethiol and ethanethiol (721, cysteamine, etc.) can act as replacing agents, but the enzyme fails to react with other thiols (including homocysteine), sulfite, and thiocyanate (Table VII).

Cysteine and 3-chloro-~-alanine (see Tables I and VIII) are adequate primary substrates for Ala(CN) synthase. Maximum reaction rates for the L-isomers of the natural amino-acid substrate and the halogenated pseudosubstrate are nearly equal (in assays with either KI4CN or HSC2H4.0H), and are almqst two decimal powers higher than those observed with most other p-replacement-specific lyases ( V values up to 43 pmol-mg-' per minute rather than per hour, as in the case of lyases I , 2, and 3).

In accordance with the high reaction rate of Ala(CN) synthesis, the reported relative affinity constants of the synthase [KZp(~-Cys) = 2.5 and 0.55 for KCN (20), KJZ) = 1.27 and K J 2 ) = 0.12 (98)] were about 20-100-fold smaller than the corresponding values of the other P-replacing lyases for their adequate amino-acid substrates and thiol cosubstrates (see Table XV).

The action of Ala(CN) synthase is irreversible under conditions similar to those for the forward reaction (66). The major physiological function of the enzyme in plants is Ala(CN) formation from

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TABLE VII Cosubstrate Specificity of p-Cyanoalanine Synthase"

Reaction rates Cosubstrate Concentration (mM) (% of rate with KCN)

["CIKCN 2.5 100 Methanethiol -25.0b PI00 Ethanethiol 25.0 45 2-Mercaptoethanol 25.0 83 Cy steamine 25.0 23 n-Butanethiol 25.0 (-6?) t-Butyl- and Benzylmercaptan; Thioglycolate; 25.0 0

Homocysteine; GSH; Dithiothreitol; Na2S03; 2.5' 0 3-Mercaptopropionate

KCNS

" From references 65 and 66. Initial rates of H2S release from L-cysteine, in percent of rate in standard assay with KCN (= 31 kmol.rnin-'/l mg enzyme).

Concentration is not exact because of the high volatility of the compound. Tests should eventually be repeated with these compounds at higher concentra-

tions (e.g., 25.0 mM).

cysteine by @-replacement with CN- (Le., detoxication of cyanide and the first step in L-asparagine biosynthesis).

The synthase unexpectedly proved capable of utilizing 3-thiocyano-L-alanine (= S-cyano-L-cysteine) as a primary substrate (28,66), we observed that lyase 4 effects a slow reaction of cystine with KCN (but with none of the thiol cosubstrates), producing hydrogen sulfide thiocyanate ion and substantially more than one equivalent of cyanoalanine. This made clear the nature of the phenomenon (Fig. 8).

Chemical cyanolysis of disulfides (including cystine residues in peptides and proteins) in alkaline media to a mercaptan and an organic thiocyanate.

RS*SR' + HCN = RSH + R'SCN

is a familiar reaction. On interaction of free cystine with cyanide, accumulation of the expected thiocyanoalanine was never detected because this unstable compound rapidly cyclizes to 2-amino-thia- zoline-Ccarboxylic acid [see ref. (66)]. In the presence of Ala(CN) synthase, cysteine and thiocyanoalanine produced by the nonenzymic cyanolysis of cystine undergo concomitant enzymic @-replacement reactions with the excess of cyanide, releasing hydrogen


a

1 2

Figure 8. Interaction of (CN)Ala synthase with L-cystine and KCN. Production of hydrogen sulfide (curve 1) and thiocyanate (curve 2) versus L-cystine concentration (from ref. 28). Ordinate: AH2S and ACNS- in pmol from 0-20 min.

sulfide and thiocyanate, respectively, at roughly equal rates. In Fig- ure 8, the yields of these products are plotted versus the concentrations of cystine. The overall process is represented by equation (9).

( C ~ S ) ~ + 3 HCN + 2 Ala(CN) + H2S + CNSH (9)

When either iodoacetate or p-chloromercuribenzoate is incorporated into the reaction mixture with Ala(CN) synthase and cystine before KCN, so as to trap cysteine produced by cyanolysis, H2S formation is depressed, yet the yield of thiocyanate remains almost unchanged [Fig. 9; see ref. (28)]. The suitability of Ala(SCN) as an amino-acid substrate for Ala(CN) synthase was corroborated in the following way (18,28). The synthase and cyanide were allowed to interact with nascent Ala(SCN), released from its chemically synthesized, somewhat unstable, Nu-acetyl derivative within the assay system by means of kidney aminoacylase 1 (EC 3.5. I . 14). Substan- tial liberation of thiocyanate in the complete test system was demonstrated by spectrophotometry of the Fe(II1)-thiocyanate (Table IX, sample 111).

Pure Ala(CN) synthase has a pH-independent spectral absorption maximum at -410 nm (18,46). It shares the absence of significant circular dichroicity in absorption bands of the coenzyme chromo-


TABLE VIII Amino-acid Substrate Specificity of p-Cyanoalanine Synthase'

Initial reaction rates (pmol from 0-10 min)

Cosubstrate: Cosubstrate: Amino acid substrate KI4CN HS*EtOH

[2.5 mM] [2.5 mM] [25 mM]

L-Cy steine 32.6 30.3 p-Chloro-DL- Alanine 17.6 18.1 L-Cystine 2.6 - p-C y ano-L- Alanine 0 0 S-Hydroxye thyl-L-Cysteine 0 L-Ser; Ser 0-sulfate; (P)Ser; L-Ala [5-20 mM1

- - 0

a From references 28 and 66. The products determined were ['4C]cyano-~-alanine (second column) and S-hydroxyethyl1~-cysteine (third column).

phore with the spectra of other p-replacement-specific lyases (Sec- tion 1II.C).

Like other PLP-enzymes, Ala(CN) synthase is sensitive to carbonyl-modifying reagents, particularly those structurally analogous to primary substrates. Although moderately susceptible to inhibition

Figure 9. Effect of p-mercuribenzoate (p-CMB) upon production of H2S (curve 1) and CNS- (curve 2) by Ala(CN) synthase (60 mu) from L-cystine (20 mM) and cyanide (50 mM). (From ref 28.) Ordinates: AHtS and ACNS- in pmol from 0-20 min.


TABLE IX Release of Thiocyanate Ion on Interaction of Ala(CN)

Synthase with Na-Acetyl-thiocyanao-L-alanine and Cyanide in the Presence of Aminoacylase Ia

Sample Ala(CN) number Substrates synthase Aminoacylase I A470b

0.06 I1 + - + 0.04

I11 + + + 0.26

a From reference 28. Samples (vol = 2 ml) contained: substrates of 50 pmol N"-acetyl-3-thiocyano]~-alanine + 50 pmol KCN; 200 pmol Tris.HCl(pH 8.0); 120 mU cyanoalanine synthase; and 0.2 ml aminoacylase I solution. Incubation: 1 hat 30°C.

A470: absorbance at 470 nm after addition of FeCls solution to filtrate.

- I + +

by some competitive pseudosubstrates such as L-serine (with apparent Ki about 5 mM), the synthase is refractory to inhibition by L- or D-cycloserine and 3- or 4-mercapto-2-amino acids such as penicillamine or homocysteine (see Sections III.E.5 and 6). Interactions of the holo- and apoenzyme 4 with coenzyme analogs are discussed in Section III.E.l (cf. ref. (93).

Galoyan et al. (68) observed that total Ala(CN) synthase activity, as well as substrate and cosubstrate affinities, are 1-2 decimal orders higher in seedlings of white (Lupinus albus) rather than blud lupine (L. angustifolius). In extracts from the mitochondria of white-lupine seedlings, limited proteolysis causes its conversion to artifactual, catalytically active multiple forms, differing slightly in M, and PI. Purification of the Ala(CN) synthase of white lupine in presence of the protease inhibitor diisopropyl-fluorophosphonate (67) resulted in a homogeneous enzyme of enhanced specific activity (>400 unitdmg, see Table VI) and considerably increased affinities for substrate and cosubstrates (ly(mapp) was 0.87 mM for cysteine, 0.18 mM for cyanide, and 0.66 mM for mercaptoethanol; see Table XV).

White lupine (a fodder plant) differs from the blue species (a gar- den flower) in its substantially higher capacity for synthesis and accumulation of asparagine. Confirmation of the close interdependence between Ala(CN) synthase activity and asparagine biosynthesis was presented by Galoyan et al. (69). Working with developing seedlings of white lupine, they found that plots of the changes in


enzymic activities versus sprout age are parallel for Ala(CN) synthase (EC 4.4.1.9) and Ala(CN) hydro-lyase (EC 4.2.1.65), which converts the 4-nitrile group to y-carboxamide (75). In white lupine the total and specific activities of this nitrile hydro-lyase were 30- to 40-fold higher than in the blue species (69).

For various other developing plants, such as Asparagus, similar observations have been reported (73,74,76), testifying to the pre- dominance in several green plants of the pathway to asparagine via cysteine and cyanoalanine.

B. RECENT STUDIES RELATING TO LYASES OF OTHER SUBGROUPS

Below, we survey newer evidence drawn from recent studies on a$- and p,y-eliminating or multifunctional subtypes of the PLP- dependent lyases (3), dwelling primarily on features which illustrate the peculiarities of P-replacement-specific lyases .

1 . Purification and Properties of Alliinase (EC 4.4.1.4)

Stoll and Seebeck (81,82) isolated alliin, an S-containing free amino acid, ( + )S-allyl-L-cysteine sulfoxide, from garlic bulbs. In damaged bulbs this compound is rapidly decomposed by a,p-elimination on interaction with alliinase (alliin allylsulfenate-lyase, EC 4.4.1.4; eqs. 10 and 11) to pyruvate NHZ , and allylsulfenic acid which, upon spontaneous dismutation and dimerization (eq. 12), is recovered as a volatile product responsible for the pungent smell and antibacterial properties of garlic.

Alliinase was shown by Goryachenkova (83) to be a PLP-dependent enzyme. In onion, garlic, and tissues of related species of the genus Allium, homologous Salkylcysteine sulfoxides (methyl, ethyl, propyl, butyl analogs, etc. of alliin) are cleaved by similar lyases (with varying substrate specificities) to yield characteristically smell- ing allicin-like antibiotics (bis-alkyl-disulfoxides) (85). Garlic alliinase (EC 4.4.1.4) was purified sixfold; some of its properties (84) included specific, partially competitive inhibition by the reduced substrate analogs (S-alkyl or alkenyl cysteines).

Using the pure natural alliin 'diastereomer isolated from garlic bulbs as substrate [( + )S-allyl-L-cysteine sulfoxide (mp 165"C)], Ka- zaryan and Goryachenkova (86) prepared electrophoretically pure garlic alliinase (70-fold purification, with 28% recovery, as shown in Table X).

Activity assays were based on the initial rates of pyruvate release

6-REPLACEMENT-SPECIFIC PLP-DEPENDENT LYASES 35

0 t

2 C H F C H ~ H ~ S H + 2 C H d ( N + H 3 + C O O - (10)

2 CHF C(N+H3+COO- + 2 H20 + 2 NH4+ t 2 CH3-CO-COO- (11)

0 0 * *

Sum:

0 t + HZO

2 C H d H 4 H 2 - S --CH&HN+H3<OO- - alliin

0 t

C H d H < H - S * S - C H 2 4 H = C H 2 t

allicin

at pH 6.5 (estimated by photometry of its 2,4-dinitrophenylhydra- zone or by spectrophotometric monitoring with NADH and lactate dehydrogenase). Specific activity of the alliin lyase (enzyme 5 ) is expressed in pmol of pyruvate per minute per milligram of enzyme protein.

Purified alliinase is quite labile, but it can be stored without substantial inactivation for - 1 month at - 5°C in a neutral buffer solution containing 2.lO-’ M PLP and 10% glycerol.

Activity optimum is at pH 6.2, which coincides with PI of alliinase, as indicated by ampholine isoelectrofocusing (86).

On equilibration in a PLP-containing buffer and gel-filtration through Sephadex G-200, an M , of 130,000 was determined for alliinase. When the enzyme was denatured by 2 hr treatment at 25°C


TABLE X Purification of Alliinase from Garlic Bulbs (60 g)

Activity" Total

protein Total Specific Recovery Fractionation step (mg) (U) (Usmg-') (%I

a. Extracts from frozen bulbs ground 1650 6600 4.0 100 in 2 x lo-' M phosphate buffer (pH 7.5) containing 10% glycerol and 1 0 P M PLP

Sephadex A-50

0.5 saturation

b. Chromatography on DEAE- 167 6O00 36.0 91

c. Precipitation with NHrsulfate at 96 5780 60.2 87.6

d. Filtration through Sephadex G-200 2980 133.2 45.1 e. Fractionation with hydroxyl-apatite 7 1872 267.1 28.4

" From reference 86. Reaction rates were measured on 2 min incubation with 2.5 pmol alliin and 0.1 pmol PLP in 2.5 ml of Na-phosphate buffer (200 pmol, pH 6.5) at 23". One U of alliinase produces 1 pmol of pyruvate per minute in this assay system.

with 6 M urea, and filtered ohrough Sephadex G-100 in a 50 mM phosphate buffer containing 6 M urea and lo-' M KCl, an M, of -65,000 was estimated. The holoenzyme is thus apparently a dimeric protein. Active alliinase preparations contain (per dimer) six equivalents of PLP, which is retained on gel filtration; most of the coenzyme is released on acid denaturation. Spectrophotometry of the pure enzyme (5) in the visible range shows one symmetrical absorption band with Amax at 430 nm, overlapping with a positive CD extremum (86); the peak is pH-independent in the range from 6.5 to 8.2. The purest alliinase preparations have absorbancy coef- ficients of A:% = 16.2 and A:% = 2.3. Thus, the subunit has a molecular absorbancy at 430 nm of 14,900 M-'.CM-' (78,86). In the presence of excess substrate, Amax at 430 nm is partially reduced, with no substantial shift in wavelength (l4,95,96). Apparently, each subunit has one catalytic center with one productively and strongly bound coenzyme molecule; two further PLP molecules can bind nonproductively but firmly, outside the catalytic site, with similar spectral parameters. In these conditions, it is not possible to titrate or calculate directly the true K,, or dissociation constant of the productively bound coenzyme. The Michaelis constant, K,,, , for al-


liin was determined graphically by the reciprocal plotting of reaction rates in the concentration range from 5 x lop5 to 5 x M alliin by the Lineweaver-Burk method; the apparent K,(alliin) value was 5 X lop4 M (86).

Apoenzyme preparations of low stability were obtained by filter- ing hydroxylamine- or hydrazine-inhibited alliinase through Seph- adex G-25 or G-50 at low temperatures. They could be rapidly recombined with either PLP or one of the activating or inhibitory coenzyme analogs (87) (cf. Section 1II.E. 1).

Native alliinase is sensitive to inhibition by typical carbonyl reagents, in particular those with substrate-like affinity to the binding site (87). Structural analogs of alliin (e.g., the deoxidized S-alkylcysteines and L-cysteine itself) are effective inhibitors (84,87). Al- liinase, and other a$-eliminating and multifunctional lyases, have a moderate sensitivity toward competitive inhibition by 3- and 4- mercapto-Zamino acids, and are highly susceptible to inhibition by L- and D-cycloserine (see Fig. 10 and Sections III.E.5 and 6). One of the most effective irreversible inactivators (Iso = 0.6 mM) of the enzyme is p-cyano-L-alanine (87).

I00 c

Figure 10. Time course of the inactivation of alliinase in the presence of M L-

cycloserine (1). lo-’ M Dcycloserine (2), ZlO-’ M p-cyanoalanine (3), 5.10-2 M hydroxylamine (4), and M aminooxyacetate (5). (See ref. 87.)


2. Observations Concerning y-Cystathionase (EC 4.4.1.1) and Some Other Eliminating or Multifunctional Lyases

This section is confined to a concise survey of data concerning several lyases possessing elimination activity, which were used or considered in our studies for comparison, for example, cystathionase (enzyme 8) and serine dehydratase (EC 4.2.1.13, enzyme 6) from rat liver and E. coli tryptophanase (EC 4.1.99.1, enzyme 7).

In 1973, y-cystathionase, or homoserine dehydratase (EC 4.4.1.1 ; formerly EC 4.2.1.15), was prepared by us in highly purified state from rat liver (36,91) mainly according to the procedure of Kato et al. (90). The last step (crystallization) was replaced by gel filtration through a Sephadex G-200 and fractionation on a column of CM- Sephadex G-50. The preparations had specific activities of 250-300 pmol per hour (4.2-5.0 pmol.min-') per milligram of enzyme protein in the homoserine deamination reaction.

Twofold higher specific activities (166-fold purification with 30% overall recovery) were recently achieved in our laboratory (1 18) by an improved procedure including cautious fractionation with ethanol at temperatures below O'C, and ion-exchange chromatog-

TABLE XI Purification of ycystathionase from 320 g of Rat LiveP

Activity Total

protein Total Specific Recovery Purification Fractionation Steps (mg) (U) (Umg-') (%) (n-fold)

(1) Cellfree extract 16,520 980 0.06 100 1

(3) 0.5 saturation with NH4- 3,120 803 0.26 83 4.3

(4) 0.5-0.8 NH4-sulfate 1,800 659 0.32 67.5 5.4

(2) Heating to 5840°C for 10 5,184 850 0.16 88 2.7 min

sulfate (supernatant)

saturation (precipitate)

sulfate saturation by equal volume of ethanol

Sephacel

(5) Precipitation at 0.28 NHr 76 354 5.48 46.3 91.3

(6) Fractionation on DEAE- 30.0 300 10.0 30.6 166.7

From reference 118. One unit (U) = 1 pmolmin- ' of a-oxobutyrate released from lo-* M DL-homoserine.

$-REPLACEMENT-SPECIFIC PLP-DEPENDENT LYASES 39

raphy on DEAE-Sephacel (Table XI). Crystallization of the enzyme was achieved from aqueous polyethylene glycol solutions buffered to pH 7.5-7.7 with 0.05-0.1 M potassium phosphate. The pure lyase sedimented on ultracentrifugation as a homogeneous protein; isoelectrofocusing showed a PI of 8.56 (1 181, in accordance with findings reported in 1979 (120). In deep freeze (at -5OOC) a 1% solution of the enzyme in K-phosphate buffer (pH 7 . 9 , stabilized with PLP, EDTA, and dithiothreitol, retains full activity for several months.

Cystathionase is a multifunctional lyase catalyzing p,y-elimination reactions (3) of cystathionine, homoserine, and homocysteine, and ol,p-elimination from cystine and cysteine (1 14-1 15a); djenkolic acid, lanthionine, and a$-diaminopropionate are also substrates (97,115). The reactions of mercaptoamino acids with this lyase account for a major part of the “cysteine desulfhydrase” activity of animal and microbial cells. Reversibility of these elimination reactions has not been studied systematically. Chatagner and coworkers (I 14,115) reported elimination-mediated. y-replacement activity of the enzyme: from L-homoserine it synthesized small amounts of L-

cystathionine on incubation with L-cysteine, and of D-allocystathion- ine with D-cysteine.

In higher plants L-cystathionine is synthesized by a cystathionine y-synthase that differs from those of microorganisms and animals in its capacity to use phosphohomoserine as a preferred C4 aminosubstrate (22).

Spectrophotometric investigation of high-purity y-cystathionase (1 18) showed a typical coenzyme-linked absorption peak with A,,, 427 nm, coincident with a positive CD extremum. Values calculated per monomeric subunit were: molar absorptivity, A4z7 = 5200 M-’.cm-’, and coefficient of molar dichroicity, at 427 nm = 10. [For cystathionase incubated with homoserine, a band with A,,, = 4 12 nm has been reported (1 14)].

As estimated from the data of ultracentrifugal sedimentation (and gel filtration), M , is -18,000 & 2,000 for native enzyme (and the apoenzyme) and 45,000 for the (identical) subunits produced by denaturation of cystathionase with Na-dodecylsulfate or in 6 M urea (118,119). The tetrameric structure of the enzyme is confirmed by electron micrography (1 19) and by the presence of four productively bound molecules of PLP in the native holoenzyme. (Nonproductive binding of additional PLP-molecules has been observed.) In the na-


tive holoenzyme, the four PLP molecules differ in ease (rate) of resolution from the active sites, as well as in reactivities with substrates, quasisubstrates, and various inhibitors (91,95,96,114-115a). These findings indicate the presence of two types of PLP-binding sites in the enzyme. Presumably, sites of one type serve to bind and activate amino-acid substrates with a C3-chain, and the other ones are involved in anchoring of C4-substrates. According to Churchich et al. (99, the bonding of PLP in cystathionase is characterized by two binding constants: K,, = 7.5 x lo5 M-I and K,, = 8.3 x lo4 M - I .

Loupou et al. (1 19) demonstrated that the main factor responsible for the stability of the enzyme's tetrameric structure is coenzyme binding. Electron micrographs of native enzyme show revalence of structures consisting of four globular particles (40-50 K diameter) arranged as an isologous square (80-90 A*). In undenatured apoenzyme (prepared by gel filtration of oximino-enzyme), the arrange- ment of subunits looks less compact in electron-micrographs, and dissociation in urea solution to dimers and monomers is facilitated.

In contrast, reduction of the internal aldimine bonds in the holoenzyme with NaBH4 tightens the subunit linkage, so that the tetrameric structure is not broken down under the same conditions. Also (119), the holoenzyme's quaternary structure is not affected by pH (in the range from 4.65 to 9.5 at ionic strength = lo-' M) or by the blocking of accessible HS groups with DTNB or p-CMB. (See refs. 14,90,91,95-97,114, and 115a for additional data concerning the properties of cystathionase.)

We used the purified enzyme in comparative studies regarding interactions of PLP-dependent lyases with diverse ligands, isotopic hydrogen exchange, and so forth. The results are presented in relevant parts of Section 111.

In 1979-1982, Dwivedi and coworkers (91a) selected, by means of DNA hybridization and cloning techniques, an E. coli strain which is able to overproduce P-cystathionase by 100-fold. Using this strain, they developed a three-step purification procedure that gives 90% pure preparations of P-cystathionase in 54% yield, with specific activity up to 263 p,mol.min-'/mg (100-fold higher than their best preparations of y-cystathionase from rat liver); but cf. Table XI, p. 38 (118). Physical, chemical, and catalytic features of the enzyme, which is responsible for an essential step in methionine biosynthesis, were presented (91a).


L-Serine (threonine) dehydratase, or deaminase (EC 4.2.1.13, enzyme 6), from rat liver (88,90) was used to examine its sensitivity to inhibition by cycloserine (Section III.E.5) and other inhibitors. The preparations were partially purified to a specific activity of 30- 50 pmol.min-'/mg.

The pure rat liver enzyme (88) has Mr - 63,500 and an absorption peak at 414 nm. It is a dimer and contains two equivalents of PLP. One-way a$-elimination reactions of L-serine, L-threonine (or al- lothreonine), and other p-hydroxy a-amino acids are catalyzed in some animal species by one lyase and in others by two or more. In microorganisms and higher plants, these enzymes occur in multiple forms (e.g., biosynthetic and biodegradative isoenzymes), widely differing in physico-chemical and regulatory properties. These are discussed in detail in reference 14.

Tryptophanase, or tryptophan indole-lyase (EC 4.1.99.1, enzyme 7) was used in the form of crude extracts from E. coli cells to study its inhibition by D- and L-cycloserine. (Other characteristics of the enzyme, described in Section 111, are from references 14 and 15.)

The pure E. coli lyase (15) is a tetramer of Mr - 220,000, containing four loosely bound molecules of PLP. Its specific activity (with PLP in excess) varies, depending on experimental conditions, from 15 to 24 pmol indole formed per minute per milligram. The enzyme has ambivalent group-specific catalytic activity. It promotes

of tryptophan and its analogs bearing CH3, HO groups, or C1 as substituents in positions 4, 5 , 6, or 7 of the indole nucleus, as well as similar reactions of cysteine and other p-substituted allosubstrates (see Table I and refs. 15 and 17). With high concentrations of pyruvate and ammonia, and a moderate supply of indole, the

enzyme can act in reverse (I) synthesizing L-tryptophan (107). Tryp- tophanase occurs in different microorganisms in a variety of multiple forms markedly dissimilar in molecular features, metabolic functions, and catalytic properties (15,105,197).

This is also true for the various forms of microbial p-tyrosinases (tyrosine phenol-lyases, EC 4.1.99.2), which are ambifunctional p- lyases incapable of utilizing tryptophan (or it analogs) as a primary substrate, but which closely duplicate most other properties of the tryptophanases, including the capacity to catalyze a$-elimination reactions reversibly'(r)- (16,108).

a,p-eliminationzand a variety of p-replacement reactions ( 1 + 11)

t

A


111. Comparative Survey of Physicochemical and Catalytic Features of f3-Replacement-Speciflc and Some Other PLP-

Dependent Lyases

In this section we examine concisely the similar and divergent features of P-replacing and other types of lyases, with emphasis on signifcant differences in their catalytic mechanism. Some properties of the enzymes were considered in the preceding sections.

A. SIZE AND STABILITY PARAMETERS: QUATERNARY STRUCTURE; OLIGOMER c) SUBUNIT AND HOLO c) APOENZYME EQUILIBRIA;

CATION EFFECTS

Both in the P-replacing and other subgroups, there is broad var- iability in size and stability parameters (Table XII). The M, ranges from 37,000 (E. coli D-serine dehydratase) to 220,000 (bacterial tryptophanases), and subunit structures are monomeric (with one PLP- containing active center) to tetrameric (with four catalytically active PLP molecules); dimeric lyases are predominant. Larger particles of mass, 3250,000 daltons, with higher numbers of protomers, rep- resent products of artifactual aggregation; they are usually inactive. Nonproductive firm binding of additional coenzyme molecules outside active centers-up to 4 PLP or more per oligomer-has been observed with some eliminating lyases (e.g., alliinase, y-cystathionase, and others).

The stability of active oligomers is variable and usually depends on a number of factors, such as the concentrations of protein and coenzyme, pH, temperature, ionic environment, and so on. In most cases coenzyme removal, increase in pH, and low protein concentration displace the equilibrium in favor of smaller species (mono- or dimers).

Variations also occur in the ease of resolution (respectively, reas- sociation) of apo- and coenzymes. K,, values are often much higher then the true dissociation constants, KO; to estimate these is not easy because apo f* holo equilibrium is only slowly reached at low commensurate concentrations of apo- and coenzyme (14,15,79,113). Thus, tryptophanase (7) from E. coli (15) is completely resolved during purification to PLP and stable apoenzyme, which exists in several interconvertible forms of variable quaternary structure. Cys- teine lyase ( I ) and alliinase (5) are largely resolved during purification, and the released apoenzymes readily undergo denaturation


unless speedily recombined with PLP or protected by stabilizing agents. However, PLP is firmly bound in serine sulfhydrase (2), cyanoalanine synthase (4, and serine dehydratase (6). In these, the internal aldimine bond remains essentially intact during the purification procedure, and must be broken by special treatment (e.g., by means of carbonyl reagents) to achieve complete liberation of the apoenzyme. Stability of the latter is low, and in some cases (e.g., with enzyme 2), optimum conditions for reconstitution of the holoenzyme (phosphate-free buffer of pH close to neutrality) differ from those favoring resolution (see Section II.A.2 and refs. 41 and 42).

In several eliminating or ambifunctional a,p-specific lyases, the mode and strength of coenzyme binding is modified by monovalent cations; relevant data and earlier literature are reviewed in references 14,15,105-109, and 112. Thus, NH4+ (2-6 mM), K + (8-20 mM), T1+ (0.35 a), or Rb+ are essential for activity of all bacterial tryptophanases, and their effects are antagonized by Na+ or Li' (15,105). Strong activation by NH4+ or K', and deactivation by Na+, was also observed with tyrosine phenol-lyase (108a), tryptophan synthase (106,112), and threonine and serine dehydratases from mammalian tissues, plants, and microbes (14). Absorption spectra, pH-activity dependence, and so forth (14,15,105,108a,109) indicate that potassium and ammonium ions lower the p K , of the imino N in the internal PLP-lysine aldimines increase the strength of inter-subunit cohesion and the affinities of the apoenzymes for PLP and of holoenzymes for substrate; opposite changes are induced by Na+ or Li'.

Interactions of the p-replacing lyases with monovalent cations have not yet been examined in detail. Currently available observations relating to serine sulfhydrase (Goryachenkova and coworkers) and to cyanoalanine synthase (67) indicate that the monovalent cations either are indifferent or moderately affect the substrate affinities and activities of replacing lyases in directions opposite to their influence upon eliminating and multifunctional lyases. Namely, K + and NHZ tend to exert a negative influence, and Na' a positive one, on the catalytic parameters, as shown in Table XIII; however, conclusive evaluation is hindered by the simultaneous inhibitory effects of anions (especially inorganic ones). (See ref. 67 for further data concerning the effects of anions and of mono- and bivalent cations upon Ala(CN) synthase.)

P

P

TA

BL

E XU

Mol

ecul

ar P

aram

eter

s of

PL

P-D

epen

dent

Lya

ses

(Sue

, Q

uate

rnar

y St

ruct

ure,

Coe

nzym

e A

ffin

ity, a

nd I

soel

ectr

ic P

oint

)

Mol

ecul

ar

Enz

yme

(Cod

e nu

mbe

r, n

ame

wei

ght

Sub-

PL

P

Apo

enzy

me

and

sour

ce)

(x 1

0V

) un

its

(mol

es)

stab

ility

f&

'pp)

or

PD

PI

Ref

eren

ces

p-R

epla

cing

1.

C

yste

ine

lyas

e (c

hick

en

120

2.

Seri

ne s

ulth

ydra

se

90

&3

Y

Olk-

SaC)

(chi

cken

live

r)

2a.

Seri

ne s

ulfh

ydra

se

60

%2

3.

Cys

tath

ioni

ne p

-syn

thas

e 11

2 (b

aker

's ye

ast)

(rat

live

r)

(ten

ds to

4.

Ala

(CN

) sy

ntha

se (

lupi

ne

52

aggr

egat

e)

seed

lings

) E

limin

atin

g or

mul

tifun

ctio

nal

5.

Aui

inas

e (g

arlic

) 13

0

2 ve

ry l

abile

2 la

bile

? la

bile

(?)

2 la

bile

1 re

lativ

ely

stab

le

2 ra

ther

( +

4 PL

P b

ound

fi

rmly

out

side

ac

tive

site

)

unst

able

larg

ely

reso

lved

dur

ing

4.8

32,3

3 pu

rifi

catio

n

reco

nstit

utio

n of

ho

loen

zym

e at

pH

7.

0)

Kp

~p

= 1

.10-

" (fu

ll 6.

0 26

,41,

91

fip

= 2

.10

-~

4.8

48,W

Kp

~p

= 4

.3 x

5.

5 26

,92a

(f

ull r

econ

stitu

tion

in

pH r

ange

7.0

-8.5

)

1.1

x 1

0v

77

K

p~

p = 4

.10-

5; K

D =

4.

8 65

,67,

68,

affi

nity

hig

h, b

ut i

ll-

6.2

86,8

7 de

fine

d

6.

L-S

erin

e de

hydr

atas

e (r

at

63.5

2

6a

. D

Ser

ine

dehy

drat

ase

(E.

40

I

7.

Try

ptop

hana

se (

E. c

oli

) 22

0 4

liver

) (m

ay f

orm

ag

greg

ates

)

coli)

8.

y-C

ysta

thio

nase

(ra

t liv

er)

160

4

2 fa

irly

sta

ble

PL

P a

ffin

ity h

igh,

?

14

incr

ease

d by

K+

ions

1 ?

=

KD

=

? 14

3.

10-*

4

stab

le

K$fp

ep,

= 2

.10-

6 ?

14,1

5 (c

ryst

aliz

es);

ex

ists

as

mul

tiple

2-

and

4-m

etri

c co

nfor

mer

s 4

rela

tivel

y bi

ndin

g of

2 o

r 3

PL

P is

8.

5 91

,93,

(m

ay b

ind

stab

le

mor

e tig

ht (

Kp

~p

=

95

%

addi

tiona

l PL

P

3.W5) an

d ra

pid

(k'

115a

. no

npro

duct

ivel

y)

= 3

700

M.

min

-')

118,

119

th

an o

f th

e fo

urth

(k'

= l

o00

Mm

in-I

) P

LP

(K

p~

p

= 1

4.10

-5

~

K'S

p) or

KP

LP

valu

es w

ere

mea

sure

d un

der

the

cond

ition

s gi

ven;

the

true

aff

initi

es a

t eq

uilib

rium

( KD

) are

in m

ost c

ases

muc

h hi

gher

.

P

ul


TABLE XI11 Effects of Cations and Anions Upon the Activity of Ala(CN)Synthase From White

Lupine Seedlings" The lyase reacted with L-CYS + NaCN in 0.1 M Tris-acetate buffer, pH 8.8, at 25"C, after 10 min preincubation with the salts tested. Activity assay was based on esti- mation of sulfide released (photometry as colloidal PbS solution at 360 nm in Beckman spectrophotometer).

Salt concentrations ~

10-3 M 5 x 1 0 - 3 ~ M

Inhibi- Activa- Inhibi- Activa- Inhibi- Activa- tion tion tion tion tion tion

salts (%) (%I (%I (%) (%I (%)

Na(CH3COO) - 11.0 - 16.0 - 5.0 NaCl 6.0 - 8.0 - 10.5 - KCI 44.0 - 47.0 - 65.0 - K2CO3 - 15.2 6.5 - 19.0 - ( N H M 0 4 36.0 - 38.6 - 40.5 - N H E l 6.5 - 16.5 - 29.5 - (NhhCO3 8.0 - - 21 .o - 26.0

" From reference 67.

B. COMPARISON OF RELATIVE REACTION RATES AND SUBSTRATE AFFINITIES

Such comparisons are roughly approximative in regard both to reaction rates (because the conditions for activity assay are widely different and often nonoptimal) and to substrate affinities-since the reported estimates are often based on Michaelis constants ( K m ) , that is, the substrate concentration at which v = V/2) , rather than on substrate constants,

which are the true equilibrium (dissociation) constants of the active complex.

Most lyases have group-specific catalytic activity toward several related substrates; the relative values of specific activities for natural substrates and their analogs (qaasisubstrates) may differ with allelic varieties of the enzymes.

Table XIV lists relative-reaction rates of lyases 1-4 with several replacing agents (cosubstrates). Their K',"pp)(K, or Ki) values for


the main amino acid substrates and cosubstrates are shown in Table XV.

Extensively purified P-replacing lyases usually displayed fairly low initial reaction rates, in the range of 10-50 p,moI.hour-'.mg-'. However, preparations of cystathionine P-synthase (3) were obtained recently with specific activities of 160 pmol-hour- 'nmg-' from human liver [estimated purification 3000-fold) (52)], and 270 pmol-hour- 'emg-' from rat liver [stated purification 2250-fold (92)], in the P-replacement reaction between L-serine and homocystein.*

TABLE XIV Relative Cosubstrate Specificities of Selectively P-Replacing Lyases"

Initial rates of H2S release from L-CYS are shown as a percentage of the rates observed with the main natural cosubstrate."

Serine Cystathionine Ala(CN) Cysteine sulfhydrase P-synthase synthase

Cosubstrate lyase (chicken liver) (rat liver) (blue lupine)

-(C y steine)' Homoc y steine' Sulfite' Cyanided 2-Mercaptoethanol' Cysteamine' Methanethiol' Ethanethiol' Benzylmercaptan' Thiogl ycollate" 3-Mercaptopropi~nate~ Thiocyanate (rhodanide)"

(43Ib 0"

100" 0

200"

78" 66a

9" 7"

-

-

0 100

0 0

85 27 9

0 6 4

0 100

0 0

71 42 24

0 5 0

100 83 23

* I 0 0 45 0 0 0 0

" From reference 18. H2S formation during the first 10 min of incubation, over and above the amount formed in absence of added cosubstrate. ' With cysteine lyase the main amino acid product is lunfhionine, that is, a second

molecule of Cys reacts as cosubstrate. This reaction is suppressed in the presence of sulfite or an adequate thiol cosubstrate. ' Concentration in incubation mix = 25 mM.

Concentration in incubation mix = 2.5 mM. ' Concentration added was -25 mM; actual concentration during incubation was

lower, owing to the high volatility of CHsSH.

* Homonymous P-replacing lyases from diverse sources (allelozymes) or differing grade of purification usually show a positive correlation between initial reaction rates (V@PP)) and affinities for aminosubstrates and cosubstrates (values of l / K m ) . This trend is evident from the data in Table XV.

P

00

1.

2.

2a. 3. 4.

TA

BL

E XV

Mic

hael

is (

K,)

and

Mi

ty

(Ki,

K,)

Con

stan

ts (in

mM

) fo

r p-

Rep

laci

ng L

yase

s

Subs

trat

e pa

irs

and

valu

es f

or s

ubst

rate

and

cos

ubst

rate

Cys

tein

e ly

ase

(chi

cken

- em

bryo

yol

k-sa

c) (3

2,33

) Se

rine

sul

fhyd

rase

(ch

icke

n liv

er)

(26,

41)

Seri

ne s

ulfh

ydra

se (

bake

r's

Yea

st) (94)

Cys

tath

ioni

ne p

-syn

thas

e (r

at li

ver)

(26)

(r

at li

ver)

(92a

)

(hum

an li

ver)

(52

) &

Cya

noal

anin

e sy

ntha

se

(blu

e lu

pine

) (20

) (b

lue

lupi

ne) (

93)

(whi

te lu

pine

) (6

7)

8.3

7.7

22.0

13

.0

[KdS

er)

= 8

.01

[&(I)

=

6.9

K

s(2)

= 3

.6

36.0

24

.0

-

-

2.5

210

-

-

-

34.0

-

8.0

2.0

[K,C

YS)

=

11 .O1

1.

15

-

-

-

-

83.0

-

18.0

?

0.59

-

-

-

2.5

0.55

[K

dL-S

er) =

11.

01

0.87

0.

18

[K, =

1.2

71

[Ks(

2) =

0.1

21

[ k",

PP(2

-rne

r-

capt

oeth

anol

) =

0.6

61


In contrast, specific activities of 300 pmol.mg-’/min or higher are readily attained on purification of a,@-eliminating or multifunctional lyases.

However, our high-purity preparations of the P-replacing cyanoalanine synthase, 4, had specific activities in the range of 40- 400 pmol-min-’.mg-’ (see Section II.A.4).

It thus appears that the differences in degree of catalytic efficiency observed in @-replacing and other PLP-dependent lyases are not an essential distinctive feature.

C. SPECTRAL FEATURES OF THE PYRIDOXAL-P-DEPENDENT LYASES IN THE VISIBLE RANGE

Like other PLP-enzymes, eliminating a,@- or @,y-active lyases have typical spectral absorption bands in the 410-430 nm range (usually associated with positive circular dichroism peaks), which are assigned to protonated “internal” PLP-lysine aldimines (14,15,110); a smaller band at 335-340 nm, also with positive CD, may be contributed by the nonprotonated internal Schiff base.

In the replacement-specific @-lyases (I, 2 , 4 ) , only a shallow, virtually nondichroic band with A,,, near 415-420 nm is seen in the absorption spectrum of the free holoenzyme (20). The position of this peak is not substantially shifted when an HS-free amino-acid substrate, substrate analog, or cosubstrate is added; although its size may decrease (e.g., in lyase I with cysteine) without the appearance of new peaks in the 325-335 nm range (PMP-ketimine or substituted aldamine forms) or above 470 nm (E.Tolosa). In serine sulfhydrase (2), no spectral change is caused by serine; however, on interaction with cysteine, the 430 nm band is sharpened and shifted slightly to the right (see Fig. 1 1 , from ref. 41). Data concerning the effects of other thiol compounds on this spectral band in lyases 2, 3, and 4 are contradictory and require detailed reinvestigation.

Spectrophotometry of complexes of elimination-active lyases with substrate-type ligands often reveals a conspicuously high absorption peak in the 490-550 nm range, coincident with a sharp usually negative CD extremum (see Figs. 8 and 9 in ref. 15 and Fig. 9 in ref. 112).

Similar peaks occur in the spectra of S.E or a1loS.E complexes of other PLP-enzymes, for example, transaminases, amino acid decarboxylases, serine hydroxymethylase, and others (4,12,14, 15,110). Such peaks, indicating an extended conjugated mystem,


z 100 L 3 A -

f

2 4

20 60E 0 I

I

Tlms lh ) Time (h

Figure 11. Incorporation by cysteine lyase (A) and serine sulfhydrase (B) of 3H into amino acid substrates and reaction products (27). Relative specific radioactivities (percentage of specific radioactivity of 'HHO in incubation medium) calculated for a-H atom in L-serine ( l ) , L-cysteine (2), and the preplacement product Shydroxy- ethyl-cysteine (3 from cysteine, and 3a from serine) in assay samples with cysteine lyase (A) and serine sulhydrase (B). Line (4) in (B) is the relative specific radioactivity of L-cysteine on incubation in the presence of sulphite. Samples (1 ml) contained 15 pmol amino acid substrate, 45 pmol cosubstrate, and 9 units of enzyme.

are attributed to deprotonated substrate- or allosubstrate-enzyme imines in tautomeric quinonoid forms (such as 2 in Fig. 3) or-when situated in the 455-480 nm range-to a$-unsaturated PLP-aldimines (14,15). They are transient in the spectra of complexes with adequate substrates, and may be persistent in certain a1loS.E complexes.

We never observed any long-wave, dichroic maxima of this kind in substrate- or allosubstrate-enzyme absorption spectra of the replacing lyases 1-4. This has cast further doubt on the occurrence of quinonoid Schiff bases (or A"*P-aldimines in equilibrium with them) in their reaction cycles (see Section IV).

D. ISOTOPIC EXCHANGE OF HYDROGEN ATOMS AND P-SUBSTITUENTS

Labilization of a-hydrogen in amino-acid substrates, and its release or isotopic exchange with aqueous solvent, occurs in several types of PLP-dependent enzymic transformations of amino acids. These include those reactions which are initiated by breaking the a- C-H bond (3,8,8a,79,1 lo), among others-all elimination and replacement reactions effected by p- or y-specific PLP-containing lyases (see Section I.B.2).

b-REPLACEMENT-SPECIFIC PLP-DEPENDENT LYASES 5 1

The liberated labeled in H atoms either enter the solvent pool or may move (in toto or in part, usually stereospecifically) to some other position in reaction participants, by acceptor-mediated transfer or direct intramolecular shift. (The mechanisms involved are discussed in refs. 99 and 100; see also refs. 78 and 92.)

In some cases (e.g., aspartate aminotransferase) (1 lo), the elimination-active lyases-cystathionine y-synthase (79), tryptophanase (15,105), and some other PLP-enzymes-the rates of isotopic a- hydrogen exchange can be severalfold higher than those of the complete enzyme-catalyzed reaction.

In the early 1960s in studies with partially purified avian enzymes: cysteine lyase (1) from chicken-embryo yolk-sac and hepatic serine sulfhydrase (2)-Sentenac et al. (35,44a) observed enzyme-catalyzed hydrogen exchange in the amino acid substrates L-cysteine and L-serine, at rates commensurate with those of the specific @-replacement reactions undergone by these substrates. The authors assigned the observed isotopic exchange to a-H of the substrate, in accordance with the theory of pyridoxal catalysis (8,Il). They also reported that lyase 2 catalyzes very slow exchange of the (3-substituent group of the substrate molecule for HO- or HS- ions from the incubation medium, at rates of approximately 2% of the a- hydrogen exchange (44a, cf. 92).

Tolosa et al. (27,33) found that cysteine lyase, 1 , in the presence of an appropriate cosubstrate catalyzes the exchange of one hydrogen atom (a-H) at practically identical rates in (excess) amino-acid substrate, L-cysteine, and in the competitively inhibitory substrate analog, L-serine. S-hydroxyethyl-L-cysteine, a @-replacement end product when incubated in the same way in the presence of homonymous P-replacing agent (Zmercaptoethanol), underwent hydrogen exchange at a slower rate.

Under these experimental conditions (27), similar results were obtained with extensively purified preparations of cysteine lyase (1) and hepatic serine sulfhydrase (2-3). Neither enzyme induced isotopic a-H exchange in close structural analogs (except L-serine) that were inactive as amino substrates (e.g., alanine, threonine, allo- threonine, 3-phosphoserine, and D-serine).

When the two lyases were incubated for 1.5 and 3 hr, with adequate cosubstrate and ~-[@-~H]serine or ~-[@-~H]cysteine, the specific radioactivity of the reaction end-products was the same as in the initial amino-substrates, and no tritium appeared in the solvent


water. The same lyases were incubated with nonlabeled L-serine and cosubstrate in 3H2'80 ("0 was 32% of the total oxygen content). When 0.5 atom-% of H had been labeled (evidently in the a-position), which required 3 h incubation with serine sulfhydrase and 24 h with cysteine lyase, analysis in a photoionization mass-spectrometer failed to reveal uptake of l8O in the hydroxyl group of serine (within the 1% sensitivity limits for detection).

In experiments with 3H-containing aqueous medium and lyases I or 2, the tritium content (atom-%) of a-hydrogen in the P-replacement product (cysteic acid, cysteine thioether) was the same as in the tritiated water of the solvent medium (Fig. 11). In addition, label was incorporated at a comparable rate into nonreacted (excess) amino acid substrate and into the inadequate inhibitory substrate analog, L-serine in experiments with lyase I ) (see ref. 27 and Table XVI).

The effects of supplementation with thiol cofactor (mercaptoethanol) or with inadequate thiol (n-butylmercaptan) upon the rates of a-hydrogen labeling are shown in Fig. 12.

Following preincubation of the lyases, without adding cosubstrate for 1 h, a slow residual tritium uptake from 'H20 into the substrate or pseudosubstrate amino acid was still observed (Fig. 12). This might be attributed to cosubstrate activity of residual mercaptoethanol retained in the enzyme preparation (which should be largely

TABLE XVI Stoichiometry of Reaction Product Formation and Incorporation of Labeled Hydrogen into Product and Nonreacted Substrate by p-Replacing Lyases"

Product of p- Substrate replacement

Amount of labeled H (pg-atom) in

(amino acid) ( wol) Product Substrate Total

Serine Sulfhydrase

L-Serine 3.0 2.9 2.8 5.7 L-C ysteine 3.8 3.8 2.2 6.0

Cysteine Lyase

L-Serine 0 - 2.1 2.1 L-C ysteine 2.5 2.5 2.3 4.8

From reference 27. Cosubstrate-2-mercaptoethanol. Incubation-3.5 h at 37°C.

p-REPLACEMENT-SPECIFIC PLP-DEPENDENT LYASES

I 5

53

rime (h ) Tlma n) Fig. 12. Effect of thiol cofactors on the rate of a-H labeling in L-serine (27). Relative specific radioactivity of the a-H atom of L-serine with cysteine lyase (A) and serine sulfhydrase (B) in the presence of 2-mercaptoethanol(3), of n-butylmercaptan (2), or without thiol cofactor ( I ) . Composition of assay samples: 20 pmol of substrate amino acid, 60 pmol of thiol cofactor (added after the first hour of incubation), and 2 mU of enzyme.

expended during the first hour of incubation), of cysteine itself (in the case of lyase I), or of water (with lyase 2; see eq. 5 ) (97).*

In experiments with Ala(CN)synthase preparations from white lupine of extremely high specific activity (see Section II.A.4), Gal- oyan, Tolosa et al. (68) observed the incorporation of small amounts of 3H from solvent water into a-hydrogen of p-chloroalanine (a very active amino acid substrate of the synthase) in the absence of ex- traneous cosubstrate (Fig. 13).

The results of the studies considered above (and additional evidence) demonstrated that both the a-hydrogen exchange associated with reactions of amino acids catalyzed by various PLP-dependent

* In 1982, Borcsok and Abeles in particular-on Table IV (92), reported findings in apparent conflict with the results (27) presented above. In experiments with approximately 50% pure cystathionine p-synthase (from rat liver), stored with 1 mM mercaptoethanol and dialyzed overnight (to eliminate the thiol) prior to actual assay, these authors observed a-’H release from m-serine at pH 7.8 in the absence of added cosubstrate at a rate (0.25 pmol/min) about 4 of that (0.75 pmoYmin) in the presence of 64 mM DL-homocysteine. The rate of a,p-elimination (pyruvate formation) was 0.01% that of inadequate p-replacement (e.g., chloroalanine t) serine interconversion) under similar conditions was -5% of the a-3H release (92). Interpretation of these findings is discussed in Section IV.


30' 60' m' rime, min

Figure 13. Time course of isotopic a-H exchange in substrate (L-cysteine, curves 1-31 and product (P-cyanoalanine, curve 4) catalyzed by P-cyanoalanine synthase, without cosubstrate (curve 1) and with 2-mercaptoethanol (curve 2) and KCN (curves 3 and 4). (From refs. 18 and 28.) Ordinate: specific radioactivities imp/min/pg-atom H (solvent 'HzO, line 5, was 1.8 x lo-').

lyases, and the substitutions in P-position effected by P-replacement-specific lyases proceed with retention of configuration. The inversionless reaction mode has been conclusively verified in thoughtful and meticulous studies by Floss and associates (98-loo), concerning the stereochemistry of pyridoxal-P-catalyzed enzyme reactions. This circumstance is of utmost importance in relation to the reaction mechanisms of PLP-dependent P-replacement, which are discussed in Sections 1II.F and IV.

It should be emphasized that in the presence of elimination-active multifunctional lyases such as tryptophanase (14,15) or cystathionase (79,97, loo), isotopic exchange of hydrogen atoms in amino acid substrates and nonreactive (inadequate) substrate analogs takes place at moderate to very rapid rates in positions a$, and y (including positions apparently not affected in the overall chemical transformation).

E. INTERACTIONS WITH ACTIVE-SITE DIRECTED LIGANDS AND GROUP-MODIFYING REAGENTS

1 . Coenzyme Analogs

Interactions of structural analogs of pyridoxal phosphate with the PLP-dependent lyases have not been studied as systematically as for other PLP-enzymes (e.g., transaminases or decarboxylases). In


a few relevant papers (15,36,41,78,91), comparative studies were reported of the apparent affinities for apoenzymes (K‘&pP)) and relative catalytic activities (e.g., competitive inhibitor constants, lyjapp), (in regard to normal PLP-enzyme) of E. coli tryptophanase (15), tryptophan synthase (107,112), y-cystathionase (91), serine sulfhydrase, and cystathionine P-synthase (36,92). The latter two enzymes are P-replacement-specific, the others are catalytically multifunctional.

Considerable differences were observed in the coenzyme analog patterns of the individual lyases, but no characteristic contrast was revealed between those of replacement-specific type and the others, with the exception of the more stringent structural requirements of serine sulfhydrase, 2, and cystathionine P-synthase 3, for catalytically active analogs. Only two analogs were found active as substitutes for PLP with these lyases, namely, 2-norPLP and 5’-MePLP (36). The catalytic parameters of PLP itself, and a number of its analogs displaying activity either as coenzyme substitutes or as inhibitory quasicoenzymes, are compiled in Table XVII for lyases 2, 3, 4, 7, and 8 (see also refs. 14,15,63, and 68).

Miles and associates synthesized two PLP analogs or derivatives, designed to inactivate irreversibly the P-subunits of bacterial tryptophan synthase, an ambifunctional lyase (1 19). These compounds are 5’-deoxyJ’-chloromethyl-pyridoxal and N4’-bromoethyl-pyridoxamine phosphate. Miles (1 12) studied and interpreted the presumably inactivation mechanisms, which involve the alkylation of certain functional apoenzyme groups in the active site. Therefore, these compounds can be regarded as specific active-site-directed modifying reagents, rather than as quasi-coenzymes. Their mode of action is comparable to that of quasisubstrates with reactive (or latent, enzymically activatable) groups modifying and blocking functional groups in the catalytic site (see Sections III.E.2, 5 , and 7). The mentioned compounds may react in a similar manner with other lyases (or PLP-enzymes of differing reaction specificity). Miles also discusses (1 12), the inactivation of the @-subunits of tryptophan synthase by two substrate analogs that act in this catalytic-site directed manner, namely, a-cyanoglycine and the microbial toxin, L-2-amino- 4-methoxy-trans-3-butenoic acid (R. Rando et al., 1974- 1976). They act either as direct, essential group-modifying reagents or as “sui- cidal,” enzyme-activated Michael addends (see Sections III.E.2 and III.E.7).

TA

BL

E X

VII

C

oenz

yme

Ana

log

Inte

ract

ions

with

the

Apo

enzy

mes

of

&R

epla

cing

Lya

ses

(2,3

,4) and

Sel

ectiv

ely

a,p-

Elim

inat

ing

or

Mul

tifun

ctio

nal L

yase

s (7

,SP

Perc

enta

ge o

f A

ctiv

atio

n (P

LP

= lW

0)

Perc

enta

ge o

f in

hibi

tion

K,,

resp

. K

i (p

W

Vita

min

Bad

eriv

ativ

elya

se:

2 3

4

7 8

2 3

4 7

8 2

3 4

7 8

P)T

idO

Xd-

5'-P

(PL

P)

Pyri

doxi

ne-5

'-P

2-no

rPL

P Y

-MeP

LPb

6-

MeP

LP

2-no

r-C

MeP

LP

2-M

ePL

P 5'

-deo

xyP

L

5-no

r-5-

pCH

2CH

2CO

OH

5-

nor-

5-pC

H=

CH

CO

OH

2'

,2'-d

iMeP

LP

2'-P

heny

lPL

P 3-

deox

y P

LP

3-M

ePL

P PM

P PL

P

LP

(N' +

0)

5'-h

omoP

LP

100

100

00

83

11

0 70

30

0

0

00

00

0

0

00

0

0

00

0

20

0

0

00

--

-

100 0 31

56

100

100 60

12

20 6 4 0 0 -

-

- 0

100 9 0 26

100

47

-

0 -

0 -

0 - 9 15

120

150

1200

12

50

430 80

100

450

-

-

1.1 -

-

3.8

--

17

8

-

80

- 800

110

80

210

-

320

-

loo0

360

-

-

--

44

31

15

22

40

70

50

20 0 0 0 -

60

56

30

27

40

71

65

33

15

13 0 -

-

120 90 3 48

80

47

-

-

82

62

31

54 6 64 0 0 0

-

24

100 57 8 53 8

- 0 - 0 0 0 -

-

65

41

5 -

500

-

~~~

a L

yase

s: 2

, se

rine

sul

fhyd

rase

(36)

; 3,

cyst

athi

onin

e p-

synt

hase

(ra

t liv

er) (

92);

4,

p-cy

anoa

lani

ne s

ynth

ase

(65,

68)

; 7,

tryp

to-

* Con

cent

ratio

ns in

dica

ted

(KC

J ar

e fo

r the

act

ive

enan

tiom

er.

phan

ase

(E. c

oli)

(15)

; 8,

y-c

ysta

thio

nase

(= h

omos

erin

e de

hydr

atas

e, ra

t liv

er) (

36, 9

1, 1

19).


2. Quasisubstrates and Nonspecific Carbonyl Reagents

In common with other PLP-dependent enzymes acting on a-amino acids, the p-reactive lyases display varying degrees of active-center- directed affinity for certain analogs of their preferred natural substrates. Affinities for these compounds are usually more pro- nounced, when the spatial and electronic structure of such a quasi- (or allo-)substrate is complementary to the enzyme’s substrate-binding site. [The structural requirements for high affhity and specific (positive or negative) effects upon enzyme activity are essentially similar, for analogs of the coenzyme and of cosubstrates (replacing agents), to those indicated below for substrate analogs. See also Tables XIV and XVII].

Prominent features desirable for an effective substrate analog include:

I. A primary a-NH2 group, required for transaldimination from internal to external PLP-aldimine.

2. Adequate configuration at a-C (L). 3. Appropriate length of the main carbon chain (preferably, but

not exclusively, C3 to C5). 4. Suitable polarity (usually electronegative) and selective chem-

ical reactivity of the p-substituent group.

Interactions of the lyases with allosubstrate molecules may differ, depending on the structure and properties of the latter:

I. The analog may act as an active substrate of the lyase, subject to adequate transformation at rates inferior, similar to, or higher than those of the normal substrate, as in the case of selena cystathionine and lyase 3 (1 17) (see Section II.A.3).

2. In many cases, as mentioned earlier, allosubstrates react with lyases as competitive or noncompetitive inhibitors, displacing normal amino substrates from their enzyme-bound external PLP-aldimines, and eventually producing apoenzyme by resolution of the 1.E complex.

3. Allosubstrates can manifest affinity to the active center if they possess a chemically reactive group, such as a double or triple carbon-carbon or carbon-nitrogen bond. Covalent addition of the unsaturated link to a particular protein side-chain (nucleophilic Mi- chael addition, see Section III.E.7) in or near the active site may obstruct access to the site or hinder the functioning of catalytic


groups. In this way, some PLP-dependent (and other) enzymes acting on amino or hydroxy acids are inactivated by substrate analogs containing an acetylenic or allenic link. The reaction mechanism has been investigated in detail by Abeles and associates (121,122), along with that of precursors of Michael addends, considered in Section III.E.7; see also below (18,20,92).

4. Certain pseudosubstrate-coenzyme imines containing no specifically reactive groups are converted into potent, irreversibly inactivating agents as a result of adequate (or atypical) enzyme- catalyzed transformation in the active site. Pseudosubstrates of this type are often appropriately designated as “suicide inactivators.” One such mechanism was revealed and elucidated in the 1960s by our colleagues Khomutov et al. (101-103), namely, the inactivation of aminotransferases by cycloserine and its analogs (the L-enantiomeric forms); see Section III.E.5.

Another important instance is mono- and poly-halogenated amino acids (and some related analogs) readily undergoing enzymic conversion to A3*4, or similar unsaturated Schiff bases by the adequate action of a$- or P,y-eliminating lyases (121,122) or abnormal elimination side-reactions in the active center of some other pyridoxal-P-dependent enzymes, for example, aminotransferases. The resulting unsaturated coenzyme imines can inactivate the enzyme by Michael addition to catalytically important functional groups, as stated above. This reaction mechanism is considered in extenso in Section III.E.7. (See also refs. 132 and 133.)

A group of relatively nonspecific ligands, resembling pseudosubstrate amino acids in reaction mechanism, is represented by typical carbonyl reagents with highly reactive amine groups. These are the free and alkyl- or acyl-substituted hydroxylamines and hydrazines (alkoxylamines, hydrazones, and hydrazides).

In the binding site of PLP-enzymes (including the p-replacing and elimination-active lyases), they displace the C4‘ aldehyde group of the coenzyme from its internal and substrate aldimines and bind it in the form of fairly stable PLP-aldoximes or (substituted) hydrazones. Derivatives with an adequately positioned carboxyl group- the 3- or 4-aminooxy and hydrazino acids-display much more selective affinity for the bound coenzyme and greatly enhanced (by one to two decimal powers) inhibitory activity. The mode of interaction of PLP-enzymes with the carbonyl reagents is time-depen-


dent; although initially competitive versus amino acid substrate, it may gradually become noncompetitive and terminate in inactivation (resolution) that can be reversed by timely supplementation with PLP in excess.

Some NH2-containing carbonyl reagents are of practical importance in enzyme research as experimental tools, and in clinical med- icine as drugs (for example, isoniazid).

3. Sodium Borohydride

Under conditions ensuring the selective reduction of >C=O and >C=N- double bonds, treatment of purified PLP-enzymes with NaBH4 in the absence or presence of aminosubstrates or of (or of NH2-containing inhibitors) can provide valuable information concerning the interactions of participants of the enzymatic reaction in the catalytic site in a given situation. (However, the experimental evidence is not absolutely reliable, due to the possibility of rapid shifts in chemical or enzyme-catalyzed equilibria during the hydrogenation operation.)

It is desirable to combine separate reduction tests with differential radioisotopic labeling of one reactant, [i.e., with sodium borotritide, I4C, 3H (or 31P)] as marker in the B6-coenzyme, and appropriate (uniform or localized) radiolabel in the substrate (or inhibitor).

Ideally, the following results can be expected, and in many cases have essentially been attained:

1 . Reduction with Na-borotritide of purified enzyme, when all active sites are in the internal PLP-aldimine form, should result in total inactivation and yield, upon denaturation and cautious hydrolysis of the protein, the free or peptide-bound secondary amine, C4'-tritiated pyrid~xinyl-~HN'-lysine, in a partially phosphorylated state (some Pi is released during the hydrogenation). The reduced nondenatured enzyme fails to regain activity on supplementation with PLP under appropriate conditions: restitution of a fraction of catalytic activity would indicate that part of the enzyme was initially in the form of apoprotein or of PMP-enzyme.

2. The apoenzyme or the pure PMP-form are neither inactivated nor specifically tritiated by reduction with labeled Na-borohydride; however, some irreversible protein denaturation may occur.

3. The reaction of PLP-enzyme with saturating concentrations of a good substrate or, preferably, an analog undergoing only the


first step (transaldimination) of the enzymic reaction, produces external PLP-substrate (or PMP-intermediate) Schiff bases. In the presence of suitably labeled PLP, aminosubstrate, or borohydride, the hydrogenation reaction, followed rapidly by enzyme denaturation, should yield nonlabeled protein and sizable amounts of radio- active free (phospho)pyridoxinyl-a-NH-acid (secondary amine). In- corporation of firmly bound labeled pyridoxyl into the denatured protein indicates that some residual P-pyridoxylidene-enzyme was present in the initial test mixture.

4. Sulfhrdryl Reagents

Titration with conventional sulfhydryl reagents (e.g., bromo- or iodoacetate, DTNB, or pCMB) has demonstrated the presence in the molecule of PLP-dependent lyases of varying amounts of thiol groups, ranging in ease of modification from readily accessible to semiburied or completely masked in the native protein (95a, 114,115a). Little research has been done and no characteristic differences have thus far been detected between P-replacing and other subgroups of the PLP-dependent lyases in regard to the number and properties of titratable HS groups.

In our experience, blocking accessible thiol groups (if any) in P- cyanoalanine synthase (4) with pCMB or bromoacetate did not significantly affect the activity of this lyase with chloroalanine as the aminosubstrate.

The most detailed studies of the significance of sulfhydryl groups, reported by Chatagner and her associates (1 l4,115a), deal with rat liver cystathionase.* Using DTNB (50-fold excess, at pH 8.0) as sulfhydryl reagent, the authors estimated 12 HS groups in the native and 20 HS groups in denatured enzyme. The same results were obtained on titration with pCMB (25-fold excess, at pH 7.0, during 1 h). Comparative titration with pCMB of the apoenzyme, and of apoenzyme + PLP, indicated that PLP does not affect either the reactivity of HS groups or the kinetics of enzyme inactivation (in the cysteine desulfhydration reaction). Two readily accessible HS- groups do not take part in the catalytic act. Among less readily reacting sulfhydryls, some of which are required for the functioning

* The existence (cf. ref. 91a) of two distinct types of cystathionases-one B,y- eliminating (involved in the formation of cysteine by transsulfuration) and the other a,&eliminating (participating in reverse transsulfuration) is not taken into account in those studies (114,115a).


of the enzyme, L-homoserine (a substrate) protects four HS groups, and L-alanine (a competitive inhibitor) protects eight HS groups (of which only two react more readily with homoserine). Two func- tionally unimportant thiol groups remain accessible to sulfhydryl reagents in the presence of alanine and homoserine. The dissociation constants of the cystathionine-alanine complex differ, depending on the substrate used-cysteine or homoserine.

The findings are interpreted by the authors as evidence indicating the presence of two active centers in cystathionase. This conclusion is supported by results obtained in their studies (1 15a) with S-car- boxyethylated and carboxymethylated analogs of C3- and C4-substrates (see also Section II.B.2).

5. Cycloserine Enantiomers and Related Compounds

The naturally occurring antibiotic, cycloserine (oxamycin), is the cyclic alkoxamide of 3-aminooxy-~-alanine; it is conventionally termed D-cycloserine, and its enantiomer is L-cycloserine. These compounds are sterically rigid structural analogs and biological an- tagonists of the corresponding alanine enantiomers (therefore the name “cycloalanines” might be more adequate). The 5-substituted derivatives (5-R-cycloserines) are cyclic analogs of the higher a- amino acids; for example, replacing 5-H with carboxyethyl (R = CH2CH2COOH) results in a mixture of threo- and erythro-DL-a- cycloglutamates, which are diastereomeric cyclic analogs of DL-glu- tamic acid (101-104).

0

a - Amino acids Cycloserine and its

5-substituted analogs

The cycloserine derivatives act as inhibitory pseudosubstrates, suppressing the enzyme-catalyzed reactions of structurally and con- figurationally cognate amino acids. Thus, the antibacterial properties of D-cycloserine result from the inhibition of bacterial enzymes catalyzing metabolic transformations of D-alanine (including D-transaminases and racemases), and from the suppression of enzyme-catalyzed incorporation of D-alanine into the bacterial cell-wall peptid- oglycans (78,103,110).

In animal and higher plant tissues, the more potent agents are the


L-enantiomers of cycloserine and its derivatives, which act as inhibitors or “suicide inactivators” of various PLP-enzymes.

Evidence revealing the chemical mechanism of irreversible trans- aminase inactivation by these and related compounds has been presented by Karpeisky et al. (101-104) and interpreted as follows. The agent-a sterically rigid pseudosubstrate-binds in the active center with PLP to form an aldimine that is rearranged to a tautomeric, unstable PMP-ketimine. The isoxazolidone ring of the latter undergoes decyclization, producing a reactive acyl residue that blocks an essential nucleophilic residue in the catalytic site (Fig. 14). PMP- ketimines and pyridoxamine were isolated after mild disintegration of the cSer-enzyme complexes (102,103). This interpretation was extended to the inactivation by L-cycloserine of other PLP-enzymes acting via PMP-ketimine intermediates, such as aspartate P-decar-

4 EIJ3a 3 Figure 14. Molecular mechanism of inactivation of an aminotransferase by L-cycloserine. (Fwm refs. 18,78,102,103.)

P-REPLACEMENT-SPECIFIC PLFDEPENDENT LYASES 63

boxylases, kynureninase, and y-cystathionase (3,8a, 1 11). PLP-enzymes presumably bypassing this reaction step, for example, amino acid a-decarboxylases, proved insensitive to cycloserine (1 8,20,78). From these facts Braunstein (3,8a) has inferred that typical “suicide inactivation” by L-isomers of aminoisoxazolidones might be a specific feature of only those PLP-enzymes whose catalytic cycle normally embodies a PMP-ketimine step. Goryachenkova et al. obtained results concordant with this hypothesis in studies of the interactions of P-specific lyases with pure preparations of L- and D-

enantiomeric cycloserines (18,20,78,87). The P-replacing lyases 1-4 (which in our opinion do not form intermediate PMP-Schiff bases) proved practically resistant to cycloserine inhibition (Table XVIII) (3,8a,78). In contrast, L-cSer inhibited the elimination-active lyases 5 , 7, and 8 (the Ki values were 10-3-10-5 M ; affinities for the D-isomer were 10- to 100-fold lower). Gel filtration resolved the 1.E complexes to native (unstable) apoenzyme and Schiff bases. In acid solution, the latter yielded mainly PMP and pyridoxamine, indicating abortive half-transamination. The fairly high sensitivity of lyases 5, 7, and 8 to L-cycloserine points to PMP-ketimines (or tautomeric quinonoid species) as essential intermediates in enzymic a- P-elimination. For further details see reference 78.

6. Mercapto-Amino Acids and Aminothiols

It is known that 1,2- and 1,3-aminothiols and especially mercaptoamino acids able to act as substrates or quasisubstrates (e.g., cysteine, homocysteine, and penicillamine) are moderately potent competitive inhibitors of many pyridoxal-P-dependent enzymes- transaminases, amino acid decarboxylases, y-cystathionase, and, among others, the p-elimination-active lyases @a, 18,20). The mercapto-L-amino acids act by binding in the active sites as affinity ligands and condensing with the carbonyl atom (C4’) of PLP to give fairly stable heterocyclic compounds with five- or six-membered rings (8,110,124,125). This is why 3- or 4-mercapto-amino-acids are often used in laboratory practice as mild reagents for the resolution of PLP-proteins and preparation (e.g., by means of gel filtration) of native apoenzymes (3,26,78).

The P-replacement-specic lyases 1-4 proved to be completely resistant to inhibition by D- or DL-penicillamine, as well as by L-

cysteine or L-homocysteine at concentrations up to lo-’ M , which

TA

BL

E X

VII

I In

hibi

tory

Eff

ects

of

the

Ena

ntio

mer

s of

Cyc

lose

rine

(cS

er) o

n PL

P-D

epen

dent

Enz

ymes

, In

clud

ing

the

Rep

laci

ng a

nd

Elim

inat

ing

Lya

ses”

O

I P

IXI v

alue

s (m

M)

of c

Ser

isom

ers

Enz

yme

L D

L

D

Mod

e of

act

ion

Ref

eren

ce

u.

Ala

: G

lu tr

ansa

min

ase

b. A

sp:G

lu t

rans

amin

aSe

(pig

hea

rt

c. A

sp p

-dec

arbo

xyla

se (b

acte

rial

) cy

toso

l)

d. G

lu a

-dec

arbo

xyla

se (E

. co

11)*

I.

Cys

tein

e ly

ase

(chi

cken

embr

yo y

olk-

2. S

erin

e su

lfby

dras

e (ch

icke

n liv

er,

3. C

ysta

thio

nine

p-s

ynth

ase

(rat

live

r)

4. &

Cya

noak

min

e sy

ntha

seb

5. A

Uiin

ase

(gar

lic)c

6. S

erin

e de

hydr

atas

e (r

at li

ver)

7.

Try

ptop

hana

se (E

. coh

’f 8.

ycy

stat

hion

ase

(rat

live

r)d

SaC

Y

bake

r’s

yeas

t)”

0.002

0.00

5 0.8

0.7

3.5

*lo

-

-0.1

?

No

inhi

bitio

n

No

inhi

bitio

n

No in

hibi

tion

No

inhi

bitio

n N

o in

hibi

tion

0.00

8 -

0.08

-

e0.5

0.5

-

0.1

-

0.01

-0.07

-0.8

L-c

Ser

inac

tivat

es, l

eavi

ng th

e en

zym

e

DcS

er in

hibi

ts, r

esol

ves

to a

poen

zym

e

L-c

Ser

inac

tivat

es, p

rodu

cing

No P

MP

ketim

ine

form

atio

n N

o PM

P ke

timin

e fo

rmat

ion

in a

min

o fo

rm

and

PLP-

oxim

e

amin

ofor

m; P

LP

rea

ctiv

ates

No

PMP

ketim

ine

form

atio

n

No P

MP

ketim

ine

form

atio

n N

o PM

P ke

timin

e fo

rmat

ion

Inhi

bitio

n pa

rtia

lly c

ompe

titiv

e; P

LP

re

activ

ates

Inhi

bitio

n by

L-c

Ser

part

ially

co

mpe

titiv

e; re

solv

es ly

ase

to P

MP

and

apoe

nzym

e

101, 1

02

102

8a, 18

8a. 18,78

3, 18

8a, 18

8a, 94

78

87

78

8a, 78

8a, 78

” Ada

pted

fro

m r

efer

ence

8a

and 78.

cSer

was

pre

incu

bate

d w

ith t

he P

LP-

lyas

es,

in c

once

ntra

tions

up

to 2

0-50

mM

, in

buff

ered

” cSer

con

cent

ratio

ns u

p to

100 m

~.

* Acc

ordi

ng to

Tur

ano

(per

sona

l com

mun

icat

ion,

1979) m

amm

alia

n D

OPA

-dec

arox

ylas

e is

likew

ise

resi

stan

t to

inhi

bitio

n by

cS

er.

solu

tion

in t

he p

rese

nce

(whe

re re

quir

ed) o

f an a

dequ

ate

cosu

bstr

ate,

(e.g

., p-

mer

capt

oeth

anol

).

Hig

h-pu

rity

lyas

es 5

-8 a

re p

artia

lly r

esol

ved;

7 a

nd 8

wer

e su

pple

men

ted

with

lo-’ M

PL

P b

efor

e th

e as

say

with

cS

er.


TABLE XIX Inhibitory Effects of I-Amino-2-Thiols Upon the Pyridoxal-P-Dependent Lyases.

Inhibitor and its Is0 (mM)b

Penicillamine

Enzyme L-Cysteine DL D Cysteamine

a,P-Eliminating

Multifunctional 5. Alliinase (garlic) 10 5 20 no inhibition

- - 7. Tryptophanase (E. coli) + + 0.2 (cysteine desulfhydration)

(homoserine deamination, cystein desulfhydration)

8. y-Cystationase (rat liver) 0.8 0.22 2.3 3 .O

@Replacing 1. Cysteine lyase (chicken yolk-

sac) 2. Serine sulfhydrase (chicken

liver) 3. Cystathionine @-synthase (rat

liver) 4. P-Cyanoalanine synthase

(lupine seedlings) [Ala(CI) + KCN = Ala(CN) + KCI]

No inhibition

No inhibition

No inhibition

No inhibition

a From references 3, 8a, and 78. IsO values-inhibitor concentrations (mM) reducing initial reaction rates by 50%

under specified assay conditions.

is far in excess of K, for Cys or K,,, for Hcy (see Table XIX). In contrast, the same compounds in concentrations of 10-4-10-2 M inhibited (and eventually resolved) the tested eliminating lyases 5, 7, and 8 (see Fig. 15).

Until recently, we believed that a#-elimination by PLP-dependent lyases proceeds by the trans-elimination mechanism, common in nonenzymic (chemical) systems. The required geometry-if fixed in E-S complexes by the binding of PLP-amino acid Schiff bases with the P-substituent in orientation anti to the a-H atom (syn to a- N)-would also be appropriate for the condensation of PLP with an inhibitory mercapto amino acid to a heterocyclic derivative.

Inverse fixed orientation of the substrate in P-replacing lyases,


CHI I I

-+ EaPLP + NH, + H,S + CO

coo- -H coo- 400-

X*CH, I I

- E . P L P + H,S + NH,-CH +HX

coo- coo- l coo- E * PLP

Figure 15. Suggested geometry of the binding and reactions of a 3-mercapto-~-amino acid (cysteine) in the active sites of a#-eliminating (a) and P-replacing ( b ) pyridoxal- P-dependent lyases. (From ref. 3.)

that is, with p-X syn to a-H and anti to a-N (or approximately so), would result in a conformation that we considered unfavorable to elimination, and that precluded condensation with PLP to a hetero- cycle (Fig. 4 in ref. 3, Fig. 9 in ref. 18).

This interpretation now appears oversimplified and needs revi- sion. The mechanism we assumed for P-replacing lyases and sche- matically visualized in Figure 4 (Section I.B.2) indicates a reaction implying steric inversion at the CP atom. However, earlier observations (44a) and our own isotopic studies (27) pointed to retention of configuration in the replacement of p-substituents by lyases 1-4. Floss and associates (98,99) have demonstrated an inversionless steric course for enzymic reactions both of a$-elimination and of p-replacement (Section I.B.2 and 1II.D). Among several possibilities for inversionless direct p-replacement (type I1 reactions), they suggested a plausible scheme involving conformational change in the enzyme protein during the catalytic act. Floss et al. (99,100) have ascertained that in a,@-elimination and p-replacement reactions


alike, the amino substrates are bound in the active site in similar geometry, with @-substituent syn to the a-H atom. Moreover, they assume, in accord with Dunathan's postulate (4), that in the reactions catalyzed by PLP-dependent lyases, all interactions at C-atoms a, @, and y take place on one side of the planar substrate-coenzyme complexes.

These and other aspects of available information concerning the catalytic events in the active site are considered in Section IV.

7 . Michael Addition and Related Reactions in the Active Center A number of concordant experimental findings and theoretical

considerations were presented above in support of the view that unsaturated Schiff base intermediates are not detectable in reactions catalyzed by the @-replacing lyases, and that even their transient occurrence is unlikely. In contrast, all elimination-active lyases produce (via PMP-ketimines or quinonoid tautomers) PLP-aldimine intermediates with either a ethylenic link (in reactions I or I + 11) or a A3,4 ethylenic link (in reactions I11 and IV).

In all reactions involving the formation of unsaturated imines, these can be trapped by nucleophilic addition of a suitable electronegative group at the C3-atom (a Michael reaction). In elimination- mediated @-replacement (I + I1 reactions) by multifunctional lyases, such as tryptophanase (3, P-tyrosinase, and threonine synthase, addition of the cosubstrate's anion adds at C3 of the unsaturated Schiff-base intermediate is a typical Michael reaction. Appropriate nucleophilic ligands may substitute for the cosubstrate and serve as site-specific Michael addends. Flavin and his associates (79,123) employed N-ethyl maleimide ( ''C-labeled NEM) to demonstrate the formation of enzyme-bound A2*3-Schiff bases: NEM traps the unsaturated intermediate by (chiral) Michael addition, yielding labeled adducts of the structure shown in Table XX, and reducing the output of the major normal reaction product (14,20). In 1978, Goryachen- kova (20,8a) applied this probe to some @-specific lyases; findings similar to those of Flavin were obtained with enzymes possessing elimination activity (lyases 6,8). Because of its rapid spontaneous reaction with SH groups, NEM cannot be used in the routine assays for @-replacing lyases (1-4), where either aminosubstrate, cosubstrate, or both are thiols. Fortunately, owing to the capacity of cyanoalanine synthase, 4 , to catalyze @-replacement between chloroalanine and cyanide, the test could be performed in the absence

TA

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of thiol-containing ligands. As shown in Table XX, NEM did not interfere with this reaction. This single negative finding does not constitute conclusive proof failure to detect a A2s3 Schiff base by the NEM test might result from the rapid transformation of the unsaturated intermediate. However, our viewpoint is enforced by an extensive body of independent findings, considered in Section III.E.2, as well as in the paragraph that follows.

We have already stated that certain enzymes acting on amino acids or hydroxy acids can be inactivated by substrate analogs containing a suitably positioned chemically reactive group (e.g., a double or triple carbon-carbon or carbon-nitrogen link), the covalent binding of which to a nucleophilic group (Michael addition) in or near the active site may obstruct access to the site or hinder its catalytic function. Potent inactivators for both eliminating and p-replacing lyases are substrate analogs with preformed unsaturated groups, especially with a highly active acetylenic or allenic link.

Of special interest are allosubstrates devoid of ready-made unsaturated groups, but capable of acquiring them as a result of enzymic transformation. Impressive examples were presented by Abeles and his associates (121,122), who showed that polyhaloal- anines, such as Ala(CI2) and Ala(F3), act on y-cystathionase (8), P-cystathionase, and other eliminating PLP-enzymes as ‘‘suicide inactivators”: they bind to the coenzyme as pseudosubstrate aldimines readily undergoing elimination of a-H and P-halogen as a molecule of haloacid. The resulting A2*3-aldiminic Schiff base is an activated Michael acceptor; it is attacked at P-C and covalently bound by a nucleophilic residue of the lyase, thus inactivating the enzyme. Another (acetylenic) “suicide” allosubstrate, propargylglycine, is converted in the active site of y-cystathionase by an intricate mechanism, involving alternating deprotonations at a-C, p-C, and tau- tomerization steps, into a different, highly activated Michael acceptor, a 2-aminopenta-A3v4, A4*5-dienoate-coenzyme Schiff base. Its covalent binding at the (allenic) C4 atom by a second nucleophilic group of the enzyme results in inactivation (122).

Abeles and associates (92,121) found that cystathionine P-synthase (our lyase 3) is refractory to inactivation by two typical ‘“I- icide” allosubstrates, Ala(C12) and Ala(F3), thus supporting our view that no unsaturated coenzyme-substrate imines are formed in the catalytic cycle of P-replacing lyases (see Sections III.E.2 and 7, III.F, IV and refs. 8a,20,78,121,122,132, and 133).


Metzler and his colleagues recently studied (1976-1982) the action on E. coli glutamate decarboxylase and on aspartate aminotransferase of certain allosubstrates with electronegative p-substituents that were susceptible to conversion in the active site, by an elimination side-reaction, into Schiff bases of a-aminoacrylate (or to a related a,p-unsaturated amino acid or amine).

They described (127,129,130) a novel mechanism of “suicide inactivation” of these PLP-enzymes by serine O-sulfate and p-chloroalanine. In the catalytic sites, a,p-elimination of such molecules is followed by release of a-aminoacrylate from its internal PLP- aldimine via “transimination. ”

In aminoacrylic acid and similar enamines, both the nitrogen and the p-C atom have nucleophilic properties. According to Metzler et al., nucleophilic attack of the C4’ atom of PLP in holoenzymes (i.e., in the internal Schiff base) is followed by the release of apoenzyme and a coenzyme derivative with an cr-imino (or a = 0xo)substituted acid with a 4- or 5-carbon, singly or doubly unsaturated (enoic or dienoic) acid side chain at position 4 of norPLP. The suggested mechanism was confirmed by chemical synthesis of the predicted coenzyme derivative. Related reaction sequences have been proposed for the inactivation of various PLP-enzymes by some toxic amino acids, such as allylglycine or propargylglycine, which also act as “suicide” inhibitors (128,129,131). In light of these findings, it is necessary to reconsider the mechanisms proposed for “suicide inactivation’’ by inhibitors forming intermediates that contain carbon-carbon double or triple bonds (or by compounds with preformed structures of that type).

The mechanism described above [or variants suggested by Metzler et al. (129)] must be considered (as alternatives to Michael addition) when interpreting the mode of “suicide inactivation” of eliminating lyases, since the formation of unsaturated Schiff bases is one of the first steps in their normal catalytic cycle.

Fortunately, this problem does not apply to the P-replacement- specific lyases, which are refractory to inactivation by “suicide” allosubstrates of this type.

F. REACTION TYPES INDICATED BY STEADY STATE KINETICS

Investigation by kinetic methods of the sequence of substrate binding to the enzyme molecule, and of end-products’ release, provides supplementary information as to the mechanism of enzyme-


catalyzed reactions. Cleland (1 13,113a) considers the following kinetic patterns as of the order of steps in enzyme reactions with one or two substrates. (To obtain minimum kinetic patterns not com- plicated by back-reactions or other intricacies, rate equations should be based on measurements under stationary conditions.)

I . When the binding of substrate(s) precedes product liberation, the mechanism is sequential. If binding of substrate(s) and release of products occurs in regular sequence, the mechanism is ordered. If there is no definite order in the binding of substrates and the release of reaction products, the mechanism is random.

2. Reactions involving two substrates can proceed by so-called Ping-Pong mechanisms, with binding of one substrate followed by release of the first reaction product prior to binding of the second substrate.

On the basis of Cleland’s classification, the minimum kinetic pattern for enzymic a,p-elimination (reaction I), as seen from the steps shown in Figure 3, should be “Uni Sequential Tri”:

S H’ HX P

i t t t E E’.S E‘*S+E”*P E

where S is a-amino acid substrate, H+ is released a-proton, HX- is deprotonated p-substituent, and P is final a-0x0 acid.

For elimination-mediated P-replacement, the stationary kinetic mechanism should be “[ordered] Ping-Pong Bi Bi,” as can be inferred from Figure 3. This pattern, represented graphically in Figure 16, has been reported for a bacterial 0-acetyl-serine sulfhydrase (1 13b).

Ping-Pong kinetics can be assumed erroneously for a two-substrate reaction if, on Lineweaver-Burk reciprocal plots of reaction rate ( lh) versus inverse concentrations of aminosubstrate [ S , ] - ’ and cosubstrate, [S,] - ’ , bunches of slightly convergent tracings are mis- taken for series of parallel lines (93). A Ping-Pong mechanism would imply that with one substrate (e.g., amino acid), and in the absence of the other (cosubstrate), the enzyme should catalyze an elimination reaction. So far as we are aware, such a reaction has never been observed with either the 0-acetylserine sulfhydrase mentioned (1 13b), or any other replacement-specific lyase.


AMBIFUNCTIONAL j?-LYASES: A P B Q

REPLACING B - LYASES: A B

B A

Figure 16. Stationary kinetic mechanisms (1 13) for elimination-mediated p-replacement by ambifunctional lyases-“Ping-Pong Bi Bi” (1)-and for reactions catalyzed by the uniquely p-replacing lyases-“Random Bi Sequential Bi” (2). (See refs. 8a,93,94)

Tolosa, Kozlov, and associates studied the stationary kinetics of P-replacement reactions of L-cysteine catalyzed (a) by Ala(CN) synthase from blue lupine, 4 (93), and ( 6 ) by serine sulfhydrase from baker’s yeast, 2a (94).

In each study, initial reaction rates were first measured in two series of experiments; in (a) at several constant concentrations of KCN(S2), variable concentrations of L-cysteine (S1), and stable cysteine with variable KCN concentrations (93). In series ( b ) the reaction rate measurements were conducted at several invariant concentrations of 2-mercaptoethanol(S2) and variable concentrations of L-cysteine (SI), and then with a set of stable L-cysteine (SI) and variable S2 (i.e., mercaptoethanol) concentrations (94).

In each of the experiments, reciprocal reaction rates (llv) were plotted as functions of l/[SI] (l/[S2] in Lineweaver Burk coordi- nates. In each of the graphs, the plots of l/v versus l/[S] initially resemble a family of parallel straight lines.

If such is the case, the equation for initial reaction rate should be:

$-REPLACEMENT-SPECIFIC PLP-DEPENDENT LYASES 73

which, according to Cleland’s system, would indicate Ping-Pong kinetics. (For figures illustrating the actual plots, and detailed mathematical derivation of the kinetic approaches used, see refs. 93 and 94).

The parallelity of lines in the graphs under study was, in fact, only apparent. Actually, in each of the plots the lines intersected at one point in the third quadrant.

Therefore, it was necessary to consider the applicability of sequential kinetic patterns for two-substrate reactions to the experimental values observed in measurements of the initial reaction rates.

Such reactions can proceed by ordered or random mechanisms (1 13,113a).

The ordered two-substrate sequential mechanism can be expressed by the following kinetic scheme (1 13):

PI PZ + SI + sz 5 $

Ks I E ESI E S I S ~ EPlP2 e EP2 e E

The reaction rate depends on the concentration of the ternary complex ESI S2:

v = kcat[ESIW

To obtain a complete kinetic formulation of the reaction rates, it is necessary to determine the values of all dissociation constants for the enzyme-substrate complexes:

(All kinetic parameters were calculated on an electronic computer by the least-squares method.)

The equation for initial rates in the ordered sequential mode is:

kcat[E~l[S~ l[S2l [SII[SZI + [SIIKS~ + KsiKsz

v =

From this expression, Lineweaver Burk graphs are constructed for fixed values of [S2], variable [ S , ] , and conversely. As recom- mended by Cleland (1 13,113a), the points of intersection of the linear

74 ALEXANDER E. BRAUNBTEIN AND ELIZABETH V. GORYACHENKOVA

plots are found, and their projections upon the ordinate and the abscissa are determined. Computation and graphic analysis shows that, given ordered sequential kinetics, intersection of the linear plots must be situated in the second quadrant (cf. the discussion in ref. 93).

The study of stationary kinetics both for Ala(CN) synthase (93) and yeast serine sulfhydrase (94) produced bunches of linear plots intersecting in the third quadrant in all graphs. This rules out for the two @-replacing lyases the possibility of ordered sequence of substrate binding.

The random binding mechanism can be depicted by the following scheme:

where

The equation for initial rates is:

Factor a indicates the interdependence of binding of the substrates to the enzyme. When a < 1, the ternary complex is preferable to the binary enzyme-substrate complexes; when a > 1, formation of the ternary complex is less favored. (Values of a > 1 result in in- creasing approximation of the linear plots in Lineweaver Burk co- ordinates to a set of parallel lines. This can lead to erroneous inferences concerning the binding of substrates.)

As in the case described above, the linear plots intersect in the second quadrant when a < 1, and in the third quadrant if a > 1.


Values of a > 1 were obtained with both Ala(CN) synthase (93) and serine sulfhydrase (94), implying random sequence substrate binding for either (and binary E-S complexes favored).

Using the following procedure, suggested by Cleland (133a), we can determine the individual kinetic constants, V, Ks, , Ksz , and a. In the case of fixed S2 concentrations, a secondary graph, plotting the values of intercepts on the ordinates from the primary Line- weaver Burk graph versus [ S 2 ] - ' , produces a straight line crossing the ordinate at 1/V and intercepting the abscissa at llaKs2; the slope is aKs2/V. If the slopes from the Lineweaver Burk graphs are plotted as a function of [S2]-', the resulting straight line intercepts the ordinate at aKs2/V; the slope is ~ K s ~ K s ~ / V . Similar constants are obtained for fixed concentrations of SI .

The values of kinetic constants for the reaction catalyzed by Ala(CN) synthase (93), [i.e., the formation of p-cyanoalanine + H2S from L-cysteine (SI) and KCN (S,)] are tabulated below.

Values for Values for Constants [S1 Iconst [ S ~ l c o n s t

V (arbitrxy units) 120.0 r 3.8 120.0 f. 5 .5 kcat 6 - l ) 83.2 r 2.6 83.2 t 3.8 Ks, f m M ) 1.27 t 0.17 1.27 f. 0.11

(m) 2.12 r 0.09 2.12 t 0.13 Ks2 (m) 0.124 r 0.015 0.124 r 0.010

aKs2 (m) 0.206 ? 0.016 0.206 f. 0.014 a 1.67 ? 0.28 1.66 ? 0.18

The coincidence of the constants obtained with fixed concentrations of S1 and of S2 proves that the model selected for the kinetic mechanism is adequate. An interesting feature of this two-substrate reaction is the 10-fold higher affinity of the lyase for S2 (the cosubstrate KCN) as compared to SI (the aminosubstrate cysteine).

The explicit inference drawn from these values is that in the reaction catalyzed by Ala(CN) synthase, binding of the substrates is mutually dependent and proceeds by the sequential random mechanism via formation of ternary aminosubstrate-PLP-enzyme-cosubstrate intermediate(s), characterized by factor a > 1.

Tolosa and associates (94) also studied the stationary kinetics of a two-substrate reaction catalyzed by highly purified serine sulfhy-


drase (2a) from baker's yeast, namely, the P-replacement reaction between L-cysteine and 2-mercaptoethanol (see Section II.A.2). The same graphic and mathematical procedures as above were used to elucidate the kinetic mechanism and to measure kinetic constants, and the conclusions reached were practically identical.

Values for the individual constants V, K s , , and KsZ, estimated from initial rates and tabulated below, were all considerably lower than with Ala(CN) synthase, whereas factor a was about 3/2-fold larger.

Values for Values for Constants [Sllconst [SZlconst

V (arbitrary units) 0.145 f 0.035 0.145 f 0.016 kcat (S-') 3.18 f 0.77 3.18 f 0.35 KSI (a) 6.86 f 1.10 6.89 f 0.65 a&, (mM) 17.64 * 4.43 17.64 * 2.06 Ks, (a) 3.60 f 0.36 3.60 f 0.27 a&, (a) 9.21 f 2.58 9.22 f 1.19 a 2.56 f 0.76 2.56 k 3.8

The implications concerning the stationary kinetic mechanism are the same as in the preceding work. As with Ala(CN) synthase, binding of the substrates to the enzyme is mutually dependent and obeys a random mechanism with formation of a ternary aminosubstrate- PLP-enzyme-cosubstrate complex; a * 1 (~2 .6 ) .

IV. Discussion of the Reaction Mechanisms and General Conclusions

In the Introduction to this chapter the existence of two significantly differing pathways for pyridoxal-P-dependent enzymic p-replacement reactions of a-amino acids was postulated (see Section I.B.2): (2) the well-known two-stage mechanism of indirect cu,p-elimination-mediated P-replacement , and (2) direct P-replacement, to all appearances requiring no intermediate elimination step, and catalyzed by a specialized subtype of PLP-dependent lyases.

Four P-replacement specific lyases have been studied and characterized, and the existence of other lyases of this type is probable.


In this section we summarize the facts and generalizations on which these inferences are based, and point out aspects in need of further investigation. A few biologically significant implications are emphasized.

The sum of experimental criteria employed indicates that the following P-replacing lyases are unable to effect typical elimination of substituents in position p (as well as y-elimination) from L-a-amino acids:

1. Cysteine lyase (EC 4.4.1.10) from avian (or chelonian) em- bryonic yolk-sac (Section 1I.A. 1).

2. Serine sulfiydrase from mammalian hepatic cells or baker's yeast. *

3. Cystathionine p-synthase from the same sources (Sections II.A.2 and 3).

4. (3-Cyanoalanine synthase (EC 4.4.1.9) isolated from lupine seedlings and present in tissues of many other plants (Section I1 .A .4).

Another recently discovered pyridoxal enzyme (1 17) specifically catalyzes reductive cleavage of 3-selenoalanine to Hz Se and alanine (Section II.A.3). The reaction results in release of the p-substituent, HSe; yet this is certainly not an ordinary a$-elimination, as it does not lead to liberation of ammonia and pyruvate via the a$-unsaturated Schiff base. The enzyme may belong to the P-replacing lyases.

It is appropriate to recall here the pathway of lyase-catalyzed a$- elimination (see Fig. 3). From enzyme-bound PLP-substrate aldimine, the a-hydrogen and p-substituent are released (in sequential rather than concerted mode) by trans-elimination (4,14,78,98- 100,113) to produce the protonated p-substituent, XH, and enzyme- bound A"*P-Schiff base intermediate. Normally, this step is followed by twofold hydrolysis, yielding NH3, Q-0x0 acid, and the free PLP- enzyme.

This reaction path is supported by the rates and stereochemistry of ligand binding-tagged H-atoms, suitable Michael addends (such as mercapto amino acids, Section III.E.6) or adequate a$-replacing agents (for example, in elimination-mediated p-substitution) [cf.

* Lyases 2, 2a, and 3 obviously are species-specific variants, or allelozymes, of one enzyme protein (EC 4.2.1.2).


Sections III.E.2 and 7, where a possible alternative to Michael addition, based on recent work by Metzler et al. (1291, is considered].

In Cleland’s system (1 13,113a) for stationary reaction kinetics, typical enzymic a &elimination is designated as a “Uni Sequential Tri” mechanism, and graphically illustrated on p. 71 (Section 1II.F.).

Elimination-mediated reactions (type f+lI) catalyzed by ambifunctional PLP-dependent p-specific lyases, such as tryptophanase, tyrosine-phenol-lyase, threonine synthase, and so on proceed according to Cleland’s “(Ordered) Ping-Pong Bi Bi” kinetics (see the graphic scheme in Fig. 16, part 0.)

Reactions in this and the preceding group are blocked by “suicide inactivator” allosubstrates, functioning in a conventional mode- as addends in nucleophilic Michael reactions.

The stationary kinetics of reactions catalyzed by the replacement- specific p-lyases, Ala(CN)synthase and yeast serine sulfhydrase, has been investigated by Tolosa et al. (93,94).

In both.cases it was demonstrated that the bonding of substrate amino acid (S1) and cosubstrate (Sz) to the enzyme is mutually dependent, and obeys a random mechanism with formation of a ternary S , .E& intermediate (see Section 1II.F). In Cleland’s nomenclature, this is “Random Bi Sequential Bi“ kinetics (Fig. 16, part 0).

Our interpretation of the direct p-replacement pathway is based on factual data considered in Sections II.A, 1II.A-E and listed below.

I . In the absence of appropriate cosubstrates (mercapto compounds, cyanide, hydroxonium ions, etc.) high-purity preparations of lyases 1-4 are unable to eliminate or replace the P-substituent of adequate amino acid substrates.

2. a,P-Unsaturated S c h 8 bases are not produced as stable or transient intermediates under the conditions studied in amounts detectable by any of the usual tests. These include long-wave spectral absorption and CD bands belonging to quinonoid tautomers of deprotonated coenzyme aldimine and trapping the unsaturated intermediate with appropriate ligands acting as addends in Michael reactions or related inactivating mechanisms, for example, by N- ethylmaleimide (Table XX and refs. 8a,20,79,123), or by “suicide inhibitors” that are converted potent inactivating ligands by enzymic p-elimination (Section III.E.7).

4


3. Lyases 1-4 (in contrast to elimination-active ones) are refractory to inactivation by DL- or L-cycloserine, suggesting failure to produce PMP-ketimine intermediates, which are essential precursors of the unsaturated Schiff bases (Section JII.E.5; Table XVIII; consult refs. 3,8a,38,101-104).

Braunstein and Shemyakin (5,6,8) interpreted direct enzymic P-replacement, bypassing elimination, as an interchange of P-substituent (anion) between primary substrate and cosubstrate bound in close vicinity, as in Figure 4. Such an S N ~ reaction implies steric inversion at the P-C atom. However, our own studies with tagged substrates (27), and observations reported by others (44a) actually indicated retention of configuration in reactions catalyzed by replacement-specific lyases. In 1978 Floss and associates (98,99) published convincing evidence demonstrating an inversionless steric course for enzymic a,p-elimination and p-replacement reactions alike, including Ala(CN)synthesis by lyase 4. These results, and experiments by Borcsok and Abeles (92) with cystathionine P-synthase, rule out the S N ~ mechanism featured in Figure 4 ~ . Possible mechanisms for inversionless P-replacement were suggested by the authors (92,98- 100). Among these, a two-step process involving conformational shifts in the catalytic center, associated with sequential reaction stages, is considered as the more plausible (8a,92).*

5. @-Replacement of type%% associated with complete isotopic exchange of a-H in the product amino acid (27,28; Figs. 11-13). However, lyases 1-4 also catalyze a-H exchange without P-replacement in amino substrates inadequate for the given lyase (e.g., serine with lyases 1 or 41, if the test sample contains an appropriate thiol cofactor. This means that the exchange of a-H and P-substituent is not concerted, but sequential (in the order indicated).

6. In our studies, on incubation with primary (amino acid) substrate or allosubstrate without cosubstrate, P-replacement-specific

4.

* For the present, a formal equation carrying no presumption as to the stereochemistry of P-replacement is preferable, such as

X H + H*OH Y H*

H doo- H coo-


lyases catalyzed very slow isotopic a-hydrogen exchange in the stationary phase, after an initial period of more rapid exchange. As shown in Figure 12, addition of adequate cofactor (mercaptoethanol) immediately causes a severalfold, steady augmentation of the slope of hydrogen exchange rate. There is some discrepancy between these results and those of Borcsok and Abeles (92), who reported (with hepatic cystathione P-synthase) rates of a-H release from C3

substrates (serine and chloroalanine) without cosubstrate (homocysteine) that amounted to 30-70% (or more) of the rates of a-H exchange and P-replacement in the complete system.

However, their measurements were taken over the initial I5 min of incubation, that is, in the nonstationary period of increased a-H exchange rates (see discussion above; and Fig. 12). The reasons for the high initial hydrogen exchange rates are not clear; it may be due in part to some residual mercaptoethanol (in dialyzed enzyme tested for comparison) or to (nonproductive) P-replacement with solvent water-a natural cosubstrate for lyase 2-3 (Section II.A.2-3). Moreover, if the reaction mode suggested (92), that is, a mechanism including transitory a$-elimination, is valid, the facilitating conformational change may have occurred earlier, with 1 m M mercaptoethanol (Sz) still present, and unstable conformation could have persisted initially in the test samples.

To account for the suggested a$-abstraction step, as well as the inhibition of eliminating lyases by P-mercapto- and y-mercapto a- amino acids, the conformation for the nonconcerted elimination reaction postulated (92) should be syn rather than anti (Fig. 16). The P-replacing lyases are refractory to inhibition by P- or y-mercapto amino acids (Section 11I.E.6; Table XIX).

7. In the experiments of Borcsok and Abeles (92) with cystathionine P-synthase, as in our earlier studies with this lyase and Ala(CN)synthase (93,94), the P-replacement reactions did not show Ping-Pong kinetics, but obeyed an intersecting line pattern, implying intermediary production of ternary complexes of the two substrates with the enzyme (Section 1II.F).

8. To account for the fact that cystathionine P-synthase does not catalyze elimination of the p-substituent in the absence of cosubstrate, Borcsok and Abeles developed one of the hypotheses proposed by Floss et al. (98-loo), which implied a reaction mechanism associated with conformational changes in the active center. They suggested (92) that the amino acid substrate is bound at the active


site with the leaving group orthogonal to the a-C-H bond-a conformation highly unfavorable to nonconcerted elimination. Binding the cosubstrate induces a conformational shift, which brings the p- C-X bond into a position parallel to that of a-C-H, a conformation (syn) required for release of the leaving group. The authors discussed the possibility of a side-reaction for the Schiff base a-carbanion, in the absence of cosubstrate, that would prevent elimination, namely, an abortive transamination half-reaction to produce PMP-ketimine. They failed to obtain evidence for such a reaction.

Braunstein (8a, 1 10,126) stated that abortive transamination has been observed in virtually all types of PLP-enzymes, including ambifunctional lyases catalyzing elimination-dependent replacement, but never, as yet, in any p-replacement-specific lyase.

9. The p-replacing lyases differ from the eliminating and multifunctional ones in another feature that is not yet understood-the evidence for contrasting effects exerted by monovalent cations by K + , NH2 and Rb+, and on the other hand by Na+ or Li+ on physicochemical and catalytic parameters of the former and latter lyase subtypes (see Section 1II.A and Table XIII).

This article is the latest and most comprehensive overview of experimental and theoretical evidence reported and partially surveyed in support of our thesis that the catalytic mechanism of p- replacement-specific lyases differs significantly from that of a-@- elimination and elimination-mediated p-replacement.

The evidence concerning the physical and chemical phenomen- ology of the differences is sufficient, and important aspects of their mechanistic basis have been revealed.

Active efforts are being made to attain better understanding of the catalytic mechanism. Some unsolved, yet important aspects may be exemplified by a few perplexing questions. For example, why are many exclusively a$-eliminating lyases such as threonine (serine) dehydratases, alliin lyases, and others unable to effect a replacement step (Michael addition)? What is the difference in dynamic topography (stereochemistry) between enzymes of this subtype and the ambifunctional p-lyases with which they share similar behavior toward L-cycloserine or aminothiol inhibitors?

Finally, it is appropriate to emphasize the significance of research in this field to several areas of general biological interest.

Many reactions of direct p-replacement are known to constitute


essential links in pathways of the biosynthesis and metabolic transformations of protein constituents and secondary (nonprotein) amino acids. Lyases responsible for metabolic reactions of nitrogen-containing compounds rank high among specific target enzymes for natural control mechanisms for toxic and medicinal agents (132,133).

Second, we were impressed to note (though this was not unknown before) how readily available cysteine is in plants (and microbes) as a privileged metabolic precursor for many biologically important p- replacement products. In contrast, this amino acid is of high metabolic value in animals (at least in vertebrates) in whose nutrition the availability of cysteine is restricted since its major precursor is the nutritionally essential methionine.

Another fascinating aspect has recently emerged from studies concerning p-cyanoalanine synthase and its metabolic significance. The major role of cyanide and cyanoalanine as intermediates in one of the main pathways of synthesis and accumulation is becoming more evident; in particular, in leguminous and cereal fodder plants of L- asparagine, an especially valuable and readily assimilateable storage form of alimentary nitrogen.

Acknowledgments

Our thanks are due to many coworkers in our Institute, to visiting scientists, and to a number of foreign and Soviet colleagues in related branches of science, who contributed to the studies surveyed above either by active collaboration, or by valuable discussions and expert advice.

We are also grateful to our laboratory assistants for their skillful technical aid in a large variety of painstaking experimental work.

We appreciate the laborious assistance of Mrs. V. S . Bukanova in preparing the text and figures.

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