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

L-ASP ARTATE-8-DECARBOXYLASE; STRUCTURE, CATALYTIC ACTIVITIES, A N D ALLOSTERIC REGULATION*

By SURESH S. TATE and ALTON MEISTER, CorneZZ University Medical College, New York

C O N T E N T S

I. Introduction 503 505 507

A. Cofactor Site 507 1. Interaction of the Apoenzyme with Pyridoxal 5'-Phosphate 508 2. Function of Pyridoxal 5'-Phosphate in Subunit Interactions 510 3. Effects of Analogues of Pyridoxal 5'-Phosphate 5 12

B. Substrate Site 517 1. Analogues of L-Aspartate 517

C. a-Keto Acid Site 522 IV. Mechanism of Allosteric Regulation by a-Keto Acids 523

533 VI. Discussion 537

References 542

11. Catalytic Versatility of the Enzyme 111. Studies on the Active Center of the Enzyme

V. Subunit Structure of the Enzyme

I. Introduction

L-Aspartate-B-decarboxylase is unusual &s compared to the other amino acid decarboxylases in that it acts on the @-carboxyl group of its amino acid substrate to yield L-a-alanine :

coo- CH, I

CH, I

CHNH,+ I coo-

l CHNH," + CO, - (400-

* The authors are indebted to Dr. Esmond E. Snell for reading this manu- script prior to publication and for his valuable criticisms of it.

503

Advances in Enzymology and Related Areas of Molecular Biology, Volume 35 Edited by Alton Meister

Copyright © 1971 by John Wiley & Sons, Inc.

504 SURESH 5. TATE AND ALTON MEISTER

Of course, aspartate is itself unique in being the only protein amino acid to possess a B-carboxyl group; nevertheless, it is curious that there is still only slender evidence for the existence of an enzyme activity that catalyzes the a-decarboxylation of aspartate to B-alanine (ref. 1) .

Aspartate B-decarboxylase is also unusual in that it is activated by low concentrations of pyruvate, a-ketoglutarate, and many other a-keto acids. The striking activation of aspartate /I-decarboxylase by u-keto acids was first observed accidentally in 1950 in the course of experiments in which Clostridium perfringens cell suspensions were used as a source of glutamate decarboxylase for the determination of glutamate formed by transamination between aspartate and a- ketoglutarate (2). In these studies carbon dioxide formation from aspartate was found to be much greater when a-ketoglutarate was also present. It was demonstrated that L-a-alanine is the product of L-aspartate decarboxylation and that addition of pyridoxal 5’-phosphate also activated the decarboxylase reaction. Understanding of the nature of the activation by a-keto acids has developed from subsequent studies on purified aspartate decarboxylase isolated from Alcaligenes faecalis (3-5) ; this allosteric mechanism is considered in detail in this chapter.

Aspartate B-decarboxylase activity was first found in Pseudo- mycobacteria (6-9), and later in a number of microorganisms including Clostridium perfringens (2,10), Desulfovibrio desulfuricans (11,12), Nocardia globerula (13), Acetobacter sp. (14), Alcaligenes faecalis (3,4,15), Achromobacter (16,17), and Pseudomonas dacunhae (18). Its occurrence has also been reported in the silkworm (19), the crayfish, and the lobster (20). Highly purified preparations of the enzyme have been obtained from Alcaligenes faecalis (3,4,15,21-25), an organism isolated by enrichment culture (4), Achromobacter (16,17,26), and Pseudomonas dacunhae (27,28) ; the last two of these have been obtained in crystalline form. These three highly purified preparations of the enzyme as well its most of the less purified preparations obtained from other sources have been shown to be activated by a-keto acids.

Most of the findings reported in this chapter were made in the course of studies on the enzyme from Alcaligenes faecalis; however, the available data indicate that the aspartate p-decarboxylases of Pseudomonas dacunhae and Achromobacter are very similar to that of Alcaligenes faecalis. Thus all three of these enzymes have molecular

L-ASPARTATE-/?-DECARBOXYI,ASE 505

TABLE I Reactions Catalyzed by L-Aspartate-B-Decarboxylase

Reaction catalyzed K , ( M ) V,,,a Ref.

L-Aspartate --f L-alanine + CO, (1 ) 6.8 x 122b 5 L-Cysteinesulfinate -+ L-alanine + SO, (3) 4.8 x loF3 99b 21

pyruvate + C1- + H+ + NH4+ (5) 6.3 x 10-5 9 34

Aminomalonate --f glycine + CO, (4) 3 x 10-3c -0.25 33 /?-Chloro-L-alanine --f

L-Aspartate-pyruvate transamination 2.4 x lo-, 0.061 5 Threo-/?-hydroxyaspartate +

Erythro-/?-hydroxysspartate +

L-serine + CO, (2) 0.89 x -0.025 32

L-serine + CO, (2) 2.4 x -0.025 32 ~~

a Micromoles per minute per milligram of enzyme; 37', pH 6.5. b In the presence of added a-keto acid. 0 Concentration of aminomalonate at which half-maximal rate of glycine formation is observed. d K,,, (pyruvate). ' K i .

weights of about 700,000 and are composed of subunits. The Alcaligenes and Pseudomonas enzymes have been shown to contain 12 subunits, each of which is supplied with a molecule of pyridoxal 5'-phosphate.

Another remarkable feature of aspartate ,!?-decarboxylase is its catalytic versatility (Table I) ; thus, in addition to the decarboxylation of aspartate (reaction l), the enzyme catalyzes desulfination of cysteine sulfinate (reaction 31, decarboxylation of &hydroxyaspartate (reaction 2), a-decarboxylation of aminomalonate (reaction 4), two ,!?-elimination reactions (reactions 5 and 6), and a variety of transamination reactions. The ability of the enzyme to catalyze these reactions has provided clues to its structure and function. The key to understanding of the allosteric activation by a-keto acids seems to lie in its transaminase activity rather than it5 subunit structure.

11. Catalytic Versatility of the Enzyme

Thanks largely to the investigations of Snell and his collaborators (29-31) it is now known that all of the activities catalyzed by vitamin B, enzymes (except y-elimination and replacement reactions and the


phosphorylase reaction) can be demonstrated in nonenzymatic model systems. However, the corresponding enzyme-catalyzed reactions proceed much more rapidly, and it is generally believed that the protein portions of the vitamin B, enzymes are responsible for the striking increase in reaction rate ; in addition, the apoenzyme contributes specificity in terms of substrate and reaction pathway. Although these concepts seem basically correct, it has become increasingly apparent that a number of vitamin B, enzymes are not as specific in terms of substrate and reaction pathway as originally thought. It is now known that several vitamin B, enzymes can catalyze more than a single reaction ; L-aspartate-b-decarboxylase was one of the first enzymes to be shown to catalyze additional reactions. The ability of the enzyme to catalyze such reactions has had certain practical advantages (for example, to make possible a more convenient method of assay) and has offered insights into both the structure and function of the enzyme. In addition, it appears that one of the additional “minor” reactions catalyzed by aspartate @-decarboxylase is of substantial physiological significance in providing the enzyme with a highly sensitive and effective control mechanism.

The following list summarizes the reactions catalyzed by the enzyme ; some of the appropriate kinetic data are listed in Table I.

1. b-Deearboxylation. I n addition to catalysis of the major physiologically significant reaction (reaction l), the enzyme also catalyzes the decarboxylation of erythro and threo-/?-hydroxyaspartate to L-serine (32) :

coo- CH,OH

I I

CHOH

CHNH,+ I I

+ co, - dHNH3+

coo- coo-

2. Desu2Jimtion. The enzyme catalyzes desulfination of L-cysteine

soo- CH3 I I

sulfinate (21) :

CHs &HNH,+ + so, I I - Aoo- CHNH,+

coo-

(3)

L-ASPARTATE-B-DECARBOXYLASE 507

This reaction proceeds almost as readily as the @-decarboxylation of aspartate and provides a convenient method of assay based on the colorimetric determination of sulfite.

3. a- Decarboxyhtion. The enzyme catalyzes a-decarboxylation of aminomalonate (33) :

coo- CHNH,+ CH,NH, + CO,

coo- (4)

I I I

Although this reaction is analogous to the typical a-decarboxylation of amino acids, studies of the stereospecificity of this reaction indicate that the enzyme acts selectively on the carboxyl group of aminomalonate which corresponds in position to the -CH,COO- moiety of L-aspartate (see Section 111).

4. @-Elimination. The enzyme catalyzes the following @-elimination reactions (34) :

COOH

CH,CI CH, I I

CHNH,+ + H,O --f C=O + NH,+ + H+ + C1- I l

CH, CH, I I

I I I l

The interaction of p-chloro-L-alanine with the enzyme also leads to the formation of an enzyme derivative which contains the carbon chain of this amino acid (see Section 111).

5. Transamination. The enzyme catalyzes a wide variety of transamination reactions. For example, reactions between a long list of L-amino acids and a-ketoglutarate have been observed. In addition, reactions involving pyruvate and oxaloacetate have been demonstrated (3,4).

111. Studies on the Active Center of the Enzyme A. COFACTOR SITE

(6)

coo- coo-

(6) CH,CI + H,O --+ CH, + NH,+ + H+ + C1-

c=o CHNH,+

coo- coo-

I n early studies on the enzyme of Clostridium perfringens it was found that addition of pyridoxal 5'-phosphate activated the enzyme. When the enzyme was irradiated with ultraviolet light, activity was

508 SURESH S . TATE AND ALTON MEISTER

lost but could be restored by addition of pyridoxal 5’-phosphate or pyridoxamine 5’-phosphate plus an a-keto acid (2). Subsequent studies on purified enzyme preparations showed conclusively that the native enzyme contains pyridoxal 5‘-phosphate. Thus determinations by microbiological and chemical procedures indicated the presence of 1 mole of pyridoxal 5’-phosphate per 53,400 and 57,100 g of protein, respectively, for the Alcaligenes enzyme (4). Similar analyses of the enzymes from Achromobacter (16) and Pseudomonas dacunhae (28) indicated 53,000 and 50,000 g, respectively, of protein per mole of pyridoxal5’-phosphate.

Treatment of the holoenzyme with sodium borohydride results in inactivation associated with reduction of the Schiff base formed between pyridoxal 5’-phosphate and an €-amino group of the enzyme, and evidence was obtained for the presence of N‘-pyridoxyl lysine in hydrolysates of the sodium borohydride-reduced Achromobacter enzyme (17). An N’-phosphopyridoxyl lysine containing peptide has been purified from the cyanogen bromide cleavage products of the sodium borohydride reduced Alcaligenes enzyme (35).

These observations indicate that the isolated enzyme contains about 1 mole of pyridoxal 5’-phosphate per 50-60,000 g of protein and that the cofactor is linked to an €-amino group of the enzyme in a manner that is characteristic of other pyridoxal 5’-phosphate enzymes.

1. Interaction of the Apoenzyme with Pyridoxal 5‘-Phosphate When the holoenzyme is incubated with L-aspartate (or with a

number of other L-amino acids) the enzyme bound pyridoxal 5’- phosphate is converted by transamination to pyridoxamine 5’- phosphate, which tends to dissociate from the enzyme (3,4). Such dissociation occurs readily in buffers of high ionic strength. Thus the holoenzyme may be conveniently resolved by dialyzing it against 1 M sodium acetate buffer containing either 0.1 M L-aspartate or 0.1 M L-glutamate (22,36). In contrast to the holoenzyme which exhibits maximal absorbance in the region of 360 mp a t values’of pH from 4 to 8 (4,16,22), the apoenzyme exhibits no absorbance in this region (Fig. 1). The characteristic spectrum is restored on addition of pyridoxal 5’-phosphate to the apoenzyme. These properties make it possible to titrate the apoenzyme with pyridoxal 5’-phosphate; spectrophotometric titration curves of the enzyme gave a value of nearly 1 mole of pyridoxal 5’-phosphate per 53,000 g of enzyme (22).

L-ASPARTATE-p-DECARBOXYLASE 509

WAVELENGTH m y

Fig. 1. Absorption spectra of several forms of L-aspartate /?-decarboxyl&se. Cuvre 1, apoenzyme; curve 2, pyridoxal 6’-phosphate enzyme (holoenzyme); curve 3, holoenzyme after reduction by sodium boroh ydride; curve 4, 4’-deoxypyridoxine 5’-phosphate form of the enzyme. From reference 22.

The binding of pyridoxal 5’-phosphate to the Alcaligenes enzyme has been investigated by measurements of the optical rotato1 y dispersion of the apoenzyme and holoenzyme (22). The optical rotatory dispersion curves for both forms are superimposable a t high and low wavelengths, but the rotation of the holoenzyme is anomalous in the region of the absorption of the chromophore which exhibits a maximum a t 360 mp; the dispersion of the apoenzyme is plain in this region. When the specific rotation of the apoenzyme is subtracted from the holoenzyme, a difference dispersion is obtained which gives the rotation associated only with the bound chromophore. The extrinsic Cotton effect exhibited by the holoenzyme exhibits a point of inflection a t 360 mp. A plot of the amplitude of the Cotton effect against the amount of pyridoxal 5‘-phosphate added gives a value of 53,000 g of protein per mole of pyridoxal5’-phosphate.

The extrinsic Cotton effect is presumably generated by the asym- metric orientation of the optically inactive chromophore on the enzyme (compare ref. 37), and it seems probable that such orientation of pyridoxal5‘-phosphate is a consequence of its multipoint attachment to the enzyme. It is notable that covalent linkage between cofactor and enzyme is not required for induction of an extrinsic Cotton effect; thus the 4’-deoxypyridoxine 5‘-phosphate enzyme derivative, which does not have a covalent bond between cofactor analogue and enzyme, exhibits a substantial Cotton effect with point of inflection at about

510 SURESH S. TATE AND ALTON MEISTER

320 mp. The pyridoxamine 5’-phosphate enzyme derivative does not show anomalous optical rotatory dispersion. In contrast, the pyridoxamine 5’-phosphate derivative of glutamate-aspartate transaminase does exhibit a Cotton effect with point of inflection a t about 340 mp (38). It thus appears that there is a significant difference in the manner in which pyridoxamine 5’-phosphate is bound in these two enzymes. It seems probable that the pyridoxamine 5’-phosphate of the transaminase is bound more tightly and with greater asymmetry than that of the decarboxylase. On the other hand, the transaminase and the decarboxylase exhibit about equal affinity for 4’-deoxypyridoxine 5’-phosphate, and extrinsic Cotton effects of about the same magnitude have been observed with both enzymes. The /3-hydroxyaspartate complex of aspartate /?-decarboxylase does not show a Cotton effect, indicating that interaction of this substrate with the enzyme disrupts the asymmetry produced by enzyme-bound pyridoxal 5‘-phosphate and suggesting that there may be an associated change in the orientation of the cofactor on the enzyme. The nature of the complex formed between @-hydroxyaspartate and aspartate P-decarboxylase is considered below (Section 1II.B).

The interaction of the apoenzyme with pyridoxal 5’-phosphate and analogues of this cofactor has also been investigated by a fluorimetric procedure. Bertland and Kaplan (39) found that the binding of pyridoxal 5’-phosphate and of pyridoxamine 5’-phosphate to chicken-heart soluble glutamate aspartate transaminase decreases the fluorescence of the tryptophan residues of the enzyme. Similar effects have been observed with aspartate ,9-decarboxylase (36). Thus excitation of the apoenzyme and holoenzyme at 280mp generates emission spectra with maxima at 345 mp; however, the tryptophan- fluorescence intensity of the holoenzyme is much lower than that of the apoenzyme (Fig. 2). This decrease in fluorescence intensity provides a sensitive method for the study of enzyme-cofactor interaction. Fluorimetric titration of the apoenzyme with pyridoxal 5’-phosphate gave a value of 54,000 g of protein per mole of pyridoxal5’-phosphate (36).

2. Function of Pyridoxal5’- Phosphate in flubunit Interactions In the course of studies on the sulfhydryl groups of the Alcaligenes

enzyme (24), it was found that resolution of the holoenzyme = 19 S) led to formation of an apoenzyme, which at pH 8 exhibited a much

L- ASPARTATE-p-DECARBOXYLASE 511

Wavelength, mp

Fig. 2. Fluorescence emission spectra of the rtpoenzyme and holoenzyme. The excitation wavelength was 280 mp. From reference 36.

lower molecular weight (a20,w = 6 S). This was first demonstrated by acrylamide gel electrophoresis (24) and subsequently confirmed by analytical ultracentrifugation (40). The holoenzyme exhibits a sedimentation coefficient ( s ~ ~ , ~ ) of 19 S at pH values of 5-8 and the apoenzyme at pH 6 exhibits the same sedimentation coefficient. However, a t pH 8, the 19-S apoenzyme undergoes dissociation to a 6-S species (Fig. 3). As discussed below, the 19-S and 6-S species have molecular weights of 675,000 and 114,000, respectively (Section V). It has also been established that the 6-S species is a dimeric protomer of the dodecameric 19-5 enzyme. There is a large difference in the mobilities of the two forms of the enzyme on acrylamide gel electrophoresis, and this procedure therefore provides a convenient method for study of association-dissociation phenomena. When the 6-S apoenzyme is treated with pyridoxal 5’-phosphate a t pH 8, prompt association to the 19-S species occurs. A number of other vitamin B, 5’-phosphate analogues also induce association of the 6-S species (see

la i P L P ; P H 6

HOLOENZYME .- * APOENZYME 1 lb

(19 S; 12 mer) (19 S; 12 mer)

5PLP ; p H 8 3b

APOENZYME I1 (6 S; dimer)

Fig. 3. Subunit interactions of ~-aspartate-8-decarboxylase. From reference 59.


below). It thus appears that pyridoxal5’-phosphate, in addition to its role in catalysis, contributes to the stability of the 19-S enzyme and thus plays a role in the maintenance of the quaternary structure of the enzyme.

3. Eflects of Analogues of Pyridoxal 5’- Phosphate The apoenzyme interacts with a number of vitamin B, 5’-phosphate

analogues (36), and some of the properties of these enzyme forms have been studied (Tables I1 and 111). Optical rotatory dispersion studies of the 4’-deoxypyridoxine 5’-phosphate enzyme have been mentioned above. As indicated in Table 111, all eight of the vitamin B, 5’- phosphate derivatives that have been studied bind to the apoenzyme to approximately the aame extent; thus almost 1 mole of cofactor analogue is bound per 57,000 grams of enzyme. In general, those analogues that form Schiff bases with the enzyme-for example, the 5’-phosphate derivatives of pyridoxal, 2-nor-pyridoxal, 2-nor-2- ethylpyridoxal, 2-nor-2-butylpyridoxa1, and 3-0-methylpyridoxal-are bound to the enzyme with high affinity. An exception to this statement

TABLE I1 Vitamin B-65’-Phosphate Derivatives&

RsO&CH20P03= “+

Rz I R,

Imax ( m p ) Vitamin B, 5’-phosphate

derivative R, R, R, R4 Free +Enzyme

Pyridoxal H 2-Norpyridoxal H

3-0-Methylpyridoxal H N-Methylpyridoxal CH3 P yridoxamine H Pyridoxine H 4’-Deoxypyridoxine H

2-Nor-2-ethylpyridoxal H 2-Nor-2-n-butylpyridoxal H

* From reference 36.

CH3 H H H C2H6 nC4Ho H CH3 CH, CH3 H CH3 H CH3 H CH3 H

CHO CHO CHO CHO CHO CHO

CH,OH CHZNH,

CH3

330,388 330,388 330,390 330,393 279,313 330,398

327 293,324 284,314

~

358 358 358 370 299

330,398 322

314

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514 SURESH S . TATE AND ALTON MEISTER

is 4'deoxypyridoxine 5'-phosphate, which exhibits the same affinity for the enzyme as does pyridoxal 5'-phosphate; pyridoxine 5'- phosphate is bound somewhat less tightly. However, the affinity for both 4'deoxypyridoxine 5'-phosphate and pyridoxine 5'-phosphate is greater than that observed for pyridoxamine 5'-phosphate. These observations seem to be of interest in relation to the observation that 4'-deoxypyridoxine 5'-phosphate and pyridoxine 5'-phosphate exert a protective effect on aspartate @-decarboxylase in the presence of aspartate and u-keto acid (41). It has been suggested that the binding of 4'deoxypyridoxine 5'-phosphate and of pyridoxine 5'-phosphate to some of the active sites of the enzyme exerts a stabilizing effect on the binding of pyridoxamine 5'-phosphate to other sites. The possibility of such cooperative effects in the binding of cofactor to the enzyme requires additional investigation. The relatively weak binding of pyridoxamine 5'-phosphate to the enzyme may possibly be ascribed to its dipolar nature. It is of interest that the curve obtained in the fluorimetric titration of the apoenzyme with pyridoxamine B'-phosphate is sigmoidal (36); an explanation of this finding is not yet available.

The presence of a 5'-phosphate group appears to be necessary for attachment of the cofactor to the enzyme; neither pyridoxal nor pyridoxal 5'-sulfate is bound. All of the vitamin B, 5'-phosphate derivatives studied induced prompt association of the 6-S apoenzyme to the 19-S form. The ability of the pyridoxine 5'-phosphate, pyridoxamine 5'-phosphate, and 4'deoxypyridoxine 5'-phosphate to induce such association indicates that this phenomenon does not require the formation of a covalent bond between the cofactor and the enzyme. In addition, N-methylpyridoxal 5'-phosphate, which as discussed below does not form 8 Schiff base with the enzyme, also promotes association. It is of interest that Morino and Snell (42) found that pyridoxal 5'-phosphate promotes the conversion of dimeric apotrypto- phanase into a tetrameric holoenzyme, and a t higher molar ratios of coenzyme to enzyme, pyridoxine 5'-phosphate, pyridoxamine 5'- phosphate, and B'-phosphopyridoxal tryptophan also promote conversion of the dimer to tetramer. It seems possible that such coenzyme- induced subunit interactions may be a general property of vitamin B, enzymes.

As indicated in Table 111, vitamin B, 5'-phosphate derivatives in which the methyl group at C-2 of the pyridine ring is replaced by a

L-ASPARTATE-,!I-DECARBOXYLASE 515

hydrogen atom, ethyl group, or butyl group, serve less effectively than pyridoxal5’-phosphate in the decarboxylase and desulfhase reactions. The 3-0-methylpyridoxal 5’-phosphate enzyme and the N-methylpyridoxal 5‘-phosphate enzyme aIso exhibit very low decarboxylase and desulfinase activities. However, the L-aspartate-pyruvate transaminase activity with 2-norpyridoxal 5’-phosphate is more than twice that found with pyridoxal 5’-phosphate, and the 2-nor-2- ethylpyridoxal enzyme is also more effective in catalyzing this reaction than the pyridoxal 5‘-phosphate enzyme. The 2-nor-2-butylpyridoxal 5’-phosphate, 3-0-methylpyridoxal 5’-phosphate, and N-methylpyridoxal 5’-phosphate derivatives of the enzyme also exhibited substantial transaminase activities. Such a difference in the effects produced by replacing pyridoxal 5’-phosphate with analogues on the activities catalyzed by the same enzyme may, a t least in part, be ascribed to the presence of a separate a-keto acid binding site in the active center of the enzyme (see Section 1II.C; Section V). Thus a cofactor analogue may be oriented in the active center in such a way as to decrease the rates of decarboxylation and desulfination, but not as to affect transamination appreciably. A detailed understanding of these phenomena must await further information about the spacial relationships between the binding sites for a-keto acid, aspartate, and cofactor within the active center.

The effects of various vitamin B, 5’-phosphate derivatives on other enzymes such as glutamate-aspartate transaminase (43-45), trypto- phanase (431, arginine decarboxylase (46), and muscle phosphorylase (47), indicate that there is no general pattern with respect to afinity of the apoenzyme for coenzyme, affinity of the reconstituted holoenzyme for substrates, or maximal velocity of the reaction catalyzed. That different enzymes behave differently when pyridoxal 5‘-phosphate is replaced by an analogue seems to reflect differences in the manner in which the coenzyme is bound and probably also differences in the mechanisms of the reactions catalyzed.

It is of interest that N-methylpyridoxal5’-phosphate does not form a Schiff base with the enzyme (36). This conclusion is substantiated by the absence of a characteristic spectral shift when this vitamin B, 5’-phosphate derivative interacts with the enzyme. Furthermore, treatment of the N-methylpyridoxal5‘-phosphate enzyme with sodium borohydride does not result in loss of enzymatic activity. Since the N-methylpyridoxal 5‘-phosphate enzyme exhibits about 70% as much


transaminase activity as the pyridoxal 5’-phosphate enzyme, it is evident that Schiff base formation between coenzyme and enzyme is not required for a substantial rate of transamination, and thus that relatively little catalytic advantage can be ascribed to a trans- Schiffization mechanism. Although the aldehyde group of N-methyl- pyridoxal5’-phosphate does not interact with an e-amino group of the

TABLE I V Decarboxylation of D-Aspartate by the N-Methylpyridoxal

5’-Phosphate Enzymea

Enzyme derivative

Alanine-I4C formed

(nM/hr)

None (N-methylpyridoxal 6’-phosphate present) None (pyridoxal 5’-phosphate present) Apoenay me Pyridoxal 5’-phosphate 2-Nor-2-ethylpyridoxal 5’-phosphate 2-Nor-2-butylpyridoxal 5’-phosphate 3-0-Methylpyridoxal 5’-phosphate N-Methylpyridoxal 5’-phosphate

4.5 3.9 1.5 2.3 2.7 2.0 1.9

44.0

a The apoenayme (0.2 mg) was incubated at 37’ for 5 min with 50 nM vitamin B, 5’-phosphate derivative and 1 pM of sodium a- ketoglutarate in 1 ml of 0.1 M sodium acetate buffer (pH 5.5). ~-Aspar ta te - l - l~C (5 p m ; 0.05 ml) was added and incubation was continued at 37’ for 1 hr. Aliquots were removed for a1anine-l4C determination by Dowex 1-acetate chromatography. (From reference 36.)

protein, it can nevertheless form a Schiff base with the substrate. An additional and most interesting property of the N-methylpyridoxal 5’-phosphate enzyme is its ability to decarboxylate and to transaminate D-aspartate. Although the rates of these reactions are much lower than the corresponding reactions of L-aspartate, they are nevertheless significantly higher than the values for the appropriate controls and those for the other vitamin B, 5‘-phosphate enzymes (Table IV). These findings suggest that the strict L-amino acid specificity of the pyridoxalEi’-phosphate enzyme is related in part to Schiff base formation between coenzyme and enzyme. The 4’ddehyde carbon atom of pyridoxal5’-phosphate is part of a rigid planar structure which includes

L-ASPARTATE-p-DECARBO XY LASE 517

the pyridine ring. Displacement of the pyridine ring on the enzyme by introduction of an N-methyl group would be expected to produce a corresponding displacement of the 4‘-aldehyde group. This might move the aldehyde carbon atom away from the position of the amino group of the enzyme to a site in which it is able to react with the amino groups of both L- and D-aspartate.

It is notable that the product of the 1-decarboxylation of D-

aspartate is L-alanine rather than D-alanine. This result is not unexpected, since asymmetry about the u-carbon atom of aspartate is lost on formation of the ketimine Schiff base. Thus the same ketimine is formed from D- and L-aspartate, and the pathways of the reactions with both isomers of aspartate lead to L-alanine. It thus appears that the substrate binding site of the N-methylpyridoxal 5’-phosphate enzyme is altered so as to allow D-aspartate to be bound, but the remainder of the catalytic process is the same L-specific pathway that is catalyzed by the pyridoxal5’-phosphate form of the enzyme.

B. SUBSTRATE SITE

1. Analogues of L-Aspartate

As outlined in Section I1 (see also Table I), the enzyme can interact with a number of substrates. The amino acids which can combine with the enzyme (except for those which participate only in transamination) possess either a negatively charged or an electronegative /?-substituent ; molecular models of these compounds exhibit interesting similarities.

Wilson and Kornberg (17) found that threo and erythro-@-hydroxyaspartate are strong competitive inhibitors of the Achromobacter enzyme and that they induce a change in the maximum absorbance of the enzyme from 360 to 325 mp. However, subsequent treatment of the enzyme with u-keto acids does not affect the absorbance, indicating that this form of the enzyme is not the pyridoxamine 5’-phosphate species. Treatment of the enzyme with sodium borohydride reduces the holoenzyme in the absence but not in the presence of b-hydroxyaspartate, indicating that the 8-hydroxyaspartate derivative is not a Schiff base. Subsequent studies (32) showed that both stereoisomers of b-hydroxyaspartate also inhibit the Alcaligenes enzyme in a similar manner, and further that such inhibition is associated with the slow decarboxylation of the substrates to yield L-serine (reaction (2). Detailed studies of the reaction indicated that the rate of carbon


dioxide liberation was greater than that of serine formation. It was also shown that the form of the enzyme that exhibits maximal absorbance a t 325 mp is inactive in the decarboxylation of L-aspartate and in the desulfination of L-cysteine sulfinate. Experiments in which the enzyme was incubated with 14C-@-hydroxyaspartate and then subjected to gel filtration showed that the inhibited form of the enzyme contains a moiety derived from carbon atoms 1-3 of @-hydroxyaspartate; this is converted to serine on further incubation. When the inhibited form of the enzyme was treated with 2-4-dinitrophenyl- hydrazine in hot acid solution, a compound was obtained which

H27- CHCOOH I

0, ,NH CH

Fig. 4. Oxazolidine derivative postulated for the b-hydroxyaspartate inhibited form of the enzyme (32).

appeared to be the osazone of glycolaldehyde. The evidence supports the conclusion that the inhibited form of the enzyme is produced in a reversible side reaction and that it is in equilibrium with one of the normal enzyme-substrate intermediates. This form of the enzyme is not reduced by sodium borohydride. Several possible structures of the inhibited form of the enzyme have been considered, including the oxazolidine derivative (Fig. 4).

It is of interest that p-methylaspartate does not exhibit inhibition of the type observed with @-hydroxyaspartate (32); neither is u- methylaspartate a substrate or an inhibitor of the enzyme (48). The latter finding suggests that the a-hydrogen atom of L-aspartate normally lies in close contact with the protein.

Aspartate B-decarboxylase from Alcaligenes faecalis catalyzes the decarboxylation of aminomalonate to glycine ; the rate of this reaction is about 0.2% of that for the decarboxylation of L-aspartate (33). Although the reaction is apparently analogous to the typical u- decarboxylation of L-amino acids catalyzed by a number of enzymes, it differs from these in that the enzyme acts selectively on the carboxyl


group of aminomalonate which corresponds to the @-carboxyl group of aspartic acid. Thus, when aminomalonate is enzymatically decarboxylated in tritiated water, (S)-glycine-2-t is formed rather than (R)- glycine-2-t, which would be the expected product of a typical a-decarboxylation (Fig. 5). Wellner (49,50) has shown that D-amino acid oxidase specifically releases one of the a-hydrogen atoms of giycine and that the other a-hydrogen atom of glycine is stereo- specifically introduced by L-specific transaminase. Wellner also showed

coo- l CHI A Aspartate Decarboxylase (PLP) I

H W C *NH,+ > H-C-NH,' . ioo- too-

S- Aspartate S-Alanine

coo- I Aspartote Decarboxylase (PLP) " I D-amino

I coo-

H-C-NH: Acid oxidast H-C=O +'H,O I I coo- coo- ' H Z O

H-C-NH:

Aminornalmate s-'H-glyclne

H Aspartate Decarboxylase (PMP) 3H-C-NH3+ I D-amino

H-C=O I m 'H-t=O + 2 0 I 3 ~ ~ 0 coo- coo- coo-

R- %-glycine

Fig. 5. Synthesis of both optical isomers of mono (tritio) glyoine catalyzed by L

aspartate /I-decarboxylase (33).

that enzymatic conversion of serine into glycine catalyzed by serine hydroxymethylase is accompanied by stereospecific introduction of a hydrogen atom into glycine; this hydrogen atom is released by D-amino acid oxidase. Thus (S)-glycine-2-t is formed in the reaction catalyzed by serine hydroxymethylase and in the decarboxylation of aminomalonate by aspartate @decarboxylase ; in the latter reaction the carboxyl group that is lost may be considered to be in a position which is analogous to the -CH,OH group of L-serine and to the -CH,COOH moiety of L-aspartate. However, it is conceivable that the carboxyl group of aminomalonate that is in the position analogous to that of the a-carboxyl group of aspartate is actually lost and that addition of the proton is accompanied by inversion of configuration.


Recently, Palekar et al. (51) carried out studies with 14C-aminomalonic acid preparations specifically labeled in each carboxyl group (prepared from the corresponding L-serines labeled either in carbon atom 1 or carbon atom 3) ; these experiments conclusively demonstrated that the carboxyl group of aminomalonic acid which corresponds to the p-carboxyl group of aspartic acid (and which is derived from carbon atom 3 of L-serine) is decarboxylated ; inversion is therefore excluded.

When the pyridoxamine 5’-phosphate form of enzyme is incubated with glyoxylate in tritiated water, (R)-glycine-2-t is obtained (Fig. 5) . The ability of the enzyme to produce both isomers of glycine-2-t reflects still another aspect of its catalytic versatility.*

When the enzyme is incubated with aminomalonate, the decarboxylation of this aspartate analogue decreases with time, indicating enzyme inhibition (33). On incubation of the enzyme with aminomalonate the absorbance maximum of the holoenzyme shifts to about 325 mp, and this absorbance change is not affected by adding u-ketoglutarate. It can be demonstrated by gel filtration that aminomalonate is bound to the enzyme under these conditions; almost 1 mole of aminomalonate is bound per 60,000 g of enzyme. Studies with amin~malonate-U-~~C and aminomalonate-l-14C gave substantially the same values; the data thus indicate that all three carbon atoms of aminomalonate are bound to the enzyme. Reduction of the aminomalonate-inhibited enzyme with sodium borohydride-t led to incorporation of tritium into the enzyme. The spectral shift to 325mp suggests formation of a ketimine derivative, and this is in accord with the uptake of tritium after treatment with sodium borohydride-t. Thus the aminomalonate enzyme complex may be a ketimine form which interacts with a group (or groups) on the enzyme in a reaction which is relatively slow as compared to the other transformations which occur on the enzyme; the strong negative charge on the ketimine might promote a conformational change in the enzyme. Aminomalonate is apparently not bound by covalent linkage, since treatment of the complex with trichloro- acetic acid or ethanol liberates aminomalonate from the enzyme. It is notable that Matthew and Neuberger (52) found that aminomalonate inhibits 8-aminolevulinate synthetase and serine hydroxymethylase but does not inhibit tyrosine decarboxylase or glutamate-aspartate transaminase.

* See related discussion by H. C. Dunathan, this volume, page 88.

L-ASPARTATE-/&DECARBOXYLASE 52 1

As indicated above, aspartate B-decarboxylase catalyzes B-elimination reactions involving 8-chloro-L-alanine and threo-@-chloro-L-a- aminobutyrate (34). The rapid /3-elimination reaction with B-chloro- L-alanine (reaction 5 ) is accompanied by a slower reaction in which the enzyme becomes irreversibly inactivated ; inactivation is aceompanied by a shift of maximum absorbance to 325 mp. Such inactivation did not take place when 1-chloro-L-alanine was incubated with the apoenzyme or with the 4‘-deoxypyridoxine 5’-phosphate enzyme. @-Chloro-D-alanine does not inhibit the pyridoxal5’-phosphate enzyme. Inactivation is associated with binding to the enzyme of the 3-carbon moiety of 8-chloroalanine, and the available data indicate that a covalent linkage is formed close to or a t the aspartate binding site of the enzyme. Quantitative studies indicate that almost 1 mole of the chloroalanine derivative binds per 57,000 g of enzyme. The inactivation process may be viewed as arising from a nucleophilic attack by a group on the enzyme on the p-carbon atom of B-chloroalanine or the analogous a-aminoacrylate Schiff base. In either case, structure I11 (Fig. 6) would be formed. Hydrolysis of I11 followed by removal of pyrido- amine 5’-phosphate would yield an a-keto derivative of the enzyme containing the 3-carbon chain of B-chloroalanine (structure VI). Tautomerization of I11 to the corresponding aldimine (V) followed by hydrolysis and loss of pyridoxal 5’-phosphate would give the analogous a-amino derivative (VII). Identification of the amino acid residue of the enzyme that is alkylated and the amino acid sequence associated with this residue should provide further information about the structure of the active center. The ,&elimination of p-chloroalanine is also catalyzed by L-serine dehydrase (53) and by rat-liver preparations (54). Glutamate-aspartate transaminase preparations catdyze dehydro- fluorination and deamination of B-fluoroaspartate (55) as well as B-elimination of p-chloroglutamate (56). Thus several vitamin B, enzymes can catalyze reactions of this type. Covalent bond formation has been observed when glutamate-aspartate transaminase is incubated with L-serine-0-sulfate (57,58).

B-Cyano-L-alanine is an effective competitive inhibitor toward L-aspartate and exhibits a Ki value of about 2.7 x lo-* (5). When the enzyme is incubated with B-cyano-L-alanine, there is a prompt shift in the maximum absorbance a t 358 mp to a spectrum which exhibits a shoulder in the region of 310-320 mp. Gel filtration or dialysis of this form of the enzyme leads to restoration of catalytic activity as well as

52 2 SURESH 9. TATE AND &TON MEISTER

i 4 I VI HzN-lys

I

II I

-:;C -C- CHI -X

Y CH HzN-lyr

Fig. 6. Proposed pathways for the conversion of 8-chloro-L-alanine to pyruvate, ammonia, H+ and C1-, and for the alkylation of the enzyme (34).

the original spectrum of the pyridoxal 6'-phosphate enzyme. It seems probable that the observed 0-cyano-L-alanine-enzyme complex is an abortive ketimine derivative. A similar complex has been observed when L-asparagine is incubated with the enzyme (48).

C. a-KETO ACID SITE

The enzyme is activated by a variety of a-keto acids (Table V), and there is now good evidence that the active center has a separate binding site for the -C-COO- moiety; see below, Section IV.

L-ASPARTATE-p-DECARBOXYLASE

TABLE V Effect of Various a-Keto Acids on Aspartic B-Decarboxylase&

523

CO, evolution a-Keto acid (p1/10 min)

None 12.1 Pyruvate 87.0 a-Ketoglutarate 92.4 a-Ketoisovalerate 69.9 a-Ketoisocaproate 62.0 D-a-Keto-B-methylvalerate 23.9 L-a-Keto-B-methylvalerate 31.6 a-Keto-6-carbamidovderate 24.2 a-Keto-6-guanidinovalerate 13.2 Phenylpyruvate 63.6 a-Ketomalonate 61.0 a-Ketophenylacetate 16.6 Trimethylpyruvate 13.1 Glyoxylate 64.9 a-Keto-y-methiolbutyate 67.6

a From Novogrodsky and Meister (4).

IV. Mechanism of Allosteric Regulation by cr-Keto Acids

In the initial studies on the aspartate 1-decarboxylase of Clostridium perfringens it was found that this enzyme is markedly. activated by pyruvate and also by pyridoxal phosphate, but that the effects of these activators are not additive (2). A possibility considered early was that the apparent decarboxylation of aspartate might be due to transamination of aspartate with pyruvate to yield alanine and oxaloacetate followed by decarboxylation of the latter compound to pyruvate. However, studies with added 14C-pyruvate excluded this mechanism because only very small amounts of 14C-alanine were formed (2). On the other hand, the observation that a very small but definite amount of labeled alanine was formed proved to be consistent with another interpretation (3,4). Thus it was shown that the enzyme catalyzes transamination between aspartate and added a-keto acid, but that this reaction takes place a t a rate very much slower than that of the decarboxylation of aspartate to alanine. When studies were carried out on highly purified preparations of the enzyme from Alcaligenes faecalis, it became evident that the enzyme acts both as a


relatively nonspecific L-amino acid transaminase and as an L-aspartate p-decarboxylase. Incubation of the enzyme with L-aspartate or any one of a number of other L-amino acids led to inactivation, which could be prevented by addition of a-keto acids and reversed by addition of pyridoxal5’-phosphate. Evidence derived from enzymatic and spectrophotometric studies indicated that the inactivation was due to conversion of enzyme-bound pyridoxal 5‘-phosphate to enzyme- bound pyridoxamine 5’-phosphate ; the latter form, inactive in decarboxylation, reverted to the pyridoxal 5’-phosphate form on transamination with u-keto acids. Under certain conditions (for example, in the

a- Keto acid

I + Aspnrate I Enzyme PLP ___f Enzyme PMP - Enzyme + PMP

t I Fig. 7. Proposed mechanism for the activation of L-aspartate /?-decarboxylase

by a-keto acid and by pyridoxal 5’-phosphate.

presence of a high concentration of sodium acetate buffer) pyridoxamine 5‘-phosphate dissociated from the enzyme ; the apoenzyme could then be reactivated by addition of pyridoxal 5‘-phosphate. According to this interpretation, the activating effects of u-keto acids and pyridoxal 5‘-phosphate can be explained in terms of the diagram given in Figure 7. Thus the enzyme is remarkable in that the same active site can participate in two reactions. One of these is capable of destroying (or regenerating) the coenzyme necessary for the other. The transamination mechanism is consistent with the experimental observations that the enzyme can catalyze a wide variety of transamination reactions between various pairs of L-amino acids and u-keto acids. Although the rate of transamination is quantitatively low as compared to the rate of the decarboxylation, the transamination reaction is of considerable significance since it effectively controls the amount of pyridoxal 5’-phosphate cofactor available for aspartate decarboxylation. Since all vitamin B, enzymes (except the transaminases) can use only pyridoxal 5’-phosphate as the active cofactor, it is evident that even a very slight amount of transamination could inhibit a vitamin B, enzyme. This type of transamination control has been suggested as a

L-ASPARTATE-p-DECARBOXYLASE 525

possible general regulatory mechanism for this group of enzymes (Fig. 8).

Later studies were directed toward determining whether the transamination mechanism alone could suffice to explain the activation by u-keto acids (5). It was possible to obtain conditions under which aspartate is decarboxylated by the enzyme a t a constant rate in the absence of added u-keto acid. Conditions were employed under which cofactor remained attached to the enzyme and did not dissociate even when it was in conditions, the

the pyridoxamine 5'-phosphate form. Under these effect of L-aspartate concentration on the rate of

I Pyridoxamine enzyme

Substrate

substrate analogue U-ZO 11 or

Pyridoxal enzyme I Substrate f products

Fig. 8. General scheme for the control of vitamin B, enzyme activity.

decarboxylation in the presence and absence of u-ketoglutarate follows Michaelis-Menten kinetics, and the double-reciprocal plots of the data indicate that, within experimental error, the K , values for L-aspartate are the same in the absence and presence of added ci-

ketoglutarate; that is, 7.2 & 0.8 x M , respectively. The results on the Alcaligenes enzyme are given in Figure 9; similar results were obtained in experiments on the enzyme from Pseudomonas dacunhae (59).* Studies in which the time course of the reaction was followed in the presence and absence of pyruvate are shown in Figure 10. The rate of decarboxylation in the absence of a-keto acid (curve 2 ) is about 20% of that observed in the presence of an optimal concentration of pyruvate (curve 1). Transamination ( p y r ~ v a t e - ~ ~ C versus aspartate) (curve 5) was determined in the

and 8.5 x M were obtained in the absence and presence, respectively, of added a-keto acid; typicaI Michaelis-Menten kinetics were observed (59). A sigmoidal relationship between decarboxylase activity and L-aspartate concentration in the absence of added a-keto acid has been reported for the Pseudomonas enzyme, and a K , value for L-aspartate of 0.1 M was derived from this sigmoidal curve (67). Studies in the authors' laboratory have failed to confirm these findings; a possible explanation for them has been presented (59).

M , and 6.4 f 0.2 x

* Apparent K , values for L-aspartate of 10 x


c I I ' 'i ' ' ' I I ' I I '

present)

2 o p - x ; i

2 4 6 8 10 12 14 [Aspartote, mM]

Fig. 9. Effect of L-aspartate concentration on the rate of decarboxylation in the presence and absence of a-ketoglutarate. Curve 1, a-ketoglutarate (1 mM) present; curve 2, a-ketoglutarate absent. The inset gives the corresponding double-reciprocal plots of the data. From reference 5.

reaction mixture used for curve 1 ; transamination was parallel to decarboxylation during the first 76% of the course of the reaction, and the ratio of the initial rate of decarboxylation to that of transamination was 2380. In an effort to determine whether the product of transamination was pyruvate ,or oxaloacetate, experiments were carried out in which lactate and malate dehydrogenases and DPNH were added. A0 shown in the experiment described in curve 4, addition of malate dehydrogenase and DPNH did not affect the reaction. However, when lactate dehydrogenase and DPNH were added, decarboxylation fell off rapidly (curve 3); when a-ketoglutarate (curve 3A) was added in this experiment, there was a substantial increase in decarboxylation

L-ASPARTATE -/~-DECARBOXYLASE

!Q 2 (No Kcto Acid

2 4 6 8 10 12 14 16 18

527

Minutes

Fig. 10. Effect of lactate and malate dehydrogenases on the decarboxylation of L-aspartate. Curve 1, decarboxylation in the presence of sodium pyruvate (1 mM); curve 2, decarboxylation in the absence of added a-keto acid; curve 3, decarboxylation in the presence of lactate dehydrogenase and DPNH or lactate dehydrogenase, malate dehydrogenase, and DPNH. At the point. indicated by the arrow, sodium a-ketoghtarate was added (curve 3A); curve 4, decarboxylation in the presence of malate dehydrogenase and DPNH; curve 5, transamination between L-aspartate and pyruvate (right-hand ordinate). From reference 5.

and a rate was achieved which is not far from that obtained when a-keto acid was added initially (curve 1). These findings indicate that pyruvate rather than oxaloacetate is formed during the decarboxylation of aspartate, and they also show that the presence of the pyruvate thus formed is required for a constant rate of decarboxylation under these conditions. However, since the rate of decarboxylation in the absence of added a-keto acid (curve 2) is only one-fifth of the rate found with added a-keto acid (curve l), it may be concluded that a substantial fraction of the enzyme must be in the pyridoxamine 5’-phosphate form. However, the amount of enzyme in the active pyridoxal 5’- phosphate form must be constant during the linear phase of decarboxylation. Furthermore, since all of the enzyme was initially in the pyridoxal 5’-phosphate form, i t would be expected that the initial rate of the decarboxylation reaction should be the same in the presence and absence of added a-keto acid. I n order to investigate this


possibility, an experiment was carried out with a relatively large amount of enzyme at 6". Under these conditions, it was possible to demonstrate that, as shown in Figure 11, during the first minute of the reaction, the rate of formation of alanine from aspartate in the absence

Minutes

Fig. 11. Concomitant decarboxylation of L-aspartate and formation of pyruvate. The reaction mixtures contained aspartate-l-14C and enzyme (0.1 mg) in a final volume of 1 ml. Curve 1, decarboxylation (alanine formation) in the presence of added pyruvate (0.1 mM); curve 2, decarboxylation in the absence of added pyruvate; curve 3, formation of l-14C-pyruvate. The inset shows the initial portions of curves 1 and 2 on an expanded scale in order to emphasize the initial burst of alanine formation in curve 2. The reaction mixtures were incubated at 6". In the experiment described in curve 3, unlabeled L-aspartate was added at the point indicated by the arrow. From reference 5.

of added a-keto acid (Fig. 11; curve 2) was indeed the same as that observed in the presence of a-keto acid (Fig. 11 ; curve 1). Thereafter, both reactions proceed linearly but a t markedly different rates, essentially as indicated by the experiments described in Figures 10 and 11. Determinations of pyruvate-14C were also carried out in the experiment described in Figure 11, curve 2. After 30 sec of incubation, about 1.3 nM of labeled pyruvate was found and, within experimental

L-ASPARTATE-8-DECARBOXYLASE 529

error, the amount of pyruvate present did not vary during the subsequent course of the decarboxylation reaction. It may be seen that the “burst” of pyruvate formation coincides with the initial rapid phase of decarboxylation in the experiment described in Figure 11, curve 2. If the amount of labeled pyuvate formed is taken as a measure of the amount of enzyme converted to the pyridoxamine 5’-phosphate species, it can be calculated that not more than one-third of the total enzyme present can be in the active pyridoxal 5’-phosphate form. This seems to be in accord with the observation that the linear portion (2-25 min) of curve 2 describes a rate of decarboxylation which is about one-third of that found in the experiment described in curve 1, in which an optimal concentration of a-keto acid was added. These experimental observations indicate that an equilibrium is established between the active form of the enzyme and enzyme-bound pyridoxamine 5’-phosphate and pyruvate so that the amount of pyridoxal 6’-phosphate available for reaction with substrate remains constant and thus serves to catalyze the decarboxylation of aspartate a t a constant rate. The findings are in accord with the scheme given in Figure 12, according to which the aspartate ketimine (111) is decarboxylated to the ketimine of alanine (IV). Tautomerization of I V to V followed by hydrolysis yields the pyridoxal 5‘-phosphate form of the enzyme and alanine, that is, the normal catalytic pathway of decarboxylation. On the other hand, hydrolysis of I V yields pyruvate and the inactive pyridoxamine form of the enzyme. To account for a constant rate of alanine formation, it is evident that the concentration of form I must be constant and that the rate of hydrolysis of IV to VI and pyruvate must be equal to the rate of formation of IV, that is, IV + H,O = VI + pyruvate. Addition of lactate dehydrogenase and DPNH would remove pyruvate and thus increase hydrolysis of IV with consequent conversion of the enzyme to the pyridoxamine form, which is inactive in decarboxylation. On the other hand, addition of pyruvate or other a-keto acids would be expected to decrease the concentration of pyridoxamine enzyme (VI) and thus increase the amount of pyridoxal enzyme (I) available for combination with aspartate. These considerations offer an explanation for the substantial increase in the V,,, value for aspartate decarboxylation that is observed in the presence of added a-keto acid (Fig. 9).

The kinetic studies indicate that a-keto acid does not bind to the aspartate site; thus the apparent K , value for aspartate is unaffected

530 SURESR 9. TATE AND ALTON MEISTER

FNZ ENZ COOH EN? COOH I I I I

H C=N - CH HzC-N=C N II NH2 7H2 NH2 y 2

+ ASPARTATE fJ AOOH 0 COOH I

/

I I1

ENZ ENZ I I

NH2 CH3 NH2 CH3 I I

COOH A

HC=N- CH

N v

ENZ EN2 I I

NH2 y 3 N II y 3 HzC-NH2 O=C HC H2N-CH

I I COOH

Yl I

Fig. 12. Proposed pathways of deaarboxylation and pyruvate formation (5).

by the presence of a-keto acid. In addition, the decarboxylation of aspartate was not inhibited when pyruvate was added even in a concentration 1000 times greater than its K , value. Direct experimental evidence for the existence of a separate binding site on the enzyme for a-keto acid was obtained by ge1 Btration studies. In such experiments almost 1 mole of pyruvate was bound per 57,000 grams of enzyme. The binding of pyruvate was substantially decreased when a-ketoglutarate was also present, indicating that these a-keto acids biad to the same enzyme site. Additional evidence that the enzyme has a separate binding site for a-keto acid comes from experiments in which the aspartate analogue j3-cyano-L-alanine was employed. As stated above (Section III.B), this compound is a competitive inhibitor


toward L-aspartate. When the binding of pyruvate to the fl-cyano- alanine form of the enzyme was determined, a substantial amount of a-keto acid was bound. This indicates that neither the aspartate site nor the vitamin B, cofactor is directly involved in the binding of a-keto acid, and indeed gel filtration studies with the apoenzyme indicate that this form of the enzyme can also bind pyruvate effectively. Evidence that the a-keto acid is not bound to the enzyme by Schiff base linkage was obtained in experiments in which mixtures of enzyme and lac- pyruvate were treated with sodium borohydride ; no radioactivity remained with the enzyme after dialysis.

Studies on the effect of pyruvate concentration on the rate of decarboxylation and transamination indicate that the enzyme exhibits a high affinity for a-keto acid. Thus data on the binding of pyruvate to the holoenzyme in the absence of aspartate lead to an estimated dissociation constant for the enzyme-pyruvate complex of about 8 x M (5). However, other studies indicate that the affinity of the pyridoxal 5'-phosphate form of the enzyme for pyruvate is much less than that for the pyridoxamine 5I-phosphate form of the enzyme. It seems probable that pyruvate can be bound by the pyridoxal form, by the pyridoxamine form, or by any of the intermediate Schiff base forms of the enzyme (Fig. 12) as well as by the apoenzyme.

It thus appears that aspartate @-decarboxylase does not decarboxylate aspartate at a maximal rate in the absence of added a-keto acid. Under these conditions there is a rapid initial formation of pyruvate and an associated formation of the pyridoxamine 5'-phosphate form of the enzyme. If these equilibrium concentrations are maintained, decarboxylation proceeds at B linear rate. Removal of pyruvate shifts the equilibrium toward the pyridoxamine form of the enzyme and thus decreases the rate of decarboxylation ; similarly, addition of keto acid converts more of the pyridoxamine form of the enzyme to the active pyridoxal form and thus increases the rate of decarboxylation. The activity of the enzyme is therefore regulated by an a-keto acid effector that can be produced by the action of the enzyme on its substrate. The a-keto acid effector does not alter the afinity of the enzyme for substrate, but participates in a chemical reaction with enzyme-bound cofactor. The a-keto acid effectors are not close chemical analogues of aspartate and are bound to a separate site on the enzyme. Thus aspartate fl-decarboxylase fulfills both the original definition of an allosteric enzyme (60) and a more recent one (61). However, the


enzyme does differ substantially in its control mechanism from a number of other enzymes that have been designated as allosteric. As discussed below (Section V), aspartate p-decarboxylase contains 12 subunits. Although one cannot exclude the possibility that such phenomena as interaction between subunits and conformational changes function in the control of aspartate /?-decarboxylase, it is not

a-KETO ACID SITE J.

0 coo- 11 / R-C

f- ASPARTATE + SITE

+ PY RI DOX AL-P SITE

Fig. 13. Schematic representation of the active center of aspartate p-decarboxylase (see the text; the double arrows represent types of Schiff base formation: double arrow 1, pyridoxal-enzyme; 2, pyridoxamine-keto acid; and 3, pyridoxal- aspartate. From reference 5.

necessary to invoke such postulates to explain the regulatory effects of a-keto acids.

The findings reviewed above therefore indicate that the active center of L-aspartate p-decarboxylase contains three closely associated binding sites : a site for the attachment of pyridoxal 5’-phosphate, one for attachment of L-aspartate, and also a site for the binding of a-keto acid. These sites must be situated within the active center of the enzyme in such a manner as to facilitate Schiff base formation between pyridoxal 5’-phosphate and the enzyme, a-keto acid, and L-aspartate. In the diagram given in Figure 13 the darker double arrows indicate the three types of Schiff base formation. Plausible points of attachment of the enzyme are indicated by single arrows.

L-ASPARTATE-b-DECARBOXYLASE 533

Since the enzyme can interact with a wide variety of a-keto acids, it seems probable that the a-keto acid site combines only with the carbonyl and carboxyl moieties of the a-keto acids.

V. Subunit Structure of the Enzyme

A variety of experimental approaches indicate that nearly 1 mole each of pyridoxal 5’-phosphate, substrate analogues (p-chloro-L- alanine, aminomalonate), and a-keto acid are bound by 50,000-60,000 g of enzyme. Amino acid analyses of the enzyme indicate that it contains 11 methionine residues and 49 lysine and arginine residues per 50,000 grams; treatment of the enzyme with cyanogen bromide yields 12 peptides; and trypsin digestion gives about 43 peptide fragments (35). These findings suggest that the enzyme possesses a unique amino acid sequence of total molecular weight about 50,000-60,000, identical with that required per coenzyme, a-keto acid, and substrate binding site.

As discussed above, resolution of the isolated holoenzyme of Alculigewes faecalis yields a 6-S component a t pH 8, exhibiting greater mobility on acrylamide gel electrophoresis than the holoenzyme. Bowers et al. (40) have carried out a series of hydrodynamic studies on the 19-S holoenzyme and on the 6-S apoenzyme; these elegant investigations have provided considerable insight into the subunit structure of the enzyme. Bowers et al. (40) also succeeded in dissociating the 6-S apoenzyme by treatment with 5 M guanidine hydrochloride to an apparently homogeneous species exhibiting a sedimentation coeflicient of 1.6 S. By application of concentration difference (AC), meniscus depletion sedimentation equilibrium, and moderate speed equilibrium methods, the data summarized in Table VI were obtained. Bowers et al. (40) used computer simulation procedures to exclude the possibility that their results might have been affected by the presence of con- taminants. They observed that the ratio of molecular weights of the 19-S and 6-5 forms of the enzyqe was 5.92, indicating that the enzyme is composed of six 6-S subunits. They obtained a ratio of 1.95 for the molecular weights of the 6-S and 1.6-5 species. Their attempts to dissociate the protein into a component of molecular weight less than 57,000 were unsuccessful. Thus it may be concluded that L-aspartate p-decarboxylase is composed of 12 identical (or nearly identical) polypeptide chains possessing a molecular weight close to 57,000, and

534 SURESH 5. TATE AND &TON METSTER

TABLE VI Ultracentrifugal Analysis of L-Aspartate-8-Decarboxylase from A. faecalia

Mawd

Enzyme form 820,u (S) AC Meniscus depletion LaBar

A. Holoenzymea 19.00 679,500 652,000 19.01 669,700 672,100 18.73 679,800 673,900

689,600 Mean 678,600 Mean

18.9 f 0.2 675,000 f 11,000

B. Apoenzymeb 6.78 113,200 5.83 118,200 5.76 111,800

Mean 6.79 f 0.04

C. Guanidine hydrochloride 61,040 dissociated apoenzymeC 58,630

59,800 1.63 -& 0.03

114,200 111,500 116,400

Mean 114,000 f 2,600

56,230 56,990 58,200

Mean 58,500 f 1,800

a In 0.05 M sodium acetate, 1 mM Na,EDTA, and 0.1 M NaCl, titrated to pH

In 0.05 M Tris, 1 mM Na2-EDTA, and 0.1 M NaCI, titrated to pH 8.0 with

OIn 6 M guanidine hydrochloride, 0.05 M Tris, and 2 mM mercaptoethanol,

5.5 with acetic acid.

acetic acid.

titrated to p H 8.0 with acetic mid. Average values obtained for each experiment. From (40).

that the 19-S form of the enzyme is assembled by association of 6 dimers of molecular weight 114,000.

An independent confirmation of the results summarized above was obtained in the course of studies in which the enzymes from Alcaligenes faecalis and Pseudomoms dacunhae were compared (59). These enzymes are remarkably similar with respect to amino acid composition, number of free sulfhydryl groups (2 per subunit), and decarboxylase, desulfinase, and transaminase activities. The sedimentation coefficient of the Pseudomoms enzyme was found to be 18.6 f 0.1 as compared to 18.9 -j= 0.2 for the Alcaligenes enzyme. The sedimentation coefficient for the Pseudomonas dimeric apoenzyme was 6.48 f 0.06 as compared

L- ASPARTATE-B-DECARBOXYLASE 535

to 5.79 & 0.04 for the Alcaligenes enzyme. The Pseudomonas enzyme can be resolved to yield a high molecular weight apoenzyme or a low molecular weight dimeric apoenzyme by application of procedures identical to those used in studies on the corresponding forms of the Alcaligenes enzyme (see above ; Section 1II.A). However, the Alcaligenes and Pseudomonas enzymes differ in mobility on acrylamide gel electrophoresis a t pH 8. This difference facilitated the performance of hybridization between the two enzymes. The results of such a study are given in Figure 14. In experiment A, the two 19-S holoenzymes were mixed and subjected to electrophoresis in the same gel; no interaction between the two enzymes was evident in this experiment or in a comparable study in which the two 19-S apoenzymes were mixed and treated with pyridoxalB’-phosphate (to preserve association during electrophoresis a t pH 8). However, when equal amounts of the two 6-8 apoenzymes were mixed and reassociation was induced either by adjustment of pH (reaction pathway 2a + l b ; Fig. 3, p. 511) or by addition of pyridoxal 5‘-phosphate (reaction pathway 3b; Fig. 3), a re- constitution mixture was obtained which migrated on the gels as 7 evenly spaced bands. The mobilities of the fastest- and slowest-moving components were identical to those of the respective parent holoenzymes. The distribution of the various forms was essentially random when association was induced by adjustment of pH to 6 (experiment C ; Fig. 14). On the other hand, when reassociation was induced by addition of pyridoxal 5‘-phosphate a t pH 8 (experiment D; Fig. 14)) certain forms are preferred. In both cases, the results obtained are those to be expected by association of 6 apoenzyme subunits (6 S ) per molecule, as indicated in Figure 15. The finding of 7 species in hybridization indicates that the 2 enzymes are homologous. These findings thus confirm the hydrodynamic studies of Bowers et al. (40), who concluded independently that the Almligenes enzyme contains 12 subunits. The hybridization studies indicate that the Pseudomonas enzyme also contains 12 subunits. It is of further interest that both enzymes exhibit the same appearance on electron microscopic examination (40,59).

Kakimoto et al. (62) have reported that the Pseudomonas enzyme exhibits a sedimentation coefficient of ( s ~ ~ , ~ ) of 20.7 S and a molecular weight of 800,000 as determined by the Archibald procedure, and they concluded that the enzyme has 16 subunits. They reported that the enzyme in 1 M guanidine exhibited a sedimentation coefficient ( s ~ ~ , ~ )


Fig. 14. Polyacrylamide gel electrophoresis of the hybrids produced by mixing the decarboxylases from Abaligenes faecalis and Psemdornonas dacunhae (see the text). (A) A mixture of the two holoenzymes. (B) The two 19-S apoenzyme forms were mixed at p H 6 and 6hen incubated with pyridoxal 5’-phosphate. (C) The two dimeric apoenzymes were mixed and incubated at pH 8 for 10 min; the p H was then adjusted t o 6.0. Pyridoxal 5’-phosphate was then added. (D) The two dimeric apoenzymes were mixed and treated with pyridoxal 5’-phosphate at p H 8. (E) The two dimeric apoenzymes were mixed at pH 8 and the p H was then adjusted to 6.0. Electrophoresis was carried out at p H 8 in Trisacetate buffer (exp. A-D) and at p H 6 in phosphate buffer (E). From reference 59.

of 5.6 S and a molecular weight of 446,000; this unlikely result is even more questionable in view of the finding of Kakimoto et al. (62) of s ~ , , ~ = 6.3 S for a species of molecular weight, 99,500 upon further dissociation in 5 M guanidine. In 5 M guanidine containing 0.1 M 2-mercaptoethanol they reported a value of szo,w = 4.2 S for a species possessing a reported molecular weight of52,600. The data obtained in

Fig. 15. Hybridization of the dimeric apoenzymes from Almligenes faecalis and Pseudomonas dacunhae. A and P represent the monomeric units of the AlcaZigefias and Pseudomom enzymes, respectively; (A)z and (P)z are the corresponding dimeric protomers. From reference 59.

L-ASPARTATE-/~-DECARBOXYLASE 537

the presence and absence of 2-mercaptoethanol in 5 M guanidine are not compatible with a random coil conformation. The unusual relationships between sedimentation coefficients and molecular weights reported by Kakimoto et al. (62) are difficult to explain, and it does not seem justifiable to accept their interpretations of the subunit structure until the primary data have been checked. Additional study might also shed light on the apparent discrepancy between the studies of Kakimoto et al. (62) and those of Tate and Meister (59) regarding the number of half-cystine residues in the enzyme; values of 3 (62) and 2 (59) per subunit have been reported, and Kakimoto et al. (62) have proposed that the monomers are linked together in the dimer by an intersubunit disulfide bond.* Although it is a t this time difficult to explain these discrepancies, the weight of evidence, especially the data on hydridization between the two enzymes, appears to strongly support the view that the two enzymes are homologous and that both the Pseudomonas and Alcaligenes enzymes are each composed of 12 subunits derived from 6 dimeric protomers.

Bowers et al. (40) have attempted to interpret the molecular weight data and electron micrographs of the Alcaligenes enzyme in terms of symmetry models. For a discussion of the subject of electron micros- copy of enzymes the reader is referred to the review by Haschemeyer (63). Bowers et al. (40) were able to exclude the dihedral(D6) hexagonal model suggested by Valentine et al. (64) for the symmetry of E. co2i glutamine synthetase, which also contains 12 subunits. On the other hand, by use of a computer program developed to simulate negative staining of macromolecules under a variety of conditions, they concluded that the most probable quaternary structure of L-aspartate 8-decarboxylase is a closed-shell tetrahedral model similar to that shown in Haschemeyer’s review (see Figs. 4 and 5 of reference 63).

VI. Discussion

The allosteric transamination control mechanism described above (Section IV) does not seem to be necessarily related to subunit interactions or conformational changes in the enzyme. Thus the experimental data indicate that the aspartate-ketimine complex (form 111,

* In very recent studies in the authors’ laboratory both the Alcaligenes and Pmdomonas holoenzymes were dissociated to the respective monomeric species in 0.1% sodium dodecyl sulfate under mild conditions in the absence of 2- mercaptoethanol.


Fig. 12) undergoes decarboxylation to yield the alanine-ketimine complex (form IV, Fig. 12) ; the subsequent transformations of this complex can lead either to the formation of enzyme-pyridoxal 5’- phosphate and alanine on the one hand, or enzyme-pyridoxamine 5’-phosphate and pyruvate on the other. If we assume that a particular conformation of the enzyme promotes the tautomerization and hydrolysis of IV a t a specific relative rate ratio, for example, 2,000, then it may be concluded that the concentrations of pyruvate and other a-keto acids present in the system determine the rate of the major physiologically significant reaction, that is, the /?-decarboxylation of aspartate. The data indicate that the enzyme is exquisitely sensitive to the concentration of a-keto acids and therefore would be expected to be subject to highly sensitive regulation in an intracellular environment containing other enzymes that can increase or decrease the concentrations of pyruvate, a-ketoglutarate, and other u-keto acids.

The enzyme catalyzes transamination reactions involving amino acids other than aspartate, and it is not yet clear as to whether these other amino acids bind to the aspartate site or to the a-keto acid site of the enzyme. It is notable that much higher concentrations of other amino acids are required to inactivate the enzyme by transamination (4) and therefore that the affinity of the enzyme for other amino acids is relatively low as compared to aspartate. Under physiological conditions it is conceivable that the keto acid-transamination mechanism of regulation might be affected by the presence of other amino acids, especially if present in sufficiently high concentration to bind significantly to either the aspartate or a-keto acid binding sites.

One may speculate as to whether, in the course of evolution, the enzyme arose by modification of a transaminase. Thus a transaminase capable of catalyzing tautomerization of the aldimine and ketimine forms (forms 11, 111, Fig. 12) might have been altered by mutation to develop a site for the decarboxylation of 111. However, one may reason in an opposite manner. Thus the transaminase activity may have developed later; since the alanine-ketimine intermediate (IV) may hydrolyze, development of a binding site on the enzyme for a-keto acid may have been favored, since this offers a mechanism for prevent- ing inactivation and a t the same time for controlling the activity of the enzyme.

In the course of studies in the authors’ laboratory it has been found that the activity of Alcaligenes aspartate decarboxylase in the absence

L-ASPARTATE-,~-DECARBOXYLASE 539

of added u-keto acid is inhibited by a variety of compounds including citrate, isocitrate, ATP, GTP, and CTP. These compounds do not inhibit the enzyme in the presence of relatively low concentrations of added pyruvate (Table VII). We have considered the possibility that

TABLE VII Inhibition of Aspartate B-Decarboxylase Activity by Citrate and

Other Compounds

Percentage Activitya

Compound added (1 mM) In presence of

0.4 mM pyruvate

None Citrate threo-DL-Isocitrate Succinate L-Malate A M P ADP ATP ITP GTP CTP Pyrophosphate Phosphate EDTA

[loop 31 61 92 91 99 7 7 41 47 46 62 47 99 98

[ 1 oop 97 97 98 97 -96 94 96 97

100 100

94 97 98

a Decarboxylase activity was measured at 2 6 O in 0.126 M sodium acetate buffer (pH 6.6) containing 1 mM ~-aspartate-4-’~C (Tate and Meister, unpublished).

Arbitrarily set at 100; actual specific activity, 20 and 53 pM, respectively, of CO, per minute per milligram enzyme in the absence and presence of 0.4 mM pyruvate.

these compounds inhibit the enzyme by altering the equilibrium between the alanine-ketimine complex and the pyridoxamine 5’- phosphate form of the enzyme and pyruvate (see Fig. 12), so as to favor the formation of pyridoxamine B’-phosphate. (The possibility that citrate and isocitrate bind to both the aspartate and keto acid sites of the enzyme has been considered, but this requires additional experimental study.) We must also consider the possibility that the activity of the enzyme might be affected by alterations of this equilibrium produced by conformational changes in the enzyme induced by


the binding of other allosteric effectors a t still other sites on the enzyme .

At the present stage of the studies on the chemical and physical properties of aspartate b-decarboxylase, some insights into the structure and function of the enzyme seem to have emerged. For example, the hydrodynamic and electromicroscopic studies of Bowers et al. (40) have led to a model for the quaternary structure of the enzyme. However, the relationship of the spacial arrangement of the 12 subunits of this enzyme to its catalytic activity is not yet clear. As yet we do not know whether an enzyme molecule with less than 12 subunits can be active. It seems evident, however, that pyridoxal 5'-phosphate plays a significant roIe in the structural integrity of the dodecameric enzyme. Thus not only pyridoxal 5'-phosphate, but a variety of analogues of this cofactor serve with virtually equal effective- ness in inducing association of the dimeric apoenzyme to the dodecameric holoenzyme. On the other hand, pyridoxal5'-phosphate does not seem to be needed for the structure of the dimer; the nature of the forces that hold the dimer together is not yet clear.

As in the case of glutamate-aspartate transaminase and other vitamin B, enzymes, there is substantial evidence that pyridoxal 5'-phosphate is bound to aspartate b-decarboxylase by multipoint attachment. The data indicate that attachment of coenzyme to enzyme requires the presence of a 5'-phosphate moiety. On the other hand, a covalent bond between cofactor and enzyme is not needed for a Cotton effect (22), cofactor analogue binding, substrate binding, a-keto acid binding, or induction of association of the dimer to the dodecamer. The formation of a Schiff base between pyridoxal 5'- phosphate and enzyme seems to be related, however, to optimal catalysis of decarboxylation and desulfination and also to the optical specificity ofthe enzyme. It may be noted also that the substi\tution of the 3'-phenolic hydroxyl and of the pyridine nitrogen atom by a methyl group does not prevent binding of cofactor t o enzyme. These considerations suggest that the 5'-phosphate moiety of pyridoxal B'-phosphate plays a major role in coenzyme- enzyme interaction, a t least in terms of binding to the enzyme. The exact positioning of the cofactor on the enzyme may be determined by interactions between the other groups of the cofactor (pyridine nitrogen atom, 3'-phenolic hydroxyl group, C,-methyl group, Schiff base formation) and the enzyme. The observation that many of the cofactor analogues can

I.-ASPARTATE-p-DECARBOXYLASE 541

replace pyridoxal 5'-phosphate effectively in transamination, but not nearly as well in decarboxylation, is in accord with the data indicating that the enzyme has a separate site for the binding of a-keto acid. These considerations emphasize the importance of the steric relationships between the binding sites for a-keto acid, aspartate, and pyridoxal 5'-phosphate a t the active center of the enzyme.

Several approaches to the understanding of the complex structure of the multisite active center of the enzyme are possible. Thus an approach based on computer-aided model building has been initiated (35) along the general lines used in studies on the active center of ovine brain glutamine synthetase (65,66). Considerations arising from these and other studies have suggested the attractive possibility of designing a bifunctional reagent capable of attaching to both a-keto acid and aspartate sites. In addition, further direct chemical information is needed about the nature of the amino acid side chains in the active center. The observation that binding of pyridoxal 5'-phosphate quenches tryptophan fluorescence indicates that there is a change in the environment of the affected tryptophan residues. It is possible that there is a conformational change induced by binding of pyridoxal 5'-phosphate or perhaps the direct involvement of tryptophan residues in the binding of pyridoxal 5'-phosphate, or both. The data on the sulfhydryl groups of the enzyme, derived from studies on both the Alcaligenes enzyme and the Pseudomonas enzyme, indicate that enzyme sulfhydryl groups are not required for the several catalytic activities exhibited by the enzyme or for binding of pyridoxal 5'- phosphate or pyridoxamine 5'-phosphate to the enzyme; however, introduction of the p-mercuribenzoate moiety can affect the conformation of the enzyme in such a manner as to increase or decrease activity. Direct chemical approaches to the amino acid structure a t the active site are clearly required. Studies now in progress on the sequences of the peptides isolated from the active center containing N'-pyrdoxyl lysine and the 3-carbon fragment derived in alkylation of the enzyme by 8-chloroalanine offer promising approaches in this direction.

Acknowledgments

* This research was snpported in part by the National Institutes of Health. the Public Health Service, and the National Science Foundation.

542 SURESH 5. TATE AND &TON MEISTER

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