Life Sciences Vol . 15, pp . 177-190Printed in the U.S.A .

(AminoAcid)AA~

MINIRSVIL~41

GLUTATHIONE ; h1ETABOLISM AND FUNCTION VIA THE r-GLUTAMYL CYCLE

Alton Meister

Department of BiochemistryComell University Medical College

1300 York Avenue, New York, N .Y . 10021

.S~umma~ The intracellular synthesis of glutathione and the broakdown of this ubiquitoustripeptide aro linked by a series of enryme-catalyzed reactions which have been collective-ly termined the y-glutamyl cycle .

Earlier work on the Y-glutamyl cycle has been review-ed (1,2); this miniroview wmmarizss the background of this aroa, the major previous find-ings, recent developments, and evidence that the r-glutamyl cycle functions in aminoacid transport .

Introduction : the r-glutamyl cycle consists of six enzyme-catalyzed reactions (Figure 1) :

glutathionej'-glu-cysH-gly

glycine

~-giy a

1%glu-cysH

AA 5-oxôproline

glutamate4

ATP ADP+P;Fig . 1 : The Y-Glutamyl Cyele : 1 .Y~luiamyl tranapeptidass, 2 . y-glutamyl eyelotrans-

fera:e, 3 . dlpeptidase, 4 . 5-oxoprolinase, 5 . r-glutamyl-cysteins synthetase, 6 . gluta-

thions synthetase .

177

Pergamon Preas

cy:t~~ j~ADP+P;

ATP

178

Glutathione and the y-Glutamyl Cycle

Vol. 15, No . 2

The synthesis of glutathione from glutamate, cysteine, and glycine is catalyzed by the

successive actions of 7-glutamyl-cysteine and glutathione synthetases (for a review, see

(3)) . A quantitatively major pathway of glutathione degradation is catalyzed by y-

glutanyl tronspeptidase, a membrane-bound enzyme that catalyzes the transfer of the y-

glutamyl moiety of glutathione (or of certain other 7-glutamyl compounds) to amino acids

(or to certain peptides) to form 7-glutamyl amino acids and cysteinyl-glycine .

The

latter dipeptide is enrymatically split to cysteine and glycine .

The y-glutamyl amino

acids, which are formed in the amino acid-dependent breakdown of glutathione, are con-

verted to the corresponding free amino acids and 5-oxoproline (pyroglutamate, pyrrolidone

carboxylate) by the action of y-glutamyl cyclotransferose . Conversion of the y-glutamyl

deriwtives of virtually all of the protein amino acids to 5-oxoproline and free amino acids

occurs in the presence of r-glutamyl trornpeptidase and r-glutamyl cyclotransferase (1) .

5-Oxoproline is converted to glutamate in an ATP-dependent reaction catalyud by 5-oxo-

prolinase .

Them is now much evidence for the existence of the 7-glutamyl cycle in a number

of different mammalian cells .

It has been suggested that the 7-glutamyl cycle functions in

the transport of amino acids across cell membranes (1,4) . A scheme has been proposed (1)

according to which (a) the amino acid to be transported binds noncovalently to o cell

membrane site, ro) a group on the membrane-bound y-glutamyl tronspeptidase interacts

with the y-glutamyl moiety of intracellular glutathione (or of other y-glutamyl compounds)

to yield a y-glutamyl enryme, (c) there is attack of the amino acid nitrogen atom on the

y-carbon atom of the y-glutamyl enzyme ~ yield o y-glutamyl amino acid .Formation ofthe

7-9lutamyl amino acid is associated with removal of the amino acid from its membrane

binding site and movement of the amino acid moiety into the cell perhaps through a space

in the membrane; this may be facilitated by a conformation change in the membrane . The

amino acid is roleased from its y-glutamyl carrier within the cell .

Thus, transpeptidase

Vol. 15, No . 2

Glutathlona and the y-Glutamyl Cycle

179

and cyclotronsferase oatalysse the "translocation" and "roleass" steps of transport,

rsspsctively .The ATP-dependent dscyclimtion of 5-oxoprolins and the synthesis of gluta-

thione aro energy-requiring rocovery steps needed for resynthesis of the carier procursor .

Bac

round: The possibility that the action of r-glutamyl transpeptidase might be involved

in amino acid transport was first suggested about 1950-1954 by several investigators (5-9),

and this idea has been mentioned again by others (10-1~ . That r-glutamyl tronspeptidase

is bound ro particles in tissue homogenates (18), and histochsmicol studies (16, 19-23)

which indicate that the enzyme is bound to cell membranes are consistent with this view .

However, this idea has apparently not been mentioned or cornidered in reviews by investiga-

tors interostsd in amino acid transport. It has also been wggested that the tronspeptidass

may function in protein rynthesis (24-2n, collagen formation (1~, and in the transport and

degradation of peptides (1,4) .

The hypothesis of the 7-glutamyl cycle was stimulated by two key observations in our

laboratory : (a) It was found that rot kidney exhibits

very high y-glutamyl-cysteine and

glutathione rynthstass activities . (b) 5-Oxoprolins was found to be rapidly metabolized by

intact animals and tissue slices, and the major initial product formed from 5-oxoproline was

found to be glutamate (28) . the two reactions inwlved in the synthesis of glutathione wero

elucidated by Bloch and collaborators (29) . The studies on glutathione synthesis in our

laboratory led to isolation of highly purified and apparontly homogeneous proparotians of

glutathione rynthetase (30) and r-glutamyl-cysteine rynthetase (31), and to studies which

showed that these roactions involve intermediate formation of acyl phosphatss(30-33) .ihs

finding in kidney of very high activities of these enzymes seemed significant to us since

this tissue, which is also the most active in catalyzing tronspsptidatlon of glutathione,

maintains a glutathione concentration of about 3-5 mM. ihat animals metabolize injected

[14C] 5-oxoproline rapidly was shown in studies on mice in our laboratory (34) and in-

dependently in experiments on rots (35) . Thus, it became evident that a pathway exists for

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Glutathione and the y-Glutamyl Cycle

Vol. 15, No . 2

the utilization of 5-oxoproline formed by the action of 7-glutamyl cyclotransferase .

Earlier work had suggested that 5-oxoproline wuld be metabolized by intact animals; how-

ever, conflicting results were observed in different laboratories . It is notable that Sroxo-

praline was found to serve in place of glutamate in the rynthesis of glutathione by liver

slices (36) . However, it is now clear that 5-ow~proline is not used directly for glutathione

synthesis, nor is it used as wch for protein rynthesis (37); the N-terminal 5roxoproline

residues of certain peptides and proteins are formed by cyclization of N-terminal glutamate

(or of o glutamate derivative) during or after protein rynthesis . Utilization of 5roxoproline

inwlves prior conversion of 5~xoproline to glutamate, a reaction catalyzed by 5-oxopro-

lipase .

the discovery of 5-oxoprolinase (38,39) was of importance because it showed that

there is a link between the enzymes that utilize glutathione and those which catalyze its

rynthesis, thus making it possible to visualize a cycle (Fig . 1) .

Function of the y-Glutamyl Cycle in Various Tiswes : The kidney is very active in amino

acid transport and the presence of high activities of the Y-glutamyl cycle enzymes in this

organ is consistent with the proposed role of the cycle in the renal handling of amino acids .

It is notable that small amounts of a number of Y-glutamyl amino acids have been found in

normal human urine (40) .

Histochemical studies (19-21) and studies on isolated kidney

brush border fractions (41) indicate that the transpeptidase (about 1 .5% of the membrane

protein (41)) is localized in the proximal convoluted tubule, the region which is thought

to be involved in renal amino acid reabsorption .

The enzymes of the y-glutamyl cycle are found also in many other mammalian tissues .

The transpeptidase is localized in the epithelial cells of the small intestinal mucosa; much

less activity is present in stomach and colon epithelium (16) . The finding of high activities

of the y-glutamyl cycle enrymes in the choroid plexus (42) and the ciliary body (43)

support the hypothesis that the r-glutamyl cycle is involved in amino acid transport in

these locations . It is generally believed that the choroid plexus functions in traropor

Vol. 15, No . 2

Glutathione and the yGlutamyl Cycle

181

phenomena at the blood-cerobrospinal fluid barrier, and it is notable that the epithelium

of the choroid plexus is similar to that of the kidney tubule . lhero appears to be an analogy

between the formation of urine by the nephron and the secrotion of cerobrospinal fluid by

the choroid plexus . The activities of the r-glutamyl cycle enrymes are much higher in

the choroid plexus than in other regions of the brain, and histochemical studies indicate

that the y-glutamyl tronspeptidase is localized in the apical portions of the epithelial cells

of the choroid plexus, which aro probably the sites at which transport occurs . It is relevant

to note that the concentrations of amino acids in the cerobrospinal fluid are much lower

than those of the blood plasma. Consistent with the function of the r-glutamyl cycle in

brain is the obserwtion that a number of y-glutamyl amino acids have been found in borin

(44-46) . Other regions of the brain also exhibit the activities of the 7-glutamyl cycle

and it has been proposed that the cycle may play a role in intracellular amino acid trans-

port associated with synaptic transmission (4~ .

The ciliary body of the eye also contains high concentrations of the enzymes of the

r-glutamyl cycle, and all of these, except for 5-oxoprolinase, ara prosent in the lens

(43) . The tronspeptidase is localized in the basal portions of the epithelial cells of the

ciliary body; the histochemical and enzyme studies suggest that the y-glutamyl cycle may

function in the transport of amino acids across the blood-aqueous humor barrier . It is of

interest that the development of cataroch from a variety of causes is associated with a

decreased concentration of glutathione in the lens . While the function of lens glutathione

is not yet understood, it is known that lea: glutathlone is continually synthesized from and

degraded ro its constituent amino acids . A high percentage of patients with congenital

ocular cataracts also exhibit aminoaciduria (e.g ., congenital galactosemia); it has been

suggested fhat decreased amino acid transport across both the ronal tubule and the ciliary

epithelium may bs aaoeiated with a similar enyeme defect inwlving the y-glutamyl

cycle (43) .

182

Glutathione and the y-Glutamyl Cycle

Vol . 15, No . 2

The available data indicate that glutathione is present and is synthesized in virtually

all cells; Y-glutamyl tronspeptidase, r-glutamyl cyclotransferase, and 5-oxoprolinase are

present at least to some extent in most mammalian tissues . these cansiderotions suggest

that the r-glutamyl cycle functions in many cells . It is not proposed that the cycle accounts

for all amino acid transport; there are undoubtedly a number of different amino acid trans-

port systems and the relative function of the Y-glutamyl cycle may vary considerably in

different cells . The mature erythrocyte contains all of the cycle enrymes except for 5-oxo-

prolinase ; these activities are of sufficient magnitude to account for most of the observed

erythrocyte glutathione turnover (48) . Certain tumors (e.g., Morris rot hepatomas and rat

kidney tumors) exhibit substantial activity of the Y-glutamyl cycle enzymes (49); the report-

ed involvement of glutathione in hepatic carcinogenesis (50-52) is interesting and requires

additional study . Studies on the enzymes of the Y-glutamyl cycle in prokaryotes are

currently sparse . However, r-glutamyl trnnspeptidase has been found in several bacteria

(53-55) ; glutathione occurs in bacteria and bacterial 5-oxoprolinase has been purified and

studied (56) .

Evidence for the function of the Y-glutamyl cycle in vivo has come from studies in

which mice were injected with L-2-imidazolidone-4-carboxylate, a competitive inhibitor

of 5-oxoprolinase (57) . When the inhibitor is administered to mice there is markedly de

creased utilization of 5-oxoproline; under these circumstances, 5roxoproline acwmulates

in a number of tissues (brain, kidney, liver, eyel and unusually large amounts of 5roxo-

proline are excreted in the urine .

In studies in which the inhibitor was given together with

one of several L-amino acids, significantly more 5-oxoproline accumulated and vras ex-

creted (58,59) . These findings on the augmentation of 5roxoproline accumulation in the

presence of L-amino acids offer strong evidence for the function of the y-glutamyl cycle

in vivo and also support the conclusion that 5-oxoproline is a quantitatively significant

metabolite of glutathione .

Vol. 15, No . 2

Glutethione end the y-Glytemyl Cycle

183

Inborn Metabolic - Emxs Involvin~the Y-Glutamyl Cycle : Thus far, two types of inborn

errors have been found in man in which them are specific enrymatic lesions of the y-

glutanyl cycle . In one type, the patients (two siblings) exhibit marked deficiency of

erythrocyte r-glutamyl-tysteine synthetase activity and of erythrocyte glutathione in

association with hemolytic anemia (60) . They have central nervous system disease (mental

rotardation, psychosis, spinocerebellar degeneration), and also decreased glutathione

levels in their leukocytes and muscles indicating that they probably have a rather generaliz-

ed deficienty of glutathione . It is of interest in relation to the proposal that the Y-glutamyl

cycle function: in amino acid transport that these patients exhibit generalized aminoaciduria

(1,61) .

The second type of inbom error is associated with 5-oxoprolinuria; three patients have

thus far been observed. the first of these to be reported, a 19 year old mentally rotarded

boy, excroted 25-35 g of 5-oxoproline per day (62) . He was at first thought to have a

defect in the urea cycle (62), but later a block in the y-glutamyl cycle was wggested (63) .

When this patient was given an intravenous infusion of a mixture of amino acids, there

was a large incroase in the urinary excrotion of 5-oxoprolins . When the patient was given

[14C] 5-oxoproline he excroted very little ~C02 in his expirod broatfi (in marked contrast

to a normal control) . While this finding suggests a block of 5-oxoprolin~e, it would be

expected that the administered [~C] 5-oxoproline would be greatly diluted by the high

blood plasma concentration of 5-oxoproline (50 mg per 100 ml or 3.9 mM (64)) ; this value

was initially reported erroneously to be 5 m8 per 100 ml (62,63,65) . Furthermore, the

cultured skin fibroblaats from this patient have considerable 5roxoproltnase activity (66) .

Two additional patients (sisters, now 3 and 0.25 yeah old) with 5-oxoprolinuria wero wb-

sequently discovered ; both have blood plasma 5-oxoproline eoneentratior~ of about 4-5 mM

(67,68) . Studies in which one of these patients was given [14C] 5-oxoprollne led to the

conclusion that about 25% of the amount of 5-oxoproline formed is exeroted, the remainder

184

Glutathione and the Y-Glutamyl Cycle

Vol. 15, No . 2

being metabolized (6~ . Similar conclusions may now be drawn from the studies on the 19

year old patient (64); the peripheral leukocytes of this patient exhibit

5-oxoprolinase

activity . The studies on these patients do not indicate a deficiency of 5-oxoprolinase, but

rather that there is an overproduction of 5-oxoproline associated with a deficiency of

glutathione synthetase as previously suggested (1) . Recent work in our laboroiory on erythro-

cytes, fibroblasts, and placenta from these patients shows that they have a marked deficiency

of glutathione synthetase, and that their 5-oxoprolinuria is secondary to this enryme defect

(69) . A block in glutathione synthesis would lead to 5-oxoprolinuria if under these circum-

stances much more than normal amounts of r-glutamyl-cysteine were formed and converted

to 5-oxoproline by the action of y-glutamyl cyclotransferase . Such overproduction of 5-

oxoproline would exceed the capacity of 5-oxoprolinase to convert it to glutamate . Y-

Glutamyl-cysteine is a good substrate for r-glutamyl transpeptidase and could serve in

place of glutathione in tronspeptidation reactions with amino acids . Thus, a modified Y-

glutamyl cycle (requiring only 4 enzymes) in which 7=glutamyl-cysteine partiçipates in

tronspeptidation may operate in such patients (Fig . 2) . Y-Glutamyl-cysteine may normally

be protected from the action of the cyclotransferose (I), but with decreased glutathione

synthetase, the unprotected dipeptide would serve as substrate for the cyclotronsferose

leading to a futile cycle of 7-glutamyl-cysteine synthesis followed by its wnversion to

5roxoproline and cysteine . Larger amounts of Y-glutamyl-cysteine would need to be

produced for transpeptidation . Normally, glutathione may regulate, diroctlyôr indirectly,

Y-glutamyl-cysteine synthesis; availability of free cysteine may also play a role .

Discussion :

Questions still remain about the role of glutathione and the way in which

its synthesis is regulated .

Earlier work showed that the liver is very active in its

nthesis and degradation; however, the en

matic ca

city of the kidne

to

nthesizee

to now avai a

e suggest t at

a cyc otrons erase

as a ower a finny or Y-glutanyl-cysteine than does glutathione synthetase .

Vol . 15, No . 2

Glutathia~ne and the y-Glutasryrl Cycle

5-oxoproline ~

ÂTP

ADP+P;

Fig . 2: Modified r-Glutamyl Cycle (see the text and Legend for Fig . I) .

185

and degrade glutathione is much higher than that of liver, and recent studies indicate that

the overall turnover time of kidney glutathione is only a fraction of that of liver (70) .

Transport of amino acids via the r-glutamyl cycle would appear to require more

energy than might be thought minimally necessary, but, as discused elsewhero (1), such

an "expensive" transport system seems not unroawnabls . the high energy requiroment may

reflect the need for high efficiency, and it is notable that other biological processes (e.g.,

synthesis of protein, glycogen, urea) also requiro much energy .

Less energy would be

required if Y-glutamyl-cysteine wero normally to function in transpeptidation; other rami-

fications and modificattona of the cycle aro conceivable, including, for example, pathways

involving wccessivs transpeptidation roactions and special transport systems for glycine

and cysteine inwlving glutathione and Y-glutamyl-cystsine synthetases, respectively .

The theory that the r-glutarryl cycle functions in amino acid trornport does not ex-

plain the apparont requiroment of Na+ for transport :inca none of the cycle enzymes seem

186

Glutathione and the

Glut

1

cle

Vol . I5, No . 2Y- ~Y

to specifically require Na+.

However, results on the Na+-dependence of amino acid

transport are inconsistent, and definite conclusions do not yet seem possible (71) . It is

possible that the cycle is involved in tronslocation rather than in recognition (1), and that

Na+ is involved in the latter phenomenon . The cycle does not account in detail for all of

the various specificity phenomena that have been observed in amino acid transport . Al-

though wch amino acids as glutamine and methionine are good substrates of the transpepti-

dose, a-aminoisobutyrote and praline are not, and it would thus appear that the imino

acids and a-substituted amino acids could not be transported by the cycle .

The significance of the finding that glycyl-glycine and certain other peptides are

good acceptor substrates for the tronspeptidase is not yet clear . the peptides may interact

with the enryme by virtue of their similarity to cysteinyl-glycine; however, one must con

eider also the interesting possibility that the transpeptidase functions in the transport,

secretion, or metabolism of certain peptides . It is notable that histochemical studies indi-

tote localization of the tronspeptidase in a variety of epithelial cells, e .g ., renal tubules,

ciliary body, choroid plexus, seminal vesicles, epididymis, pancreatic ducts, bile ducts,

small intestine, bronchi, bronchioles, fallopian tubes; this finding is consistent with a

transport or secretary function . However, histochemical studies also indicate various intra-

cellular (including neuronal) and capillary localizations; the significance of these observa-

tions may become evident after further histochemical and biochemical study .

In conclusion, the Y-glutamyl cycle accounts for the synthesis of glutathione and for

a significant fraction of its utilization . Several lines of evidence indicate that the cycle

functions in the kidney and in other mammalian tiswes . These studies also demonstrate that

free 5roxoproline is a quantitatively significant metabolite of glutathione . the proposed

function of the cycle in amino acid transport is supported by a number of observations and

considerations . the glutathione molecule has two interesting structural features, a wlfhydryl

group and a y-glutamyl linkage; many studies on glutathione have centered on its sulfhydryl

Vol . 15, No . 2 Glutathione and the y-Glutamyl Cycle 187

group.

The findings and ideas reviewed hero may serve to stimulate further interost in the

biological function of the Y-glutomyl moiety of this tripeptide which occurs in substantial

concentrations in virtually all cells .

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Glutathione and the Y-Glutamyl Cycle

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Glutathione and tha y-Glutamyl GYcle

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Glutathione and the y-Glutamyl Cycle

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