THE .JOU~NAL OF BIOLOGICAL CHEMISTRY

Vol. 253, No. 5, Issue of March 10, PP. 1574-1581, 1978

Prrnred m U.S.A.

y-Glutamyl Transpeptidase DETERMINATION OF SPECIFICITY IN THE PRESENCE OF MULTIPLE AMINO ACID ACCEPTORS*

(Received for publication, April 18, 1977, and in revised form, October 24, 1977)

ABRAHAM M. KARKOWSKY AND MARIAN ORLOWSKI

From the Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029

The mechanism of action of y-glutamyl transpeptidase involves the formation of a y-glutamyl-enzyme intermediate which reacts either with an amino acid to form a transpep- tidation product, or with water to yield free glutamate. When two or more amino acid acceptors are incubated with the enzyme and a y-glutamyl donor, the relative ratio of the rates of formation of any two transpeptidation products (u,,,/u,~,) is linearly dependent on the ratio of the concentra- tions of the precursor acceptors (B,,,/B,,,) (i.e. u (1) lu (2) = k,,,lk,,,.B,,,/B,,,). The ratio, k,,,/k,,,, is a constant, and re- flects the relative abilities of the amino acids to interact with the y-glutamyl-enzyme intermediate. This ratio, de- fined as the reactivity ratio, is independent of the concentra- tions of the enzyme, the r,-y-glutamyl donor and the amino acids. Reactivity ratios are a measure of the relative effi- ciencies with which equimolar concentrations of amino acids would serve in the transpeptidation reaction. When amino acids are not present at equimolar concentrations, the relative amount of any two transpeptidation products can be calculated from the reactivity ratios and the relative concentrations of the precursor amino acids.

The reactivity ratios of several amino acids toward y- glutamyl transpeptidase were studied relative to L-alanine in a mixture of plasma amino acids under conditions of pH similar to those existing in uiuo. The affinities, in decreas- ing order, were as follows: glutamine = cystine > methio- nine > alanine > aromatic amino acids > branched chain amino acids = basic amino acids > acidic amino acids. Our results suggest that at physiological concentrations of amino acids found in serum, 30 to 50 times as much glutamine would serve as a substrate in the transpeptidation reaction as any of the branched chain or aromatic amino acids.

Reactivity ratios were found to be unchanged when any of several L-y-glutamyl derivatives were used as the y-glu- tamyl donor. This finding is consistent with the formation of an identical L-y-glutamyl-enzyme intermediate from sev- eral L-y-glutamyl donors. Reactivity ratios are changed when an cu-methyl-y-glutamyl derivative, or r)-y-glutamyl

* This work was supported in part by a grant from the National Institute of Neurological and Communicative Disorders and Stroke (NS 11018). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘hduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

derivatives are used as the y-glutamyl donors, suggesting that the change in structure of the y-glutamyl-enzyme intermediate affects the interaction of the enzyme with amino acids.

y-Glutamyl transpeptidase (1, 2) is the only known enzyme capable of the in uiuo degradation of glutathione. This enzyme catalyzes the transfer of the y-glutamyl group of glutathione to an amino acid or peptide acceptor to yield the t,ranspepti- dation product and cysteinylglycine. The enzyme is also capa- ble of transferring the y-glutamyl group to water, to yield the hydrolysis products glutamate and cysteinylglycine (3). The transpeptidation reaction predominates with increasing con- centrations of amino acids and increasing pH.

There is presently renewed interest in the mechanism of action and physiological function of y-glutamyl transpepti- dase. This interest has been stimulated, in part, by the finding that the activity of this enzyme in serum is one of the most sensitive indicators for liver disorders (4-7). Moreover, interest has also been stimulated by recent findings concern- ing the metabolism of glutathione (8-12) and by speculations concerning the physiological significance of this tripeptide. The localization of y-glutamyl transpeptidase in the brush border of the proximal tubules of the kidney, and other sites with high transport activity (13, 14) suggested the possibility that the transfer reaction might function in the translocation of amino acids across the brush border membrane (15-17). Other functions, such as mercapturic acid formation and even hydrolysis of extracellular glutathione (18-20) and ammoni- agenesis (21-24) have also been proposed for this enzyme.

Initial velocity kinetic data have shown that the mechanism of action of y-glutamyl transpeptidase is consistent with a ping-pong mechanism modified by a hydrolytic shunt. That this is the mechanism of action of this enzyme has been shown by us for sheep kidney and by others for y-glutamyl transpeptidase obtained from various tissues (19, 25-29). On the basis of kinetic studies it has been postulated that an activated y-glutamyl-enzyme intermediate is formed in the reaction and that this intermediate reacts with an amino acid or water to yield the reaction products.

Recent studies in this laboratory have clearly demonstrated

that transfer of the y-glutamyl group to appropriate acceptors in uiuo is a significant function of the enzyme (30,311. Indirect evidence supporting this conclusion has also been obtained by

1574

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Specificity of y-Glutamyl Transpeptidase 1575

Mechanism I

showing that the administration of several amino acids to- gether with an inhibitor of 5oxoprolinase caused an increase in tissue levels of Soxo-L-proline (32). Thus, the determination of the amino acid specificity of the enzyme under conditions similar to those in uiuo is of importance. All previous studies on the interaction of y-glutamyl transpeptidase with amino acids or peptides have been limited to experiments with optimal concentrations of a single amino acid or peptide (3, 25, 29, 33-36). The conditions in these experiments did not reflect the in uiuo situation, where a complex mixture of amino acids would be expected to interact with the enzyme.

We report here experiments on the interaction of y-glutamyl transpeptidase with a y-glutamyl donor and a mixture of amino acids normally occurring in serum. The affinity of the amino acids toward the enzyme has been determined, reflect- ing the likely specificity of the enzyme under conditions similar to those existing in Co. Experiments are also re- ported which support the conclusion derived from kinetic studies that a y-glutamyl-enzyme intermediate is formed as a transitory complex in the reaction catalyzed by the enzyme.

EXPERIMENTAL PROCEDURES

Materials and Methods’

Kinetic Considerations

A scheme representing the mechanism of action of y-glutamyl transpeptidase in the presence of several amino acids is shown as Mechanism I. This scheme is consistent with the ping-pong mecha- nism modified by a hydrolytic shunt postulated for the enzyme. Glutathione (A) binds to y-glutamyl transpeptidase (E), with the subsequent release of cysteinylglycine (P), and the simultaneous formation of a x,-y-glutamyl-enzyme intermediate (F). This inter- mediate’can react with any of the amino acids in the solution (B,,,, B,,, B,,,), to form the corresponding transpeptidation product (Q(,,, Qc2, Q&, or can be degraded by water to form the hydrolytic product (R). Degradation of the intermediate by either of these routes regenerates the native enzyme (E).

The initial velocity equations for Mechanism I which describe the formation of the first product, no,) (Equation 1) and the various transpeptidation products, oqci) (Equation 2) are:

’ “Materials and Methods” are presented as a miniprint supple- ment immediately following this paper. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No. 77M-558, cite author(s), and include a check or money order for $1.00 per set of photocopies.

where the subscript in parentheses denotes the amino acid to which the constant of Mechanism I applies; E,, refers to the concentration of the enzyme and:

v = k&+&

a k, + k, @a)

K = Mk,, + km,) mu(1,

km,. km

K = (km + k,i,Kk, + k,) tar,

kw& + kni,~

(24

(2e)

The relationship between the rates of formation of the individual transpeptidation products (e.g. otil) and u& can conveniently be expressed by Equation 3.

VdJ&,, VP(l)- K bl<l, “u(2) V,M2,%,

K fltw

Equation 3 alternatively can be written as:

%I) = 41, %I = (amino acidd Reactivity ratio

u,,z, k,z, 4n (amino acid,,,)

where

(3)

(4)

(da)

and

The constants V,,,, and Kblclj are the respective measureable

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1576 Specificity of y-Glutamyl Transpeptidase

quantities V,,, and K, for the transfer reaction of the y-glutamyl donor (Substrate A) in the presence of saturating concentrations of the amino acid acceptor (Substrate B,,,). The ratio Vnb(l,/Kh,~,,, therefore, is equal to the slope of the Lineweaver-Burk plot for this reaction in the absence of any contribution from the autotranspepti- dation reaction.

Equations 3 and 4 show that the relative velocities of formation of the transpeptidation products are linearly dependent on the relative concentrations of the precursor amino acids. Upon substi- tuting the definitions of V,,,, (Equation 2b) and K,,,,, (Equation Be) into Equation 3 the following expression is obtained (Equation 51:

(5)

This last equation shows that the constant of proportionality (k,,,/k,,,) of Equation 4 is related to the interactions of the amino acids with the y-glutamyl-enzyme intermediate. The interactions are: (a) the relative ability of the amino acids to bind to the intermediate (k5,,)/k5& and (5) the relative ability of the bound amino acids to successfully consummate the transpeptidation reac- tion and allow release of the transpeptidation products:

k k ?(I).-- ~2) + km k WI) + km km

The proportionality constant k,,,/k,,, is independent of terms related to the binding of the L-y-glutamyl donor. In view of the fact that the constant reflects the relative interactions which occur either during, or after, the binding of the amino acid to the enzyme, we have defined this constant as the reactivity ratio of the amino acids.

Measurement of Reactivity Ratios

Reactivity ratios can be determined when two or more amino acid acceptors are incubated with y-glutamyl transpeptidase and a y-glutamyl donor. Two experimental methods were used to deter- mine the ratios of transpeptidation product formation.

Method 1 -This procedure allows the simultaneous determination of several transpeptidation products using an amino acid analyzer (for details of Method 1 see miniprint).

Method 2 -This procedure allows the determination of the rate of formation of any one of several transpeptidation products which are produced when a y-glutamyl donor is incubated with a mixture of amino acid acceptors. The transpeptidation product is measured by isolating on a Dowex 1 (acetate) column and counting the y-glutamyl “C-amino-acid formed in an incubation medium containing the precursor 14C-amino-acid of known specific activity. A second trans- peptidation product can similarly be measured by utilizing a parallel incubation medium of identical composition as the first, with the exception that a second ‘%-amino-acid acceptor is used while the concentration of the first amino acid, although unlabeled, remains the same. In a similar manner any number of transpeptidation products can be simultaneously measured in an incubation medium by performing an equivalent number of parallel incubations, each containing a different labeled amino acid. The relative rates of formation of the transpeptidation products can be used to calculate the reactivity ratios of the precursor amino acids according to Equation 5. (For details of Method 2 see miniprint.)

Measurement of Amino Acid Reactivity Ratios in Serum

The procedure used to determine the reactivity ratio of serum amino acids was based on the principle outlined under Method 2. The reactivity ratios of all amino acids were measured relative to L- alanine arbitrarily set as 1. (For details of the measurement of amino acid reactivity ratios in serum see miniprint.)

RESULTS

The reactivity ratio is a useful parameter to assess the interactions of amino acids with y-glutamyl transpeptidase. As can be seen from Equation 4, all that is necessary to determine the reactivity ratio of amino acids is a knowledge of the concentration of the amino acids, and the velocity of formation of the corresponding transpeptidation products. The reactivity ratio is independent of most other conditions in the reaction mixture, such as the concentration of the enzyme and both the concentration of amino acid acceptors and the y- glutamyl donor.

That the reactivity ratio is independent of the concentration of enzyme was shown when the concentration of the solubi- lized y-glutamyl transpeptidase was varied over a 30-fold range (3.6 to 108 ng of protein) in the presence of methionine (5 mM), alanine (15 mM) and a y-glutamyl donor (7 mM N’y- L-glutamyl-N”-benzyloxycarbonyl-L-lysine), no change in the methionine/alanine reactivity ratio (2.26 to 2.38) was detected. A crude, particle-bound enzyme preparation gave a methio- nine/alanine reactivity ratio (2.29) identical with that found in experiments in which the purified enzyme was used. The process of solubilization of the particle-bound enzyme (which includes deoxycholate extraction and trypsin treatment) (36) apparently does not affect the amino acid binding site of y- glutamyl transpeptidase.

Although the actual velocity of transpeptidation product formation markedly changes as the concentration of one of the amino acids is varied, the reactivity ratio remains unaf- fected. This can be seen from the data presented in Tables I and II (Experiments 1 to 6). In Table I the methionine concentration was varied while the concentrations of glycyl- glycine, a-aminobutyrate, and glutathione were held con- stant. The effect of the concentration of methionine on the velocity of formation ofy-glutamylmethionine, y-glutamylgly- cylglycine, y-glutamyl-a-aminobutyrate, and y-glutamylglu- tathione is tabulated. As the concentration of methionine is increased, the rate of formation of y-glutamylmethionine is increased. At the same time, the rate of formation of the other y-glutamyl compounds is decreased. The various amino acid reactivity ratios, however, remain unchanged.

Table II (Experiments 1 to 6) describes a similar experi-

TABLE I

Effect of varying concentration of methionine on rate of formation of transpeptidation products and on reactivity ratios

The reaction mixture (final volume, 1 ml) contained 1.8 mM glycylglycine, 18 mM L-o-aminobutyrate (a-AB), 6 mM glutathione, 7.5 rnM dithiothreitol, 10 mM MgCl,, 0 to 24 mM methionine, 117 ng of enzyme in a Tris/HCl buffer (100 mM, pH 8.6). Incubation time was 12 min. The formation of the transpeptidation products was measured on an amino acid analyzer (see Method 1 under “Materials and Methods”). The data represent the mean of three experiments.

concentra- tion of methi-

Rate of formation of y-glutamyl derivatives of Amino acid reactivity ratios

onine GlyCly Met a-AB GSH GlyGlylMet GlyGljla-AB GlyGly/GHS Met/a-AB MetlGSH GSH/a-AB

lTlM nmollll7 ng protein/l:! mill

0 250 0 307 94 0 8.86 8.83 0 0 1.01

1.8 233 118 275 90 1.96 8.95 8.65 4.42 4.29 1.03 3.6 209 238 261 91 1.81 8.45 7.68 4.68 4.24 1.11

6.0 204 353 244 83 1.98 8.78 8.07 4.43 4.08 1.09 12.0 172 552 191 67 2.13 9.39 8.29 4.42 4.13 1.13 24.0 127 796 140 44 2.18 9.35 9.12 4.30 4.19 1.03

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Specificity of y-Glutamyl Transpeptidase 1577

TABLE II

Effect of amino acid concentrations on methioninelalanine reactivity ratios for sheep kidney y-glutamyl transpeptidase

The incubation mixtures (final volume, 0.2 ml) contained 7 rn~ N*-y-L-glutamyl-NG-benzyloxycarbonyl-L-lysine, 35 ng of enzyme, and the amino acids listed in the table in a Tris/HCl buffer (pH 8.7, 140 mM). Tracer quantities of either L-[“Qmethionine or L-[‘Vlalanine were added to the incubation mixtures in order to determine the rate of formation of the y-glutamyl amino acids (for details see “Materials and Methods”).

Experiment No. Amino acids

1 Met (1 mM) + Ala (15 mM) 2 Met (2 mM) + Ala (15 mM) 3 Met (3.5 mM) + Ala (15 mM) 4 Met (5.0 mM) + Ala (15 mM) 5 Met (7.0 mM) + Ala (15 mM) 6 Met (10 mM) + Ala (15 rnM) 7 Met (5 mM) + Ala (15 rnM) + GlyGly (0 mM) 8 Met (5 mM) + Ala (15 mM) + GlyGly (1 mM) 9 Met (5 mM) + Ala (15 mM) + GlyGly (2 mM)

10 Met (5 mM) + Ala (15 mM) + GlyGly (4 mM) 11 Met (5 mM) + Ala (15 mM) + GlyGly (8 mM)

ment. The concentration of methionine was varied in the presence of constant concentrations of alanine and N+L- glutamyl-N6-benzyloxycarbonyl-L-lysine, as the y-glutamyl donor. The experiments show that a lo-fold increase in the concentration of methionine results in a 590% increase in the rate of formation of y-glutamylmethionine. Concurrently, the rate of formation of y-glutamylalanine drops 43%. The methi- onine/alanine reactivity ratios, however, remain unchanged. It is thus apparent that the amino acid reactivity ratios are independent of concentration of amino acid acceptors.

Several experiments have confirmed the independence of reactivity ratios from the number and concentration of the acceptors in the incubation mixture. For example, Table II (Experiments 7 to 11) shows the effect of various concentra- tions of glycylglycine on the rate of formation of y-glutamyl- methionine and y-glutamylalanine. In the presence of glycyl-

glycine, there was a decrease in velocity of formation of these y-glutamyl derivatives. This velocity decrease is to be ex- pected since glycylglycine competes with alanine and methio- nine for reaction with the y-glutamyl-enzyme intermediate. The methionine/alanine reactivity ratios, however, remain constant and are independent of the presence and concentra- tion of glycylglycine. Furthermore, when in addition to glycyl-

glycine (2 mM), other amino acids such as tryptophan, phen- ylalanine, and a-aminobutyrate (each at 5 mM) (data not shown) were added to the incubation mixture, a 25% decrease was observed in the rate of formation of the transpeptidation products. There was still, however, no change in the methio- nine/alanine reactivity ratio. Amino acid reactivity ratios are thus independent of the presence and concentration of other amino acids in the solution.

That the reactivity ratio is independent of the concentration of the L-y-glutamyl donor was confirmed in an experiment summarized in Fig. 1. When glycylglycine, cY-aminobutyrate, and methionine were incubated with increasing concentra- tions of glutathione and the initial velocity for the formation of the three y-glutamyl products was measured, an increase in the formation of the y-glutamyl derivatives with the in- crease in the concentration of glutathione was observed. At the highest concentration of glutathione used in these experi- ments, however, some decrease in the formation of transpep- tidation products may have occurred. This decrease was prob- ably due to interference caused by the autotranspeptidation

y-Glutamyl- y-Glutamylme- M,ethionine/al-

alanine formed thionine formed an’ner~t~~tlvlty

n??Z0llliZin

10.2 1.48 2.21 9.11 2.64 2.22 8.70 4.37 2.19 7.65 5.85 2.33 7.06 7.37 2.27 5.86 8.68 2.25 7.64 5.85 2.33 7.39 5.38 2.22 7.12 5.14 2.20 6.18 4.80 2.37 5.24 3.86 2.23

kdk 3 , 1 2 GSli(mY), a a

2 4 6 GSH (mM)

FIG. 1. Effect of varying the concentration of GSH on the trans- peptidation product formation and on the reactivity ratios of the amino acids. The reaction mixtures (final volume, 1.0 ml) contained 1.5 mM glycylglycine, 5 mM methionine, 15 mM a-aminobutyrate, 10 mM MgCl,, 11.2 mM dithiothreitol, 32 ng of enzyme, 100 mM Tris/ HCl (pH 8.7), and 1.2 to 7.8 mM glutathione. The incubation time was 10 min. The velocities for the formation of the transpeptidation product were measured on an amino acid analyzer (see Method 1 under “Materials and Methods”). Five experiments were performed and the values shown represent the mean k SE. The initial velocities for the formation of y-glutamylmethionine (m), y-gluta- myl-a-aminobutyrate (A), and y-glutamylglycylglycine CO), as the concentration of glutathione was varied, are shown. Velocity is expressed as nanomoles of product formed per min per 32 ng of enzyme. The inset shows reactivity ratios of glycylglycinelmethio- nine (k,lk,), glycylglycinelwaminobutyrate (k,lk,), and methioninel waminobutyrate (k,lk,) as the concentration of glutathione was varied.

reaction of glutathione and the formation of y-glutamylgluta- thione. The various amino acid reactivity ratios (glycylgly- cinelmethionine, glycylglycinela-aminobutyrate, and methio- nine/a-aminobutyrate) are, however, unaffected by these glu-

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1578 Specificity of y-Glutamyl Transpeptidase

tathione concentration changes (see inset, Fig. 1). In a similar experiment when various concentrations of glutathione (2.8 to 21 mM) were incubated with methionine (5 mM) and alanine (15 mM) the resultant methionine/alanine reactivity ratios were also found to be independent of the concentration of the y-glutamyl donor used.

According to the mechanism of action of y-glutamyl trans- peptidase (Mechanism I) the same y-glutamyl-enzyme inter- mediate would be expected to be formed from any L-y-glutamyl donor. The structure of such an intermediate should be inde- pendent of the other part of the peptide (i.e. the leaving group). This intermediate would, furthermore, be predicted to display the same pattern of reaction with the amino acid acceptors of the incubation medium, independent of the struc- ture of the parent donor. To test this hypothesis we incubated y-glutamyl transpeptidase with L-methionine and L-alanine and a number of L-y-glutamyl donors (Table III). Following incubation the reactivity ratio of the amino acids was deter- mined as described under “Materials and Methods.” As pre- dicted the reactivity ratios obtained with all the L-y-glutamyl

compounds were almost identical (2.21 to 2.45). This finding is consistent with, and supports, the view that all L-y-gluta- my1 donors yield the same L-y-glutamyl-enzyme intermediate.

Previous studies have shown that the specificity of y-gluta- my1 transpeptidase is not limited to L-y-glutamyl compounds as donors of the y-glutamyl group. The enzyme can interact with n-y-glutamyl compounds (29, 37). The enzyme can also interact with L-cw-methyl-y-glutamyl derivatives although at a significantly reduced rate (26, 37). The mechanism of reac- tion with these substrates is identical with that with L-Y-

glutamyl derivatives, an activated y-glutamyl-enzyme being postulated as an intermediate in each of the reactions. The structure of the n-y-glutamyl-, and L-cu-methyl-y-glutamyl-, enzyme intermediates, however, would be expected to be somewhat different from that formed with L-y-glutamyl deriv-

atives. It was therefore of interest to determine whether a change in the structure of the y-glutamyl-enzyme intermedi- ate would affect the interaction of this complex with amino acids. Table III shows that when n-y-glutamyl compounds were used as the y-glutamyl donors (i.e. n-y-glutamyl-p-ni-

TABLE III

Effect of y-glutamyl donor structure on methioninelalanine reactivity ratios

The reaction mixtures (final volume, 0.2 ml) contained 5 rn~ I,- methionine, 15 rn~ L-alanine, 32 to 9’7 ng of sheep kidney y-glutamyl transpeptidase, 140 mM Tris/HCl buffer (pH 8.71, and a tracer amount of either c-l’4C1methionine or L-l’4Clalanine and one of the y-glutamyl donors (7 mM) listed in the table. When glutathione was the y-glutamyl donor 7.5 rnM dithiothreitol was included in the incubation mixtures. Data are mean values * S.E. The number of experiments is given in parentheses. For details of the determina- tion of the labeled y-glutamyl product see under “Materials and Methods.”

y-Glutamyl donor Methionine/alanine reactivitv ratio

L-y-Glutamyl-O-benzyl ester L-y-Glutamyl-L-a-aminobutyrate A”-y-L-Glutamyl-NG-benzyloxycarbonyl-L-ly-

sine GSH L-y-Glutamyl-p-nitroanilide n-y-Glutamyl-p-nitroanilide n-y-Glutamyl-L-phenylalanine L-a-Methyl-y-glutamyl-L-a-aminobutyrate

2.36 (3) 2.45 (3) 2.33 t 0.02 (7)

2.31 f 0.05 (IO) 2.21 (4) 1.70 t 0.02 (8) 1.76 (2) 1.89 * 0.03 (7)

troanilide or n-y-glutamyl+phenylalanine) the resulting me- thionine/alanine reactivity ratios were approximately the same for the two n-y-glutamyl donors. These ratios were, however, different from those observed with the L-y-glutamyl donors (Table III). The similarity in reactivity ratios for the two n-compounds apparently results from the identical inter- mediates generated by both D donors. This n-y-glutamyl- enzyme intermediate is, as with the L-intermediate divided at a consistent rate ratio, between methionine and alanine to form the corresponding transpeptidation products.

In a similar experiment, when L-a-methyl-y-glutamyl-n-a- aminobutyrate was used as the y-glutamyl donor, the methio- nine/alanine reactivity ratio differed from those obtained with the L-y-glutamyl-, and n-y-glutamyl-, donors. These results suggest that modifications of the structure of the y-glutamyl donor influence the interaction, and thus the specificity of the enzyme toward amino acid acceptors.

Reactivity Ratios of Serum Amino Acids-Although the amino acid reactivity ratios are independent of the concentra- tion of the enzyme, the concentration of amino acids and the concentration and structure of the L-y-glutamyl donor, they may still be dependent on other factors such as the concentra- tion of cations and the pH of the medium. In order to obtain a better understanding of the amino acids which are primarily involved in the degradation of glutathione under physiological conditions, the reactivity ratios of several serum amino acids were measured.

When N”-y-L-glutamyl-NG-benzyloxycarbonyl-L-lysine and sheep kidney y-glutamyl transpeptidase were incubated with pooled normal serum (pH 7.3), the reactivity ratios relative to alanine listed in Table IV were obtained. The methionine/

alanine reactivity ratios under these conditions (2.76) are somewhat different than those observed at pH 8.7 in Tris/HCl buffer (2.31).

TABLE IV

Reactivity ratios of serum amino acids toward y-glutamyl transpeptidase

The incubations were carried out as described under “Materials and Methods,” with 57 to 70 ng of sheep kidney y-glutamyl transpep- tidase. The serum was preincubated 1 h with a mixture of 95% O,, 5% CO,, and the pH adjusted to 7.3. The incubation time was 10 to 25 min. Data are mean values f S.E.

Amino acid SeNmconcen- Relative utiliza- tration Reactivity ratio tion ;i,\mino

Cystine Glutamine Methionine Alanine Serine Histidine Glycine Leucine Tyrosine Arginine Phenylalani Glutamate Valine

ne

FTlM

0.041 0.405 0.358” 0.470 0.185 0.078 0.386 0.149 0.075 0.121 0.129 0.276 0.221

3.66 f 0.54 0.32 3.66 f 0.76 3.15 2.76 2 0.30 0.06b

1 1 0.66 2 0.07 0.26 0.53 k 0.06 0.09 0.42 k 0.03 0.34 0.32 -t 0.02 0.10 0.32 T 0.02 0.05 0.31 f 0.07 0.08 0.30 2 0.03 0.08 0.14 T 0.02 0.08 0.04 f 0.02 0.02

u Methionine was added to the incubation mixtures measuring the reactivity ratio of this amino acid. The final concentration of this amino acid is given.

h Normal serum methionine concentrations were approximately 0.010 mM. This value was used to calculate the relative utilization of this amino acid.

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Specificity of y-Glutamyl Transpeptidase 1579

Of the amino acids in serum which were tested, the reactiv- ity ratios of glutamine, cystine, and methionine were the highest. The small, neutral amino acids: alanine, serine, and glycine were good substrates, but had reactivity ratios of approximately 20% of the previously mentioned amino acids. The aromatic, branched chain, acidic (glutamate) and basic (arginine) amino acids which were tested had low reactivity ratios when compared with cystine or glutamine.

In order to determine the most probable substrate for y- glutamyl transpeptidase, the concentration of the amino acid, in addition to the reactivity ratio, must also be considered. The product of the reactivity ratio and the amino acid concen- tration is the relative (in this experiment relative to alanine)

amount of that particular amino acid which serves as the substrate in the transpeptidation reaction. The data in Table IV clearly suggest that under conditions that prevail in serum, glutamine is the amino acid most likely to react with y-glutamyl transpeptidase. Alanine would also seem likely to account for a significant portion of glutathione degradation via y-glutamyl transpeptidase.

DISCUSSION

Understanding of the physiological role of y-glutamyl trans- peptidase requires knowledge of its specificity and mechanism of action. At pH 8.0 to 9.0 the main reaction catalyzed by the enzyme is transfer of the y-glutamyl group of glutathione to appropriate acceptors. Lowering the pH toward more physio- logical values, however, decreases the rate of this reaction and increases the relative significance of the hydrolytic reac- tion. This observation has led some investigators to postulate that the main function of the enzyme is the hydrolysis of extracellular glutathione (19, 20). Recent studies, however, in which the in uivo metabolism of y-glutamyl derivatives was studied have clearly indicated that the transfer reaction catalyzed by y-glutamyl transpeptidase is a significant part of the metabolism of these compounds (30, 31). Since the transfer reaction of -,+glutamyl transpeptidase can be demonstrated in uiuo it seems relevant to obtain a clearer understanding of the amino acid specificity of this reaction.

The amino acid specificity of this enzyme has been usually studied under conditions in which an optimal concentration of a single amino acid acceptor (usually 10 to 30 mM) (3, 33) was incubated with the enzyme and a y-glutamyl donor such as glutathione (36) or a model substrate such as y-glutamyl- p-nitroanilide (37) at a pH optimal for the transfer reaction (usually 8.2 to 9.0). The specificity of the enzyme has been evaluated from the ability of an amino acid to accelerate the release of the first product (i.e. cysteinylglycine from gluta- thione, or p-nitroaniline from y-glutamyl-p-nitroanilide). These studies have led to a general consensus that the enzyme has a broad amino acid specificity and that it can transfer the y-glutamyl group to almost any amino acid, although methio- nine, glutamine, and cystine activated the reaction to a greater extent than other amino acids (25, 35-37).

There are several drawbacks in such an approach. The results are dependent on the concentration of the amino acid acceptor and the concentration of the y-glutamyl donor and

do not provide any quantitative information concerning the ability of an amino acid to compete with other amino acid acceptors for the y-glutamyl-enzyme intermediate. Further- more, such experiments do not reflect the conditions in uiuo where some 20 or more amino acids with widely differing concentrations are expected to compete in the reaction for the

y-glutamyl-enzyme intermediate, in an ionic environment greatly different from a simple buffer solution.

The method used by us in studying the specificity of the enzyme is based on kinetic considerations. It determines reactivity ratios which express the ability of any amino acid relative to a reference amino acid (in our case alanine) to react with the y-glutamyl-enzyme intermediate. By using this ratio the efficiency of any amino acid to function as an acceptor for the y-glutamyl group can be compared with any other amino acid. Furthermore, the reactivity ratio is inde- pendent of the concentration of the enzyme and both the concentration of the amino acid acceptor and the y-glutamyl donor. It provides therefore a true measure of the ability of a particular amino acid to bind to the enzyme, react with the y- glutamyl-enzyme intermediate and form the y-glutamyl prod- uct.

This approach provides new quantitative insight into the specificity of y-glutamyl transpeptidase. Thus although the enzyme can interact with almost any amino acid, its specificity is by no means as broad as was previously suggested (3, 25, 34-37). Our determination of the reactivity ratios under con- ditions found in normal human serum show (Table IV) that although at equimolar concentrations of amino acids the enzyme would be expected to react approximately equally well with glutamine, cystine, and methionine, the reaction with phenylalanine, tyrosine, leucine, and arginine would be ten times less favored, and the reaction with glutamate and valine would be even less significant. In addition, knowledge of the reactivity ratios and the concentration of amino acids in serums can be used to calculate the likelihood of a reaction between glutathione and any of the serum amino acids as catalyzed by y-glutamyl transpeptidase. These calculations show that since glutamine is the most predominant serum amino acid, this amino acid would be 30 to 50 times more favored as substrate in the transpeptidation reaction than the aromatic, branched chain, acidic and basic amino acids. Of the other amino acids, only alanine, and to a lesser extent glycine, cystine, and serine would be expected to significantly interact with the enzyme. Although these reactivity ratios were obtained with an enzyme isolated from sheep kidney, the amino acid acceptor specificities as determined by activa- tion of first product release, seem to be similar for enzymes isolated from various sources. It would, therefore, seem that the observations made here should also be valid for other y- glutamyl transpeptidase preparations.

The specificity of y-glutamyl transpeptidase toward gluta- mine is consistent with the finding of significant y-glutamyl- glutamine concentrations in mammalian tissues including brain, intestine, and kidney (38). The high y-glutamylgluta- mine concentrations found are of interest when it is noted that y-glutamylglutamine is an excellent substrate (39) for y- glutamyl cyclotransferase (40). This enzyme catalyzes conver- sion of y-glutamyl amino acids to pyrrolidine carboxylate and free amino acids. The rate of formation of y-glutamylgluta- mine would, therefore, have to be significant in order to maintain such concentrations of this compound in the pres- ence of an enzyme which readily causes its degradation. These findings suggest that glutamine is the preferred accep- tor in uiuo in the reaction between glutathione and amino acids.

It is of interest that the reactivity ratio of amino acids remained unchanged when several different L-y-glutamyl do- nors were used as the substrate. This is consistent with the formation of a y-glutamyl-enzyme intermediate in the reac-

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1580 Specificity of y-Glutamyl Transpeptidase

tion catalyzed by the enzme. An identical L-y-glutamyl-en- zyme intermediate would be expected to be formed from all L-

y-glutamyl derivatives and its structure would be independent of that part of the peptide which is attached to the y-glutamyl residue. The structure, however, of the y-glutamyl-enzyme intermediate apparently influences the specificity of the en- zyme toward amino acids. This conclusion is derived from the observation that the reactivity ratios obtained with n-y-gluta- my1 and L-cy-methyl-y-glutamyl derivatives markedly differed from those obtained with L-y-glutamyl derivatives. The ori- entation of the y-glutamyl group in the active site of the enzyme would be expected to be different for each of these derivatives. These differences apparently are sufficient to alter either the binding or the reaction of the amino acid acceptor with the y-glutamyl-enzyme intermediate. It is of interest that a change in the structure of the acyl-enzyme intermediate formed in the reaction catalyzed by guinea pig liver transglutaminase, was also found to markedly influence the relative abilities of two second substrates to bind and to react with this intermediate (41).

There are many enzymatic reactions in which an acyl-

enzyme intermediate or some other activated enzyme inter- mediate is formed. When this intermediate is capable of reacting with two or more second substrates to yield the final product or products, the measurement of reactivity ratios may be applied for the quantitative evaluation of the specific- ity toward these substrates.

1.

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A M Karkowsky and M Orlowskimultiple amino acid acceptors.

gamma-Glutamyl transpeptidase. Determination of specificity in the presence of

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