©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chemical Modification of Active Site Residues in -Glutamyl Transpeptidase
ASPARTATE 422 AND CYSTEINE 453 (*)

Terry K. Smith , Alton Meister (§)

From the (1) Department of Biochemistry, Cornell University Medical College, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

-Glutamyl transpeptidase, an enzyme of central significance in glutathione metabolism, is inactivated by iodoacetamide, which esterifies an active site carboxyl group identified here as that of Asp-422. Treatment of the inactivated enzyme with hydroxylamine leads to de-esterification and to restoration of enzymatic activity. N-Acetylimidazole, which also inactivates the enzyme, acetylates several amino acid residues. Acetylation exposes Cys-453, which is buried in the native enzyme, to reaction with iodoacetamide. Incubation of the acetylated enzyme with glutamine produces a stabilized -glutamyl-enzyme form which is (a) located exclusively on the light subunit, (b) more labile to base than to acid, (c) destabilized by denaturation of the enzyme with guanidinium ions, and (d) reactive with hydroxylamine to form -glutamylhydroxamate. Stabilization of the -glutamyl-enzyme appears to be associated with acetylation of lysine residues (including Lys-99). These and other findings suggest that the -amino group of the -glutamyl substrate is linked electrostatically to Asp-422 so as to facilitate reaction of the -carbonyl of the substrate with an enzyme hydroxyl group to form a -glutamyl-enzyme.


INTRODUCTION

-Glutamyl transpeptidase is a glycoprotein consisting of two nonidentical subunits (1, 2, 3, 4, 5) . The light subunit is bound to the heavy subunit by noncovalent interactions; the heavy subunit has a hydrophobic N terminus that anchors the enzyme to the extracellular membrane. The enzyme catalyzes cleavage of the -glutamyl bond of glutathione and related compounds, to form a postulated -glutamyl-enzyme intermediate, which may be cleaved by water to form glutamate (hydrolysis), or the -glutamyl moiety may be transferred to acceptors such as amino acids (transpeptidation). Definitive evidence for formation of a -glutamyl-enzyme intermediate is still needed. It has been suggested that the -glutamyl moiety attaches to an enzyme hydroxyl group (1) , but other possibilities exist; studies on the nature of this bond led to the conclusion that the -glutamyl group is attached by amide linkage to an enzyme amino group (6) . Szewczuk and Connell (7) found that the enzyme was irreversibly inhibited by treatment with iodoacetamide, that this inhibition was prevented by a mixture of serine and borate, and that acid hydrolysis of the inhibited enzyme led to formation of glycollic acid, which was postulated to be derived from an alkylated group at the active center of the enzyme. Elce (6) found that treatment of the enzyme with N-acetylimidazole, followed by treatment with iodo[C]acetamide led to labeling of two amino acid residues at the active site; one has a carboxyl group and the other is a cysteine.

Studies carried out after the primary structure of the enzyme was determined (8, 9, 10, 11, 12) led to identification of Thr-523 of the rat enzyme as the site of attachment of the inhibitor acivicin (13).() Although previous studies suggested (1, 14, 15) that the active site is situated on the light subunit, selective labeling experiments on the rat enzyme with [C]phenylglyoxal showed labeling of Lys-99 and Arg-111 (on the heavy subunit) (16) , and site-specific mutagenesis studies of the heavy subunit of the human enzyme suggested that Arg-107 plays a significant role in binding of -glutamyl substrate and that Glu-108 participates in acceptor substrate binding and catalysis (17) .

To ultimately understand the structure and mechanism of action of the enzyme, more information is needed about the nature of the active site residues and their locations. In the present work, in which the interaction of the enzyme with iodoacetamide and N-acetylimidazole was probed further, we have obtained evidence for the specific amino acid residues of the enzyme that react with these compounds. We also found that the inactivation of the enzyme that occurs when it is incubated with iodoacetamide can be reversed by treatment with hydroxylamine. We have observed a stabilized -glutamyl enzyme form that exhibits properties that are consistent with this postulated intermediate in the reactions catalyzed by the enzyme.


EXPERIMENTAL PROCEDURES

Materials

-Glutamyl transpeptidase was isolated (specific activity, 1100 units/mg) from frozen rat kidneys (Pel-Freez Biologicals) and solubilized by papain digestion (1) . Endoproteinase Glu-C (EC 3.4.21.19) and endoproteinase Lys-C (EC 3.4.21.50) were purchased from Boehringer Mannheim. Other chemicals were purchased from Sigma except as noted. L-(S,5S)--Amino-3-chloro-4,5-dihydro-5-[3-C]isoxazole acetic acid (acivicin, AT-125) with specific radioactivity of 31 mCi/mmol and unlabeled acivicin were kindly supplied by Dr. R. S. Hsi of the Upjohn Company. Iodo[2-C]acetic acid and iodo[2-C]acetamide (specific radioactivity, 55 mCi/mmol) and L-[U-C]glutamine (specific radioactivity, 270 mCi/mmol) were obtained from Amersham Corp. HPLC() solvents were obtained from VWR Scientific Corp. Imidazole and methylamine was obtained from Eastman Kodak Co., and acetic anhydride was purchased from Aldrich.

N-Acetylimidazole was prepared according to the method of Reddy et al.(18) ; N-L[-C]acetylimidazole was prepared by slight modification, as follows. [1-C]Acetic acid (100 µCi; specific radioactivity, 12.4 mCi/mmol) was added to acetic anhydride (200 µl; 3.2 mmol) and stirred for 5 min; with further stirring, imidazole (108.8 mg, 1.5 mmol) (recrystallized thrice from ethyl acetate) was added, and the mixture was placed for 1 h at 20 °C. Excess L-[C]acetic anhydride was removed by vacuum distillation, and the remaining white solid was crystallized from ethyl acetate. The resulting N-[1-C]acetylimidazole (101 mg, 1.5 mmol, 31 µCi/mmol) was stored in a desiccator at -20 °C.

Fisher was the source of boric acid and dialysis tubing (molecular weight cut off 6-8 kDa) and Mallinckrodt for hydroxylamine and guanidinium hydrochloride from Schwarz-Mann. Pierce Chemical Co. supplied the constant boiling 6 M HCl, amino acid standards, and phenylisothiocyanate that were used for Pico-Tag amino acid analysis. National Diagnostics was the source of Monofluor scintillation liquid, and S-carboxymethyl-L-cysteine was obtained from Chemical Dynamics Corp. Low molecular weight standards for gel-electrophoresis and protein assay dye were purchased from Bio-Rad.

Methods

Gel Electrophoresis

The gels were 10% SDS-polyacrylamide, prepared according to Weber and Osborn (19) and run in duplicate on a Mighty Small II (SE 250) gel-electrophoresis apparatus (Hoefer Scientific) at a voltage of 100 V for 1 to 1.5 h. Estimates of the molecular weights of the subunits were made using low molecular weight standards; staining was done with Coomassie Blue R-250. The lanes of identical gels were cut out and each was cut into five equal parts, which were shaken with 0.5 ml of Tris-HCl (0.1 M, pH 8) containing 0.1% SDS for 24 h. Liquid scintillation fluid (4 ml) was added, and the radioactivity was determined with a LKB 1218 RackBeta liquid scintillation counter.

Enzyme Activity Assay

Standard -glutamyl transpeptidase activity assays were performed with L--glutamyl-p-nitroanilide (1 mM) and glycylglycine (20 mM) in Tris-HCl (0.1 M, pH 8) at 37 °C (20) . The rate of release of p-nitroaniline was monitored at 410 nm ( = 8800 M cm) (Varian Cary 219 spectrometer). Portions (5-10 µl) taken at appropriate times after chemical modification and reactivation were assayed (1 unit is equivalent to the release of 1 µmol of p-nitroaniline/min.).

Inactivation Studies

The enzyme (125 units) was incubated at 37 °C in a solution (100 µl) of Tris-HCl (0.1 M, pH 8), containing the various inhibitors and other compounds. At intervals, portions (5 µl) were assayed for activity.

Chemical Modification Experiments

The chemical modifications were carried out at 37 °C in Tris-HCl (0.1 M, pH 8) with between 550 and 600 units of enzyme per experiment.

Dialysis using tubing having a molecular weight cutoff of 6-8 kDa was carried out to remove excess modifying agent when necessary, against three changes of 4 liters each of Tris-HCl (5 mM, pH 8) buffer, for 36-48 h at 4 °C. The presence of both L-serine and borate (S/B), each at a final concentration of 50 mM, which acts as a competitive inhibitor (21, 22) , was used to protect the active site of the enzyme. Solid iodoacetamide and iodoacetate (in the presence of maleate (50 mM)) were added as indicated to a final concentration of 100 mM; the pH values of the solutions were readjusted to 8 with NaOH or HCl (0.1 M). After 6 h at 37 °C, excess 2-mercaptoethanol was added for 30 min before dialysis. N-Acetylimidazole was added as a solid to a final concentration of 100 mM and incubated for at least 12 h; the pH values of these solutions were not readjusted; the final values were usually 7.8.

In the experiments described in , the final volumes were brought to 4 ml by speed vacuum lyophilization. Each experiment was then split into four equal parts (1 ml each), and one of the following was added to each; (a) iodo[2-C]acetamide (2.75 mCi/mmol; final concentration, 100 mM); (b) iodo[2-C]acetic acid (2.75 mCi/mmol; final concentration, 100 mM), in the presence of maleic acid (50 mM); (c) [3-C]acivicin (3.1 mCi/mmol; final concentration, 1 mM); and (d) L-[U-C]glutamine (5.4 mCi/mmol; final concentration, 1 mM). These solutions were then incubated at 37 °C for 24 h, and the radiolabeled enzymes were separated from excess radioactive compounds by rapid gel filtration using a Sephadex G-25 (fine) column (0.5 5 cm) equilibrated with sodium phosphate (5 mM, pH 8) (23) . The pooled fractions were concentrated to 100 µl, and the following analyses were carried out.

The bound radioactivity was determined by counting the radioactivity in 10 µl (10%) of the solution. To ascertain to which subunit the radiolabel was bound, 25 µl (25%) of the solutions were subjected to gel-electrophoresis as described above. SDS-polyacrylamide gel electrophoresis of the enzyme showed two bands corresponding to molecular masses of 42 and 22 kDa for the heavy and light subunits, respectively.

The remainder of the solutions (65 µl) were treated with guanidinium hydrochloride (8 M) and acetic acid (1 M) at 37 °C for 18 h. (final volume, 250 µl). The subunits were separated by reversed-phase HPLC on a Vydac C column (0.46 25 cm) using a Waters system and 0.1% trifluoroacetic acid as solvent A and 95% (v/v) acetonitrile/0.1% trifluoroacetic acid as solvent B. A linear gradient from 20 to 60% solvent B for 40 min was used with a flow rate of 1.5 ml/min. The effluent was monitored at 214 nm, and fractions were collected each minute; the separated subunit fractions were pooled and concentrated. Portions of the eluted subunits were taken for liquid scintillation counting.

Amino Acid Analysis

The enzyme preparations were hydrolyzed with 6 M HCl at 110 °C under N for 20-24 h. The hydrolysates were neutralized, and the amino acids present were converted to the corresponding phenylthiocarbamyl amino acids and analyses were performed as described by Bidlingmeyer et al.(24) . Fractions (1 min) of the eluent were collected for liquid scintillation counting.

Proteolytic Digestion

Several chemically modified enzyme samples were subjected to proteolytic digestion at 37 °C for 24 h using endoproteinase Glu-C (10% by weight) in NaHPO (50 mM, pH 7.8) or endoproteinase Lys-C (10% by weight) in Tris (100 mM, pH 8.2) containing urea (2 M) and methylamine (20 mM). Digestion with -chymotrypsin (5% by weight) was carried out for 6 h in Tris-HCl (50 mM, pH 7.8) containing CaCl (5 mM).

Separation of the peptides generated was conducted on a Waters system with a µBondapak C ODS (3.9 mm 30 cm) using trifluoroacetic acid (0.1%) as solvent A and 95% (v/v) acetonitrile (0.1% trifluoroacetic acid) as solvent B. A flow rate of 0.7 ml/min. was used in conjunction with a linear gradient between 100% A and 60% B after 60 min and, thereafter, 100% B for 10 min. Elution of the peptides was monitored at 214 nm. Fractions (1 min) of the eluent were collected and assayed for radioactivity. Analysis of [C]Inhibitor-Enzyme Linkage Stability-Several chemical modification experiments were carried out to investigate the stability of the [C]inhibitor-enzyme linkage under various conditions; hydroxylamine (200 mM, pH 8), dithiothreitol (25 mM), guanidinium ion (8 M, pH 8), and combinations thereof, in sodium phosphate (50 mM, pH 8), as well as in NaOH (0.1 M) and HCl (0.1 M). The solutions (total volume, 100 µl) were incubated at 37 °C for 24 h. A 10-µl portion (10%) was assayed for enzyme activity and compared with the controls.

The protein was precipitated after addition of bovine serum albumin (1 mg), by adding trichloroacetic acid to a final concentration of 20%. After incubation at 0 °C for 30 min, the samples were centrifuged in a Beckman Microfuge B for 5 min at 4 °C. The supernatant was removed carefully, and the precipitated protein was washed twice with 100-µl portions of 5% trichloroacetic acid. Portions of the precipitated protein and of the combined washings were analyzed for radioactivity. The remainder of the precipitated protein was subjected to gel-electrophoresis to separate the subunits, whose content of radioactivity was determined.

Protein Determination

Bovine serum albumin was used as a standard for protein concentration estimations using the Bio-Rad protein assay dye (25) . Chemical Modification of the Enzyme by N-[1-C]Acetylimidazole-N-[1-C]Acetylimidazole (0.5 mmol; specific radioactivity, 31 µCi/mmol) was added to the enzyme (4 mg, 58 nmol) in 200 µl of Tris-HCl (0.1 M, pH 8) and incubated at 37 °C for 12 h. The excess [C]reagent was removed by use of a Penefsky column (23) ; the C-acetylated enzyme was lyophilized and redissolved in 2 ml of sodium phosphate (50 mM, pH 8). Analyses of the [C]acetyl-enzyme linkages were made under various conditions as described above.


RESULTS

Inhibition of the Enzyme by Iodoacetamide and by N-Acetylimidazole

Incubation of the enzyme with 50 mM iodoacetamide at pH 8 led to 50% inactivation after 30 min and to complete inactivation after 5 h. Iodoacetate (50 mM) had little effect on activity, but in the presence of 50 mM maleate, about 50% of the activity disappeared within 90 min. Maleate also increased inhibition by iodoacetamide; under these conditions, maleate itself had no effect on activity. Maleate enhances hydrolysis of a -glutamyl donor and decreases transpeptidation, apparently by binding to the acceptor site (26). Inactivation of the enzyme by iodoacetamide (and by iodoacetate) was prevented by addition of L-serine plus sodium borate (50 mM each), but not by adding either component alone. These findings are in general agreement with earlier work.

In the present studies we made the interesting observation that the iodoacetamide-inhibited enzyme can be reactivated; thus, when the iodoacetamide-inhibited enzyme was incubated with hydroxylamine (200 mM, pH 8) at 37 °C, about 75-80% of the initial activity was restored after 12 h at 37 °C. Only slight (<5%) reactivation was observed when the inactivated enzyme was incubated in sodium phosphate buffer (50 M, pH 8) in the presence or absence of dithiothreitol (25 mM), glycylglycine (0.1 M), or GSH (0.2 M).

Incubation of the enzyme with N-acetylimidazole also led to inactivation. Serine and borate did not protect against inactivation. The activity was not restored by incubation of the inactivated enzyme with hydroxylamine, dithiothreitol, or substrates.

Modification of the Enzyme by Treatment with Iodoacetamide, Iodoacetate, and N-Acetylimidazole

summarizes studies on the effects of iodoacetamide and N-acetylimidazole on labeling with iodo[C]acetamide. In these studies, samples of the enzyme (550-600 units) were treated with serine plus borate (S/B), acivicin (1 mM), iodoacetamide (unlabeled), N-acetylimidazole (unlabeled), and dialyzed as indicated; then the treated enzyme samples were incubated with labeled iodoacetamide as stated in . (Since the results with iodoacetate (50 mM) plus maleate (50 mM) were very similar to those found with iodoacetamide, these data are not shown.) Experiment 1 shows that treatment of the enzyme with iodo[C]acetamide led to labeling of both subunits; protection with S/B led to decreased labeling of the light subunit equivalent to about one residue (experiment 2). Experiment 4 shows that protection of the active site by S/B, followed by treatment with unlabeled iodoacetamide, dialysis, and treatment with iodo[C]acetamide, led to labeling of about one residue of the light subunit. In experiment 9, the enzyme was treated with N-acetylimidazole (after iodoacetamide treatment in the presence of S/B); here, treatment with iodo[C]acetamide led to labeling of about two residues of the light subunit. A similar result, labeling of two residues, was obtained in experiment 12. In experiment 11, S/B protected about two residues of the N-acetylimidazole-treated enzyme from reaction with iodo[C]acetamide. Notably, labeling equivalent to only about one residue was found in experiment 8 in which the unprotected enzyme was treated with iodoacetamide prior to treatment with N-acetylimidazole and iodo[C]acetamide.

Addition of acivicin to the S/B-protected enzyme (experiment 3) resulted in the same degree of labeling as the control (experiment 1). Treatment with N-acetylimidazole (experiment 10) shows that about eight residues (about three on the heavy subunit and about five on the light subunit) are not modified by iodo[C]acetamide as compared with the control. Protection of the active site with S/B (experiments 5 and 10) does not alter the number of residues modified by N-acetylimidazole, consistent with the finding that S/B does not protect the enzyme against inactivation by N-acetylimidazole.

To identify the residues modified by iodo[C]acetamide, portions of the reaction mixtures of these experiments were subjected to hydrolysis with 6 M HCl for 20-24 h at 110 °C; and derivatization and amino acid analysis (see ``Methods'') were carried out. S-[C]Carboxymethylcysteine was found only in experiments 8, 9, and 12; 0.96 ± 0.09 (n = 9) mol/mol of enzyme was found, and an additional quantity (0.91 ± 0.11 (n = 9) mol/mol) of C equivalent appeared in the fractions collected from these experiments. This C material was not a phenylisothiocyanate derivative, but was apparently [C]glycolate, formed by hydrolysis of the corresponding aspartate -ester (see Ref. 7 and below). The labeled enzyme obtained in experiment 4 was subjected to enzymatic digestion by endoproteinase Glu-C; separation of the peptides obtained by chromatography on a C column gave a single labeled component (retention time, 11 min), which was presumably the aspartate -ester. Amino acid analysis after acid hydrolysis of this component showed aspartate to be the only derivatized residue. Under the conditions used here, endoproteinase Glu-C digestion cleaves the carboxylate peptide bonds of both aspartate and glutamate residues, so that the resulting -aspartyl-[C]glycollamide ester could only arise from Glu-Asp or Asp-Asp. These sequences occur throughout the enzyme, but are found only twice in the light subunit; 388,389:ED and 421,422:DD. A similar digestion of the labeled enzyme obtained in experiment 4 (Table l) was carried out with endoproteinase Lys-C, and a labeled peptide was obtained; the amino acid composition of this peptide corresponded to residues 407-443 of the light subunit (data not shown) (and not to residues 381-406), thus indicating that the modified aspartyl is Asp-422.

The C-labeled enzyme obtained in experiment 4 () was treated with various reagents (such as phosphate buffer, pH 8, 8 M guanidinium hydrochloride, hydroxylamine; see ``Methods''). Under these conditions, a large fraction (73-84%) of the radioactivity was released on treatment with 0.2 M hydroxylamine or 0.1 M sodium hydroxide; about 30% was released on treatment with 0.1 M hydrochloric acid. Treatment with dithiothreitol, substrate, or guanidinium ions released less than 8% of the radioactivity. These findings are consistent with an aspartate -ester.

Modification of the enzyme with N-[1-C]acetylimidazole led to >99% inhibition and binding of 4.89 ± 0.21 (n = 3) mol of [C]acetyl/mol of the enzyme, (i.e. modification of five residues). Treatment of the C-acetylated enzyme under various conditions (see ``Methods'' and Ref. 27) showed that about of 20% of the label, equivalent to about one molar residue, was released by dithiothreitol and 40% (equal to about two molar residues) was released by treatment with hydroxylamine, consistent with release of [C]acetyl from a cysteine residue and two tyrosine residues. SDS-gel electrophoresis studies indicated that the presumed acetylated cysteine was on the heavy subunit, whereas one each of the tyrosine residues was located on the heavy and light subunits. C-Acetylated enzyme, after treatment with dithiothreitol and hydroxylamine, was still fully inhibited and contained label equivalent to two acetyl groups; probably these are acetylated lysines; one was attached to each subunit. This acetylated enzyme was subjected to endoproteinase Glu-C digestion; fractionation on a C column separated two radiolabeled peaks. Acid hydrolysis of these revealed that the amino acid composition of one of these corresponded to residues 66-101, which contains only one lysine; lysine 99. This was confirmed by treatment of the peptide with -chymotrypsin followed by separation of the oligopeptides produced; the label was associated with a peptide corresponding to residues 93-101. The other labeled peak from the endoproteinase Glu-C digestion contained two peptides which could not be separated; these were identified on the basis of the mixed amino acid composition as VKRAVE and SRKGGE; both contain a lysine (Lys-495 and Lys-561).

Binding of Acivicin to the Enzyme

Chemical modification experiments were carried out in which [C]acivicin was added in place of the iodo[2-C]acetamide used in . Virtually all of the bound radioactivity found in these experiments () was associated with the light subunit. Treatment with S/B of the native enzyme (; experiment 2), and of the N-acetylimidazole-modified enzyme (experiment 4), prevented acivicin from binding to the enzyme. In the absence of S/B, acivicin bound to both the native enzyme (experiment 1) and the acetylated enzyme (experiment 3) at about a 1:1 molar ratio. [C]Acivicin bound to enzyme that had been pretreated with iodoacetamide (experiment 5); iodoacetamide was previously shown to modify an aspartyl residue in the -glutamyl binding site (see above). When the N-acetylimidazole modified enzyme was treated with iodoacetamide (experiment 7), both an aspartyl and a cysteine residue at the -glutamyl binding site were modified (). Experiments 5 and 7 () show that acivicin binds to the enzyme light subunit in such a way as to not be affected by either of the modified aspartyl and cysteine residues in the -glutamyl binding site. However, acivicin is prevented from binding by S/B (experiment 2). Thus, it appears that the binding domains of S/B overlap those of acivicin. Experiment 6 also shows that iodoacetamide treated enzyme after addition of S/B can still interact with acivicin. Apparently the S/B complex cannot protect the -glutamyl binding site because the iodoacetamide modified aspartyl residue interferes. Inactivation of the enzyme by acivicin does prevent modification of the aspartyl and cysteine residues at the -glutamyl binding site. The binding of acivicin also prevents the formation of a stabilized -glutamyl-enzyme form (see next section).

-Glutamyl-Enzyme Formation

Evidence for formation of a -glutamyl-enzyme was obtained in studies in which chemically modified enzyme was incubated with [C]glutamine (I). In all of the experiments in which binding of [C]glutamine was observed, the label was present only on the light subunit. In the absence of chemical modification (experiment 1, control), there was no binding of [C]glutamine. After treatment of the enzyme with N-acetylimidazole, the equivalent of about 1 mol of [C]glutamine was bound (experiment 2). Treatment of the [C]labeled enzyme with 20 mM hydroxylamine led to >90% formation of -[C]glutamylhydroxamate, as identified previously (29). Protection of the active site by adding S/B (experiment 3), or modification with iodoacetamide before or after N-acetylimidazole treatment (experiments 4 and 5, respectively), prevented formation of -glutamyl-enzyme. Treatment with S/B before iodoacetamide treatment, which prevents modification of the aspartyl and cysteinyl residues at the -glutamyl binding site, did not prevent formation of -glutamyl-enzyme (experiments 6 and 7). Treatment of the N-acetylimidazole-modified enzyme with acivicin prevented [C]glutamine binding (experiment 8). Preincubation of the N-acetylimidazole-modified enzyme with either unlabeled glutamine or glutathione (experiment 9) led to a marked decrease of binding of [C]glutamine. This was not observed on preincubation with glutamate (experiment 10).

Other evidence indicating formation of a stabilized -glutamyl-enzyme came from enzyme assays carried out in experiments 2, 6, and 7 (I), in which the enzyme was modified by N-acetylimidazole. In these studies, monitoring at 410 nm for the release of p-nitroaniline from -glutamyl-p-nitroanilide showed activity within the first 10 s after mixing the substrates with enzyme. Thereafter, the activity decreased, and no further release of p-nitroaniline was found after the initial 20 s. In a typical experiment, the change in absorbance between time 0 and 20 s was equivalent to the formation of 0.6 µmol of p-nitroaniline. The amount of enzyme assayed here (500 units, 0.45 mg) would be expected to release 6.6 nmol of p-nitroaniline, in a single enzyme turnover. Therefore, the enzyme turns over about 90 times before forming a stabilized -glutamyl-enzyme.

The stability of the -[C]glutamyl-enzyme formed from the N-acetylimidazole-modified enzyme was examined under various conditions (see ``Methods''). In sodium phosphate (50 mM, pH 8), with or without glycylglycine (0.1 M), the -[C]glutamyl-enzyme was relatively stable. Release of radioactivity increased substantially upon denaturing the enzyme with guanidinium ions (8 M, pH 8), suggesting that this form is stabilized by its environment on the enzyme. Relatively little radioactivity was released on incubation with dithiothreitol. Most of the enzyme-bound radioactivity was released on treatment with hydroxylamine, which led to formation of -[C]glutamylhydroxamate. This finding, and the observation that the linkage between the -[C]glutamyl moiety and the enzyme was more susceptible to base than acid, are consistent with an ester. The -[C]glutamyl-enzyme was subjected to endoproteinase Glu-C digestion. However, a peptide containing radioactivity was not found; only free [C]glutamate was identified.


DISCUSSION

The present studies indicate that the active site carboxyl group of -glutamyl transpeptidase that reacts with iodoacetamide is Asp-422. In this reaction the -carboxyl group of Asp-422 is esterified to form O&cjs0808;C-OCHCONH, which is associated with loss of enzymatic activity. Treatment of the inactivated enzyme with hydroxylamine restores most of the initial enzymatic activity. Reactivation is associated with cleavage of the ester linkage; the details of this reaction still need study. Asp-422 may provide an essential electrostatic binding site, probably for the -amino group of the -glutamyl substrate. This subject is also considered elsewhere (28) .

Another amino acid residue at the active site (cysteine) was exposed by treatment of the enzyme with N-acetylimidazole so that it became available for reaction with iodoacetamide (cf. Ref. 6). This is the only cysteine residue on the light subunit and is therefore identified as Cys-453. Although it is located in the active site region, this thiol is unreactive in the native enzyme in which it is apparently ``buried'' (see Ref. 28).

The data indicate that about five amino acid residues are acetylated by treatment of the enzyme with N-acetylimidazole and that residues of both subunits are acetylated. It seems significant that one of these is Lys-99, which previous studies (16) suggested is at or near the binding site for acceptor substrates. Interestingly, Lys-99 was found to be modified by phenylglyoxal (16) , which is generally regarded as an arginine reagent. Acetylation evidently affects one or more of the amino acid residues needed for catalysis; since it stabilizes the -glutamyl-enzyme, it apparently affects amino acid residues that are involved in the breakdown of the -glutamyl enzyme. Acetylation is probably associated with conformational change in the enzyme; the number of residues that can be modified by iodoacetamide decreases after acetylation ().

The -glutamyl enzyme formed here by reaction of the acetylated enzyme with glutamine (I) has properties expected of an intermediate that might be formed in catalysis. It is highly reactive with hydroxylamine and is not very stable. The conditions under which it may and may not be formed are consistent with such an intermediate -glutamyl enzyme. Previously, evidence was reported for formation (by a different procedure) of an apparent -glutamyl enzyme form in which the -glutamyl moiety was linked to an enzyme lysine amino group (6) , but the enzyme form obtained in that work was found to be stable to performic acid and to hydroxylamine, and thus its properties were markedly different from those found here.

In summary, the present studies have identified two active site residues of rat -glutamyl transpeptidase: Asp-422 and Cys-453. Asp-422 appears to be required for enzyme activity, whereas Cys 453, which is buried, is probably not.() Acetylation seems to open and expose the active site region, which, nevertheless, retains the reactive -glutamyl binding site. This enzyme site, which probably has a hydroxyl group, is not identical to the acivicin binding sites (Ser-405, Thr-523) previously identified (13, 29). We tentatively suggest that the -amino group of the -glutamyl substrate is linked electrostatically to Asp-422 in such a manner as to facilitate reaction of the -carbonyl of the substrate with a specific enzyme hydroxyl group thus forming a -glutamyl-enzyme, which may participate in the transpeptidation and hydrolysis reactions that are catalyzed by the enzyme.

  
Table: Labeling of the light and heavy subunits of the enzyme by iodo[C]acetamide after treatment of the holoenzyme with unlabeled iodoacetamide (IA) and N-acetylimidazole (N-Ac-I)

See ``Methods'' for experimental details. After completion of the chemical treatments indicated, the samples were incubated for 24 h at 37 °C with iodo[2-C]acetamide. Excess C reagent was removed by rapid gel filtration (23), and the C present in the light and heavy subunits was determined after their separation by SDS-gel electrophoresis.


  
Table: Binding of acivicin to the light subunit

Each of the experiments was carried out in sequential order as described under ``Methods.'' [3-C]Acivicin was incubated for 24 h at 37 °C with the modified enzymes; excess [3-C]acivicin was removed by use of a Penefsky column (23). Portions were subjected to gel electrophoresis, and the mean molar ratio of C-compound to light subunit are given ± S.D. (n = 6) (<0.02 molar ratio of C-compound was associated with the heavy subunit).


  
Table: Formation of a stabilized -glutamyl-enzyme on the light subunit

The chemically modified enzymes were incubated with [C]glutamine for 24 h at 37 °C as indicated above (see ``Methods''). Excess C-compound was removed by gel filtration and portions of the enzyme were subjected to gel electrophoresis. The molar ratios (C-compound to light subunit) are given as means ± S.D. (n = 3).



FOOTNOTES

*
This research was supported in part by National Institutes of Health Grant 2 R37 DK12034 from the United States Public Health Service (NIDDK). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Deceased April 6, 1995.

The numbering of the amino acid residues of the human and rat -glutamyl transpeptidases differ by one residue. Thus, Asp-422 and Cys-453 in the rat enzyme are equivalent, respectively, to Asp-423 and Cys-454 in the human enzyme.

The abbreviation used is: HPLC, high performance liquid chromatography; S/B, serine and sodium borate; IA, iodoacetamide; N-Ac-I, N-acetyl-imidazole.

Independent studies on the human enzyme involving site-specific mutagenesis of conserved aspartate residues and the cysteine residue of the light subunit strongly support these conclusions (28).


ACKNOWLEDGEMENTS

We thank Dr. Einar Stole and Dr. Yoshitaka Ikeda for very helpful discussions and Edith Perryman, Selma Haschemeyer, and Linda Gilbert for their assistance in preparing this manuscript for publication.


REFERENCES
  1. Tate, S. S., and Meister, A.(1985) Methods Enzymol. 133, 400-419
  2. Meister, A.(1989) in Glutathione: Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulson, R., and Avramovic, O., eds) pp. 367-474, John Wiley & Sons, New York
  3. Tate, S. S.(1980) in Enzymatic Basis of Detoxification (Jakoby, W. B., ed) Vol. 2, pp. 95-120, Academic Press, New York
  4. Meister, A., Tate, S. S., and Ross, L. L.(1976) in The Enzymes of Biological Membranes (Martinosi, A., ed) Vol. 3, pp. 315-347, Plenum Publishing Corp., New York
  5. Meister, A., and Anderson, M. E.(1983) Annu. Rev. Biochem. 52, 711-760 [CrossRef][Medline] [Order article via Infotrieve]
  6. Elce, J. S.(1980) Biochem. J. 185, 473-481 [Medline] [Order article via Infotrieve]
  7. Szewczuk, A., and Connell, G. E.(1965) Biochim. Biophys. Acta 105, 352-367 [Medline] [Order article via Infotrieve]
  8. Laperche, Y., Bulle, F., Aissani, T., Chobert, M. N., Aggerbeck, M., Hanoune, J., and Guellean, G.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3155-3159 [Abstract]
  9. Colomo, J., and Pitot, H. C.(1986) Nucleic Acids Res. 14, 1393-1403 [Abstract]
  10. Suzuki, H., Kumagai, H., Echigo, T., and Tochikura, T.(1989) J. Bacteriol. 171, 5169-5172 [Medline] [Order article via Infotrieve]
  11. Papandrikopoulou, A., Frey, A., and Gassen, H.(1989) Eur. J. Biochem. 183, 693-698 [Abstract]
  12. Sakamuro, D., Yamazoe, M., Matsuda, Y., Kangawa, K., Taniguchi, N., Matsuo, H., Yoshikawa, H., and Ogasawara, N.(1988) Gene (Amst.) 73, 1-9 [CrossRef][Medline] [Order article via Infotrieve]
  13. Stole, E., Seddon, A. P., Wellner, D., and Meister, A.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1706-1709 [Abstract]
  14. Tate, S. S., and Meister, A.(1977) Proc. Natl. Acad. Sci. U. S. A. 74, 931-935 [Abstract]
  15. Gardell, S. J., and Tate, S. S.(1980) FEBS Lett. 122, 171-174 [CrossRef][Medline] [Order article via Infotrieve]
  16. Stole, E., and Meister, A.(1991) J. Biol. Chem. 266, 17850-17857 [Abstract/Free Full Text]
  17. Ikeda, Y., Fujii, J., and Taniguchi, N.(1993) J. Biol. Chem. 268, 3980-3985 [Abstract/Free Full Text]
  18. Reddy, G. S., Mandell, L., and Goldstein, J. H.(1963) J. Chem. Soc.1414-1421
  19. Weber, K., and Osborn, M.(1969) J. Biol. Chem. 244, 4406-4412 [Abstract/Free Full Text]
  20. Orlowski, M., and Meister, A.(1963) Biochim. Biophys. Acta 73, 679-681 [CrossRef]
  21. Revel, J. P., and Ball, E. G.(1959) J. Biol. Chem. 234, 577-582 [Free Full Text]
  22. Tate, S. S., and Meister, A.(1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4806-4809 [Abstract]
  23. Penefsky, H. S.(1977) J. Biol. Chem. 252, 2891-2899 [Abstract]
  24. Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L.(1984) J. Chromatogr. 226, 93-104
  25. Bradford, M. M.(1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  26. Thompson, G. A., and Meister, A.(1980) J. Biol. Chem. 255, 2109-2113 [Free Full Text]
  27. Means, G. E., and Feeney, R. E.(1971) in Chemical Modification of Proteins, Holden-Day, Inc., San Francisco
  28. Ikeda, Y., Fujii, J., Taniguchi, N., and Meister, A.(1995) J. Biol. Chem. 270, 12471-12475 [Abstract/Free Full Text]
  29. Stole, E., Smith, T. K., Manning, J. M., and Meister, A.(1994) J. Biol. Chem. 269, 21435-21439 [Abstract/Free Full Text]

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