Proc. Nati. Acad. Sci. USA Vol. 87, pp. 1706-1709, March 1990 Biochemistry Identification of a highly reactive threonine residue at the active site of y-glutamyl transpeptidase (glutathione/L-(aSSS)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid/glycoprotein/p-hydroxyglutamic acid) EINAR STOLE, ANDREW P. SEDDON, DANIEL WELLNER, AND ALTON MEISTER Department of Biochemistry, Cornell University Medical College, 1300 York Avenue, New York, NY 10021 Contributed by Alton Meister, December 13, 1989 ABSTRACT y-Glutamyl transpeptidase [(5-glutamyl)- cDNA (9-11). These sequence studies support the conclusion peptide:amino-acid 5-glutamyltransferase, E0 2.3.2.2], an en- that a 21-amino acid peptide moiety at the N terminus of the zyme of major importance in glutathione metabolism, was heavy subunit is closely associated with the cell membrane inactivated by treating it with L-(aS,5S)-a-amino-3-chloro- (12, 13). There are as yet no studies that identify specific 4,5-dihydro-5-[3-"4C]isoxazoleacetic acid. This selective re- amino acid residues of the enzyme that function in catalysis agent binds stoichiometrically to the enzyme; more than 90% at the active site. Previously, evidence was obtained sug- of the label was bound to its light subunit. Enzymatic digestion gesting that the y-glutamyl moiety of the substrate and ofthe light subunit gave a 14C-labeled peptide that corresponds various inhibitors bind to a hydroxyl group on the enzyme to amino acid residues 517-527 of the enzyme and two incom- (14). Findings that suggest involvement of an enzyme amino plete digestion products that contain this labeled peptide moi- group (15), a histidine moiety (16), a carboxyl group (15, 17), ety. The radioactivity associated with this peptide was released a cysteine residue (15, 17), and an arginine residue (18, 19) with threonine-523 during sequencing by the automated gas- also have been reported. phase Edman method. The light subunit contains 14 other In the present work, the enzyme was inactivated by threonine residues and a total of 19 serine residues; these were incubation with L-(aS,5S)-a-amino-3-chloro-4,5-dihydro- not labeled. Threonine-523 is situated in the enzyme in an 5-[3-14C]isoxazoleacetic acid, a selective irreversible inhibi- environment that greatly increases its reactivity, indicating tor (20-22). Inactivation was associated with binding of 1 mol that other amino acid residues of the enzyme must also ofinhibitor per mol of enzyme and the inhibitor was found to participate in the active-site chemistry of the enzyme. be attached to a specific threonine residue (Thr-523) of the light subunit. y-Glutamyl transpeptidase [(5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2], the enzyme that cata- EXPERIMENTAL PROCEDURES lyzes the cleavage of the y-glutamyl bond of glutathione and related 'y-glutamyl compounds, plays a key role in metabo- Materials. 'y-Glutamyl transpeptidase, isolated from frozen lism by virtue of its function in the cleavage and formation of rat kidneys (Pel-Freez Biologicals) (1), had a specific activity y-glutamyl bonds (1-4). Such reactions are involved in the of 1100 prmol/min per mg when assayed at 370C with 1 mM processing of various S-conjugates of glutathione (formed L-y-glutamyl-p-nitroanilide (23) and 20 mM glycylglycine in from endogenous and exogenous compounds), in the trans- 60 mM Tris-HCI buffer at pH 8.0. L-(aS,5S)-a-Amino- port of amino acids as y-glutamyl amino acids, and in the 3-chloro-4,5-dihydro-5-[3-14C]isoxazoleacetic acid (acivicin, cellular recovery of cysteine moieties as y-glutamylcystine. AT-125; specific activity, 67,800 cpm/nmol as determined by The reactions catalyzed by y-glutamyl transpeptidase are PICO-TAG amino acid analysis and comparison with an thought to involve formation of a y-glutamyl enzyme and authentic sample of acivicin standard) was supplied by Rich- transfer of the y-glutamyl moiety to acceptors such as amino ard S. P. Hsi (Upjohn). Endoproteinase Lys-C purified from acids to form the corresponding y-glutamyl compounds. Lysobacter enzymogenes (specific activity, 30 units/mg) Hydrolysis of the y-glutamyl donor occurs when water is the (24), pyroglutamate aminopeptidase purified from calf liver, acceptor. y-Glutamyl transpeptidase is widely distributed and endoproteinase Glu-C purified from Staphylococcus and has been found, for example, in kidney, pancreas, aureus V8 were purchased from Boehringer Mannheim. epididymis, jejunal mucosa, biliary epithelium, ciliary body, Trifluoroacetic acid, trifluoromethanesulfonic acid, Tris, gly- and choroid plexus (5). The enzyme is typically membrane- cylglycine, Sephadex G-25 fine, L--glutamyl-p-nitroanilide, bound and is extensively glycosylated. L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated Rat kidney y-glutamyl transpeptidase consists of two sub- trypsin, and peptide:N-glycosidase F (PNGase F) isolated units (heavy subunit of Mr 51,000 and light subunit of Mr from Flavobacterium meningosepticum were obtained from 22,000). The heavy subunit is linked to the cell membrane Sigma. Acetonitrile (ChromAR HPLC) was purchased from through its N-terminal segment, whereas the light subunit is Mallinckrodt. Guanidinium hydrochloride and urea (ultra attached to the heavy subunit by noncovalent interactions pure; recrystallized before use) were products of Schwarz/ (1). The two subunits of rat kidney y-glutamyl transpeptidase Mann. Methylamine was obtained from Eastman Kodak, and are synthesized as a single peptide chain, and glycosylation triethylamine, constant-boiling HCl, and phenyl isothiocya- and membrane insertion take place as cotranslational events nate were obtained from Pierce. Compounds used in peptide (1, 6-8). The enzyme subunits, which are formed by cleavage sequencing were obtained from Applied Biosystems. Mono- of the proenzyme, are encoded by a common mRNA. The fluor scintillation liquid was purchased from National Diag- complete amino acid sequence of the two subunits has been nostics (Somerville, NJ). deduced from the nucleotide sequence of the corresponding Methods. y-Glutamyl transpeptidase (6 mg; 91 nmol) was incubated with [14C]acivicin (910 nmol) in a final 0.3-ml volume of 50 mM at 370C. Portions (1 The publication costs of this article were defrayed in part by page charge NaH2PO4 (pH 7.5) pul) payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: PNGase F, peptide:N-glycosidase F. 1706 Downloaded by guest on September 25, 2021 Biochemistry: Stole et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1707 were withdrawn at various times and assayed (1) to monitor inactivated labeled enzyme was separated by HPLC into light the degree of inactivation. The inactivated enzyme was and heavy subunits, which were examined by SDS/PAGE; recovered by gel filtration on Sephadex G-25 (0.7 cm x 40 92% of the radioactivity bound to the recovered protein was cm) (0.1 M NH4HCO3 at pH 7.5). associated with the light subunit. The subunits of the denatured enzyme were separated by Digestion of the PNGase F-treated labeled light subunit reverse-phase HPLC on a protein C4 column (5,um; 0.46 cm with endoproteinase Lys-C, followed by fractionation on a x 25 cm) (Vydac, Hesparia, CA) with a Waters system and C18 column, gave three peptide fractions (I, II, and III), each 0.1% trifluoroacetic acid as solvent A and 95% (vol/vol) containing about one-third of the total peptide-bound radio- acetonitrile/0.1% trifluoroacetic acid as solvent B. After the activity recovered. Amino acid analysis indicated that the enzyme was denatured by treatment at 370C with 8 M composition of fraction I agrees closely with the expected guanidinium hydrochloride in 1 M acetic acid for 15 hr, the composition of a specific peptide corresponding to amino sample was injected into the column and eluted with a linear acid residues 517-527. Automated Edman degradation of gradient run from 20-60% (vol/vol) solvent B over 40 min at fraction I confirmed this and gave the sequence Asn- a flow rate of 1.5 ml/min. The effluent was monitored at 214 Ile-Asp-Gln-Val-Val-Thr-Ala-Gly-Leu-Lys (Fig. 1). Radioac- nm. Portions of the eluted peptides were taken for liquid tivity was eluted from the sequencer with residue 7 offraction scintillation counting on an LKB-1218 Rackbeta scintillation I, which corresponds to Thr-523; small amounts were carried counter. The separated peptides were examined by sodium over into cycles 8 and 9. That radioactivity carried over into dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/ later cycles suggests that either the Edman cleavage reaction PAGE) (25). Samples (1.5 mg) of isolated light subunit were or the extraction of the cyclized acivicin-derivatized threo- treated with PNGase F (6 units) in 0.3 ml of0.25 M NH4HCO3 nine complex is less efficient than for threonine. The peptide (pH 8.6) at 370C for 24 hr. The neutral carbohydrate content was estimated in several separate sequencing runs to be ofthe treated enzyme was determined by the phenol/sulfuric 90-100% pure. No evidence was obtained for the presence of acid method using D-galactose as a standard (26); 60% of the other peptides derived from y-glutamyl transpeptidase. carbohydrate was removed by treatment with PNGase F. There is a gradual loss of label from the protein during the The labeled light subunit (5 mg/ml) was digested at 370C several procedures applied, reflecting the lability of the with 10%o of its weight of endoproteinase Lys-C in 100 mM acivicin-enzyme linkage under the conditions used. The Tris HCl, pH 8.5/2 M urea/20 mM methylamine for 24 hr. acivicin-enzyme bond is presumably formed through reac- Peptides generated from endoproteinase Lys-C digestion of tion at C-3 of acivicin.