Uncoupling the Enzymatic and Autoprocessing Activities of Helicobacter pylori γ-Glutamyltranspeptidase
2006; Elsevier BV; Volume: 281; Issue: 28 Linguagem: Inglês
10.1074/jbc.m603381200
ISSN1083-351X
AutoresGina Boanca, Aaron Sand, Joseph Barycki,
Tópico(s)Pneumocystis jirovecii pneumonia detection and treatment
Resumoγ-Glutamyltranspeptidase (γGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the γ-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori γ-glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported γGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the γ-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian γGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the γ-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions. γ-Glutamyltranspeptidase (γGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the γ-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori γ-glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported γGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the γ-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian γGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the γ-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions. Helicobacter pylori is a Gram-negative bacterial pathogen that colonizes the gastric mucosa. Infection puts the individual at greater risk for developing gastritis, peptic ulcer disease, and gastric cancer (1Bjorkholm B. Falk P. Engstrand L. Nyren O. J. Intern. Med. 2003; 253: 102-119Crossref PubMed Scopus (65) Google Scholar, 2Sharma P. Vakil N. Aliment. Pharmacol. Ther. 2003; 17: 297-305Crossref PubMed Scopus (100) Google Scholar). H. pylori γ-glutamyltranspeptidase (Hp-GT) 3The abbreviations used are: HpGT, H. pylori γ-glutamyltranspeptidase; γGT, γ-glutamyltranspeptidase; GNA, l-glutamic acid γ-(4-nitroanilide). is a glutathione-degrading enzyme that has been shown to be a virulence factor in infection (3Chevalier C. Thiberge J.M. Ferrero R.L. Labigne A. Mol. Microbiol. 1999; 31: 1359-1372Crossref PubMed Scopus (162) Google Scholar, 4McGovern K.J. Blanchard T.G. Gutierrez J.A. Czinn S.J. Krakowka S. Youngman P. Infect. Immun. 2001; 69: 4168-4173Crossref PubMed Scopus (81) Google Scholar). H. pylori lacking γ-glutamyltranspeptidase has been shown to grow normally in vitro, but exhibits diminished growth rates within the gut in animal model systems. Bacterial loads of the HpGT-deficient strains are reduced by nearly 70% relative to the parental strain. Although not essential for colonization, HpGT clearly confers a growth advantage to the bacteria in vivo by mechanisms that remain unclear. HpGT has also been shown to up-regulate COX-2 and epidermal growth factor-related peptides in human gastric mucosal cells (5Busiello I. Acquaviva R. Di Popolo A. Blanchard T.G. Ricci V. Romano M. Zarrilli R. Cell. Microbiol. 2004; 6: 255-267Crossref PubMed Scopus (50) Google Scholar) and to induce apoptosis in human gastric epithelial cells (6Shibayama K. Kamachi K. Nagata N. Yagi T. Nada T. Doi Y. Shibata N. Yokoyama K. Yamane K. Kato H. Iinuma Y. Arakawa Y. Mol. Microbiol. 2003; 47: 443-451Crossref PubMed Scopus (85) Google Scholar). Both these activities are abolished by inactivation of the enzyme with mechanism-based inhibitors. Despite its demonstrated involvement in H. pylori colonization, persistence, and disease progression, biochemical characterizations of HpGT have been limited. The reclamation of extracellular glutathione and its conjugates is initiated by γ-glutamyltranspeptidase (γGT). The enzyme cleaves the γ-glutamyl amide bond to liberate cysteinylglycine, and the catalytic mechanism proceeds via a γ-glutamyl-enzyme intermediate (7Tate S.S. Meister A. Mol. Cell. Biochem. 1981; 39: 357-368Crossref PubMed Scopus (409) Google Scholar, 8Allison R.D. Methods Enzymol. 1985; 113: 419-437Crossref PubMed Scopus (82) Google Scholar, 9Taniguchi N. Ikeda Y. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 239-278PubMed Google Scholar, 10Ikeda Y. Taniguchi N. Methods Enzymol. 2005; 401: 408-425Crossref PubMed Scopus (68) Google Scholar, 11Keillor J.W. Castonguay R. Lherbet C. Methods Enzymol. 2005; 401: 449-467Crossref PubMed Scopus (86) Google Scholar). The γ-glutamyl group can be transferred to water (hydrolysis) or to an amino acid or short peptide (transpeptidation). Whereas mammalian γGTs are embedded in the plasma membrane by a single N-terminal transmembrane anchor and are heterologously glycosylated, bacterial homologs are soluble and localized to the periplasmic space. Overall, the γGTs are highly conserved, with mammalian and bacterial homologs often sharing >25% sequence identity. A required post-translational modification is the maturation of the precursor protein. γGT is synthesized as a 60-kDa polypeptide, and cleavage of the proenzyme yields a heterodimer composed of a 40- and a 20-kDa subunit (see Scheme 1) (3Chevalier C. Thiberge J.M. Ferrero R.L. Labigne A. Mol. Microbiol. 1999; 31: 1359-1372Crossref PubMed Scopus (162) Google Scholar, 12Suzuki H. Kumagai H. Echigo T. Tochikura T. Biochem. Biophys. Res. Commun. 1988; 150: 33-38Crossref PubMed Scopus (50) Google Scholar, 13Carter B.Z. Shi Z.Z. Barrios R. Lieberman M.W. J. Biol. Chem. 1998; 273: 28277-28285Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Kinlough C.L. Poland P.A. Bruns J.B. Hughey R.P. Methods Enzymol. 2005; 401: 426-449Crossref PubMed Scopus (38) Google Scholar). Processing of γGT is thought to be an intramolecular autocatalytic event, and a mechanism has been proposed in which processing proceeds via a nitrogen → oxygen acyl shift, with a conserved threonine residue serving as the nucleophile (15Suzuki H. Kumagai H. J. Biol. Chem. 2002; 277: 43536-43543Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Based on its enzymatic function and autoprocessing activity, γGT has been classified as an Ntn (N-terminal nucleophile) hydrolase (15Suzuki H. Kumagai H. J. Biol. Chem. 2002; 277: 43536-43543Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Members of the Ntn hydrolase family contain an αββα-core structure, are autocatalytically processed to yield an active enzyme, and catalyze amide bond hydrolysis (16Brannigan J.A. Dodson G. Duggleby H.J. Moody P.C. Smith J.L. Tomchick D.R. Murzin A.G. Nature. 1995; 378: 416-419Crossref PubMed Scopus (544) Google Scholar, 17Oinonen C. Rouvinen J. Protein Sci. 2000; 9: 2329-2337Crossref PubMed Scopus (208) Google Scholar). The new N-terminal residue of the processed enzyme, typically a serine, threonine, or cysteine residue, then serves as a nucleophile in the catalytic mechanism. Although the general features of the function of HpGT can be inferred based on its classification as an Ntn hydrolase, many mechanistic details of the autoactivation and catalytic function of HpGT have not been addressed. In this study, we isolated and biochemically characterized recombinant H. pylori γ-glutamyltranspeptidase. In contrast to previous reports of γGT purified from other organisms, the solubly expressed protein was isolated primarily as the 60-kDa precursor. This allowed us an opportunity to examine the autoprocessing of the protein. The rate of maturation was maximal at neutral pH, as was the enzymatic activity of the mature α2β2-heterotetramer. We used biochemical measurements of enzymatic activity in conjunction with site-directed mutagenesis and a coexpression system to investigate the involvement of a conserved threonine residue in the processing and catalytic activities. Generation of Wild-type and Mutant HpGT Expression Constructs—The isolation of HpGT has been described (3Chevalier C. Thiberge J.M. Ferrero R.L. Labigne A. Mol. Microbiol. 1999; 31: 1359-1372Crossref PubMed Scopus (162) Google Scholar), and its sequence corresponds to a predicted open reading frame within the H. pylori genome (KEGG Data Base entry HP1118) (18Alm R.A. Ling L.S. Moir D.T. King B.L. Brown E.D. Doig P.C. Smith D.R. Noonan B. Guild B.C. deJonge B.L. Carmel G. Tummino P.J. Caruso A. Uria-Nickelsen M. Mills D.M. Ives C. Gibson R. Merberg D. Mills S.D. Jiang Q. Taylor D.E. Vovis G.F. Trust T.J. Nature. 1999; 397: 176-180Crossref PubMed Scopus (1596) Google Scholar, 19Tomb J.F. White O. Kerlavage A.R. Clayton R.A. Sutton G.G. Fleischmann R.D. Ketchum K.A. Klenk H.P. Gill S. Dougherty B.A. Nelson K. Quackenbush J. Zhou L. Kirkness E.F. Peterson S. Loftus B. Richardson D. Dodson R. Khalak H.G. Glodek A. McKenney K. Fitzegerald L.M. Lee N. Adams M.D. Hickey E.K. Berg D.E. Gocayne J.D. Utterback T.R. Peterson J.D. Kelley J.M. Cotton M.D. Weidman J.M. Fujii C. Bowman C. Watthey L. Wallin E. Hayes W.S. Borodovsky M. Karp P.D. Smith H.O. Fraser C.M. Venter J.C. Nature. 1997; 388: 539-547Crossref PubMed Scopus (3021) Google Scholar). Although encoded by a single gene, the protein was isolated as two polypeptides of ∼40 and ∼20 kDa. Primers were designed to amplify HpGT, excluding a 26-amino acid signal sequence that targets the enzyme to the periplasmic space (3Chevalier C. Thiberge J.M. Ferrero R.L. Labigne A. Mol. Microbiol. 1999; 31: 1359-1372Crossref PubMed Scopus (162) Google Scholar). Using H. pylori genomic DNA (American Type Culture Collection) as a template, a 1.6-kb DNA fragment was amplified by PCR and inserted into a pET-28a expression vector (Novagen) incorporating a thrombin-cleavable N-terminal histidine tag. The resulting expression construct (pET-28/HpGT) was sequenced at the Genomics Core Facility of the University of Nebraska (Lincoln, NE) and confirmed to be identical to HP1118 excluding the signal sequence. Expression constructs for individual subunits of the processed enzyme were generated. The gene sequence for the N-terminal 40-kDa subunit was amplified by PCR, incorporating SpeI and SalI restriction sites. The insert was digested with the appropriate restriction enzymes and ligated into the pET-28a expression vector digested with NheI and SalI. Similarly, the gene sequence for the C-terminal 20-kDa subunit was amplified by PCR, incorporating NdeI and SalI restriction sites. The resultant 0.6-kb PCR product was digested with the relevant enzymes and ligated into a similarly digested pET-24a expression vector (Novagen), thus incorporating a C-terminal histidine tag. A bicistronic construct was also generated to express the N-terminal 40-kDa and C-terminal 20-kDa subunits separately but concurrently. The full-length HpGT expression construct was used as a template, and the HpGT sequence corresponding to the 20-kDa subunit was amplified by PCR, incorporating NdeI and XhoI restriction sites. The product was digested with the relevant enzymes and ligated into a similarly digested the pETDuet expression vector (Novagen). The dual expression vector containing the 20-kDa coding sequence was amplified and isolated. Next, the coding sequence for the 40-kDa subunit was excised from the above 40-kDa expression vector using NcoI and SalI. The resultant 1-kb fragment was gel-purified and ligated into a similarly digested pET-Duet expression vector containing the 20-kDa subunit sequence. The completed construct (HpGT-Duet) contained an N-terminal histidine tag on the 40-kDa subunit and an N-terminal methionine on the 20-kDa subunit. Point mutations were generated using the QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's protocol, and all constructs were verified by sequencing at the Genomics Core Facility of the University of Nebraska. Expression and Purification of HpGT—The pET-28/HpGT expression construct was used to transform Escherichia coli strain Rosetta(DE3)pLysS (Novagen). Cultures grown at 30 °C to A600 nm = 0.6–0.8 in 2× YT medium containing 34 mg/liter chloramphenicol and 30 mg/liter kanamycin were induced for 4 h by the addition of isopropyl β-d-thiogalactopyranoside to a final concentration of 500 μm. The cells were harvested by centrifugation and stored at –80 °C. Cells were resuspended in lysis buffer (50 mm NaH2PO4 (pH 8.0), 300 mm NaCl, and 10 mm imidazole), lysed by sonication, and centrifuged. The supernatant was treated with 0.35% polyethyleneimine at 4 °C and centrifuged to separate precipitated nucleic acids from the protein-containing supernatant. The protein was purified by affinity chromatography using a nickel-chelating column (Novagen) following the manufacturer's protocol. Recombinant HpGT was concentrated, dialyzed against 20 mm Tris (pH 7.4), and stored at 4 °C. Protein concentrations were estimated using a calculated extinction coefficient based on aromatic residue content (A280 nm of 1 mg/ml solution = 0.766) (20Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3452) Google Scholar). The histidine tag used for affinity purification was removed by proteolytic cleavage with thrombin (1 unit of thrombin/mg of HpGT, incubated overnight at 18 °C), which left an additional seven residues (GSHMASA) on the N terminus of HpGT. Following thrombin incubation, >50% of HpGT was found in the processed form. To ensure complete maturation of the enzyme, it was incubated at 37 °C for 6 h. The sample was centrifuged to remove precipitated proteins, concentrated, and dialyzed against 20 mm Tris (pH 7.4). A similar protocol was followed for each of the expression constructs with the following modifications. After ascertaining that the N-terminal histidine tag did not impact the processing or enzymatic activity of the wild-type enzyme, the thrombin cleavage step was omitted from all subsequent enzyme purifications. The HpGT-Duet construct confers resistance to ampicillin, and kanamycin was thus replaced with 100 mg/liter ampicillin. For HpGT generated using the pETDuet expression construct (HpGT-Duet), the enzyme was isolated in its mature form, and additional incubations were not required. Kinetic Characterizations of HpGT—To determine the rate of processing and its effects on catalytic activity, unprocessed HpGT was purified as described above with the following modifications. After concentration and dialysis of the enzyme, it was flash-frozen and stored at –80 °C to limit processing. It is important to note that 2 h). The absorbance at 340 nm was measured, and a standard curve for glutamate (0–100 μm) was determined. Similar experiments were performed using glutamine as the HpGT substrate. For routine enzymatic characterizations, the substrate analog l-glutamic acid γ-(4-nitroanilide) (GNA) was used (11Keillor J.W. Castonguay R. Lherbet C. Methods Enzymol. 2005; 401: 449-467Crossref PubMed Scopus (86) Google Scholar, 22Tate S.S. Meister A. Methods Enzymol. 1985; 113: 400-419Crossref PubMed Scopus (264) Google Scholar). The release of 4-nitroaniline can be monitored continuously at 412 nm, and concentrations can be determined using the reported extinction coefficient of 8800 m–1 cm–1. For standard assays, hydrolysis activity measurements were made in 0.1 m Tris (pH 8.0) containing 1 mm GNA at 25 °C using a Cary 50 spectrophotometer. The pH profile of the enzymatic activity was assessed using a citrate/phosphate buffer system of constant ionic strength (21Elving P.J. Markowitz J.M. Rosenthal I. Anal. Biochem. 1956; 28: 1179-1180Google Scholar). To determine the apparent kinetic constants, HpGT activity was assayed at various concentrations of GNA ranging from 1 to 1000 μm. The reaction exhibited saturation kinetics with respect to GNA, and the data were fit using the Michaelis-Menten equation to determine Km and Vmax values. To assess transpeptidation to an acceptor substrate, 20 mm glycylglycine was added to the assay mixture. Pre-steady-state kinetic studies were also performed. HpGT (17 μm final concentration) was rapidly mixed with GNA (1 mm final concentration) at 10 °C, and the absorbance at 412 nm was monitored using an Applied Photophysics stopped-flow apparatus (23Keillor J.W. Menard A. Castonguay R. Lherbet C. Rivard C. J. Phys. Org. Chem. 2004; 17: 529-536Crossref Scopus (21) Google Scholar). Molecular Mass Determination of HpGT—Purified HpGT was loaded onto a Superdex 200 HR 10/30 gel filtration column (Amersham Biosciences) and separated by fast protein liquid chromatography in 50 mm NaPO4 (pH 7.0) containing 150 mm NaCl at a flow rate of 0.25 ml/min. Size determination was made by comparison with molecular mass standards (Amersham Biosciences) chromatographed under the same conditions. The molecular mass standards used were as follows: thyroglobulin, 699 kDa; ferritin, 416 kDa; catalase, 219 kDa; aldolase, 176 kDa; albumin, 67 kDa; ovalbumin, 47 kDa; chymotrypsinogen A, 20 kDa; and RNase A, 15 kDa. Dynamic light scattering experiments were performed on a DynaPro MSXTC instrument (Proterion Corp., Somerset, NJ). Protein samples (1 mg/ml) were centrifuged at 20,000 × g for 15 min to remove particulates and loaded into the sample cuvette. Data were analyzed using the manufacturer's DYNAMICS software package, and the molecular mass was estimated based upon the hydrodynamic radius of the protein assuming a spherical model. Electrospray mass spectrometric analyses were performed at the Nebraska Redox Biology Center Metabolomics Core Facility of the University of Nebraska (Lincoln). Protein Expression and Purification—HpGT is expressed as an ∼60-kDa precursor that undergoes autocatalytic processing to an ∼40 kDa α-subunit and an ∼20-kDa β-subunit (Scheme 1). HpGT has been implicated as a virulence factor in colonization (3Chevalier C. Thiberge J.M. Ferrero R.L. Labigne A. Mol. Microbiol. 1999; 31: 1359-1372Crossref PubMed Scopus (162) Google Scholar, 4McGovern K.J. Blanchard T.G. Gutierrez J.A. Czinn S.J. Krakowka S. Youngman P. Infect. Immun. 2001; 69: 4168-4173Crossref PubMed Scopus (81) Google Scholar), an inducer of apoptosis (6Shibayama K. Kamachi K. Nagata N. Yagi T. Nada T. Doi Y. Shibata N. Yokoyama K. Yamane K. Kato H. Iinuma Y. Arakawa Y. Mol. Microbiol. 2003; 47: 443-451Crossref PubMed Scopus (85) Google Scholar), and a modulator of COX-2 and epidermal growth factor-related peptides (5Busiello I. Acquaviva R. Di Popolo A. Blanchard T.G. Ricci V. Romano M. Zarrilli R. Cell. Microbiol. 2004; 6: 255-267Crossref PubMed Scopus (50) Google Scholar). However, the autoprocessing activity, enzymatic profile, and quaternary structure of HpGT have not been rigorously characterized. To examine these properties, we generated a prokaryotic expression vector containing the coding sequence of HpGT and overexpressed the protein in E. coli. HpGT was purified by affinity chromatography exploiting an engineered N-terminal hexahistidine tag, with typical yields of 50 mg of purified protein/liter of bacterial culture. The enzyme was >95% pure based on SDS-PAGE, and the vast majority of HpGT exhibited an apparent molecular mass of ∼60 kDa (Fig. 1A, lane 1), corresponding to the inactive precursor. The precursor could be induced to catalytically cleave itself to produce the active heterodimer composed of an ∼40- and an ∼20-kDa subunit (Fig. 1A, lane 8). Autocatalytic Processing of HpGT—To characterize the autoprocessing of the enzyme, we monitored the conversion of precursor HpGT to its mature form as a function of time (Fig. 1). At the indicated times, an aliquot of HpGT was removed and denatured. Samples were analyzed by SDS-PAGE (Fig. 1A), and the percent processing was plotted versus time (Fig. 1B). Under the indicated conditions, the processing of HpGT exhibited t½ = 1.73 ± 0.22 h (Table 1). Catalytic activity was also plotted as a function of processing (Fig. 1B, inset). Hydrolysis activity was strongly dependent on autocatalytic processing of the enzyme, exhibiting a nearly 1:1 relationship. Extrapolation to the completely unprocessed enzyme suggested that the uncleaved enzyme exhibited ∼7% activity. However, the uncertainty in the densitometric measurements may underestimate the extent of cleavage in the early time points. Subsequent studies of HpGT mutants (discussed below) with diminished processing activities indicated that processing is required for enzymatic activity. Thus, it is likely that the unprocessed wild-type enzyme is also completely inactive.TABLE 1Comparison of the apparent kinetic constants for HpGT and human γGTEnzymeHydrolysisTranspeptidationt½ (processing)GNA KmVmaxGNA KmVmaxμmμmol/min/mgμmμmol/min/mgHuman γGTaThe kinetic constants for human γGT were reported previously (24). In these studies, saturation kinetics were observed with glycylglycine as the varied substrate with an apparent Km of 2.5 ± 0.3 mm.7.2 ± 0.54.3 ± 0.21000 ± 30800 ± 12Not observedHpGT12.5 ± 1.25.81 ± 0.1310.9 ± 0.5bSaturation kinetics were not observed with glycylglycine as the varied substrate, and therefore, measurements were made using 20 mm glycylglycine as reported for the human enzyme.6.81 ± 0.08bSaturation kinetics were not observed with glycylglycine as the varied substrate, and therefore, measurements were made using 20 mm glycylglycine as reported for the human enzyme.1.73 ± 0.22 hT380S31.0 ± 1.70.49 ± 0.0127.0 ± 0.7bSaturation kinetics were not observed with glycylglycine as the varied substrate, and therefore, measurements were made using 20 mm glycylglycine as reported for the human enzyme.0.77 ± 0.01bSaturation kinetics were not observed with glycylglycine as the varied substrate, and therefore, measurements were made using 20 mm glycylglycine as reported for the human enzyme.5.75 ± 0.82 daysa The kinetic constants for human γGT were reported previously (24Ikeda Y. Fujii J. Anderson M.E. Taniguchi N. Meister A. J. Biol. Chem. 1995; 270: 22223-22228Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In these studies, saturation kinetics were observed with glycylglycine as the varied substrate with an apparent Km of 2.5 ± 0.3 mm.b Saturation kinetics were not observed with glycylglycine as the varied substrate, and therefore, measurements were made using 20 mm glycylglycine as reported for the human enzyme. Open table in a new tab The maturation of HpGT was further characterized to gain insights into the autoprocessing reaction. To verify that maturation of HpGT is an intramolecular event, the rates of HpGT cleavage were determined over a range of protein concentrations (0.1–10 mg/ml). Comparable processing rates were observed at each protein concentration. Similarly, the addition of mature HpGT to unprocessed protein at various ratios (1:10, 1:1, and 10:1) did not impact the rate of precursor maturation (data not shown). These observations strongly support an autocatalytic intramolecular maturation mechanism. The pH dependence of HpGT processing was also examined and found to be most efficient in the neutral pH range (data not shown). Because of the inherent uncertainty in the densitometric measurements and prolonged incubation times, considerable variability in rates was observed. Most notably, reliable rates for processing could not be determined below pH 5.0 and above pH 9.0, as incomplete maturation of the enzyme was observed. At these extreme pH values, HpGT was prone to aggregation as judged by dynamic light scattering experiments. Determination of the Oligomeric State of HpGT—To examine the quaternary structure of HpGT, the apparent molecular mass of the native enzyme was determined by gel filtration (Fig. 2). Compared with molecular mass standards, mature HpGT exhibited an estimated molecular mass of ∼90 kDa, suggesting that the enzyme is either an extended heterodimer (αβ, 60 kDa) or a compact heterotetramer (α2β2, 120 kDa). To differentiate between these two possibilities, dynamic light scattering experiments were performed. Measurements of mature HpGT indicated a molecular mass between 109 and 122 kDa, suggesting that the enzyme is an α2β2-heterotetramer. Although SDS-PAGE analysis suggested equivalent concentrations of 20- and 40-kDa subunits, the possibility of an unequal combination of α- and β-subunits in the mature enzyme, such as α1β2 or α2β1, was excluded by studies with the unprocessed precursor and an HpGT mutant incapable of forming the α- and β-subunits (T380A; discussed below). Both of these proteins ran nearly identically to mature HpGT on the gel filtration column, and both had comparable molecular masses (110–120 kDa) as judged by dynamic light scattering experiments. Kinetic Characterization of HpGT—A coupled assay system was used to detect the release of glutamate from potential physiological substrates. HpGT exhibited a specific activity of 3.59 ± 0.17 μmol of glutathione hydrolyzed per min/mg of enzyme. Similar experiments were performed using glutamine as the substrate, and a comparable rate of hydrolysis (2.77 ± 0.04 μmol of glutamine hydrolyzed per min/mg of enzyme) was observed. Cleavage of the γ-glutamyl bond in both glutathione and glutamine was verified by mass spectrometry. The coupled assay system is cumbersome, and therefore, most studies of γGT have employed the substrate analog GNA. To obtain kinetic constants (Km and Vmax) for the artificial substrate, the steady-state release of 4-nitroaniline was monitored by its absorbance at 412 nm. Measurements to determine the dependence of the reaction on substrate concentration were done using purified HpGT incubated with increasing concentrations of GNA. Saturation kinetics were observed, and data were fit to the Michaelis-Menten equation to obtain Km and Vmax for the enzyme-catalyzed reaction (Table 1). The apparent Km for GNA was 12.5 ± 1.2 μm, an
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