Mapping of the active site of rat kidney gamma-glutamyl transpeptidase using activated esters and their amide derivatives

The enzyme gamma-glutamyl transpeptidase (GGT), implicated in many physiological processes, catalyses the transfer of a gamma-glutamyl from a donor substrate to an acyl acceptor substrate, usually an amino acid or a peptide. In order to investigate which moieties of the donor substrate are necessary for recognition by GGT, the structure of the well-recognized substrate L-gamma-glutamyl-p-nitroanilide was modified. Several activated esters and their amide derivatives were synthesized and used as substrates. Kinetic (K(m) and V(max)) and inhibition constants (K(i)) were measured and reveal that almost the entire gamma-glutamyl moiety is necessary for recognition in the binding site of the donor substrate. The implied presence of certain complementary amino acids in this substrate binding site will allow the more rational design of various substrate analogues and inhibitors.     

Human kidney gamma-glutamyl transpeptidase. Catalytic properties, subunit structure, and localization of the gamma-glutamyl binding site on the light subunit.

Human kidney gamma-glutamyl transpeptidase has been purified by a procedure involving Lubrol extraction, acetone precipitation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-150. The final preparation is a glycoprotein (molecular weight of approximately 84,000) composed of two nonidentical glycopeptides (molecular weights of 62,000 and 22,000). The isozymic forms, separable by isoelectric focusing, have different contents of sialic acid. The utilization of L-glutamine (which is both a gamma-glutamyl donor and acceptor) is stimulated about 3-fold by maleate in contrast to 10-fold stimulation of glutamine utilization by the rat kidney enzyme. The gamma-glutamyl analogs, 6-diazo-5-oxo-L-norleucine (DON) and L-azaserine inactivate the human kidney enzyme with respect to its transpeptidase and hydrolase activities. Inactivation is prevented by gamma-glutamyl substrates (but not by acceptor substrates) and is accelerated by maleate. [14C]DON reacts covalently and stoichiometrically at the gamma-glutamyl site, which was localized to the light subunit of the enzyme. The light subunit of human transpeptidase closely resembles that of rat kidney enzyme in having the gamma-glutamyl binding site, and similar molecular weight and amino acid composition. The heavy subunits of the two enzymes are markedly different in both molecular weight and amino acid content; this may account for differences observed in acceptor amino acid specificity and in the magnitude of the maleate effect.     

Induction of gamma-glutamyl transpeptidase by hexachlorocyclohexane

Hexachlorocyclohexane (BHC) induced gamma-Glutamyl transpeptidase in rat liver. The enzyme was partially purified from normal BHC fed and fetal liver and also from hepatoma. The gel filtration and electrophoretic properties of the BHC-induced enzyme was compared against that of the other three. Chemical induced hepatoma showed an additional peak of activity in Sephadex G-200 filtration. The other enzymes could be cleaved by papain to give a fraction which cochromatographed with the additional peak of hepatoma enzyme. BHC-induced enzyme and normal enzyme had similar electrophoretic mobility but differed from that of hepatoma and fetal liver enzyme which showed a slightly slower movement.     

Slow-binding inhibition of gamma-glutamyl transpeptidase by gamma-boroGlu

gamma-Glutamyl transpeptidase (gammaGTase) catalyzes the transfer of the gamma-glutamyl moiety of gamma-glutamyl-derived peptides, such as glutathione (gammaGlu-Cys-Gly), and anilides, such as gamma-glutamyl-7-amido-4-methylcoumarin (gammaGlu-AMC), to acceptor molecules, including water and various dipeptides. These acyl-transfer reactions all occur through a common acyl-enzyme intermediate formed from attack of an active site hydroxyl on the gamma-carbonyl carbon of gammaGlu-X with displacement of X. In this paper, we report that gammaGTase is potently inhibited by the gamma-boronic acid analogue of L-glutamic acid, 3-amino-3-carboxypropaneboronic acid (gamma-boroGlu). We propose that gamma-boroGlu adds to the active site hydroxyl of gammaGTase to form a covalent, tetrahedral adduct that resembles tetrahedral transition states and intermediates that occur along the reaction pathway for gammaGTase-catalyzed reactions. Our studies demonstrate that gamma-boroGlu is a competitive inhibitor of the gammaGTase-catalyzed hydrolysis of gammaGlu-AMC with a K(i) value of 35 nM. Kinetics of inhibition studies allow us to estimate the following values: k(on) = 400 mM(-1) s(-1) and k(off) = 0.02 s(-1). We also found that gamma-boroGlu is an uncompetitive inhibitor of Gly-Gly-promoted transamidation of gammaGlu-AMC. This observation is consistent with the kinetic mechanism we determined for gammaGTase-catalyzed transamidation of gammaGlu-AMC by Gly-Gly to form gammaGlu-Gly-Gly. To probe rate-limiting transition states for gammaGTase catalysis and inhibition, we determined solvent deuterium isotope effects. Solvent isotope effects on k(c)/K(m) for hydrolysis of gammaGlu-AMC and k(on) for inhibition by gamma-boroGlu are identical and equal unity, suggesting that the processes governed by these rate constants are both rate-limited by a step that is insensitive to solvent deuterium such as a conformational fluctuation of the initially formed E-S or E-I complex. In contrast, the solvent isotope effect on k(c) is 2.4. k(c) is rate-limited by hydrolysis of the acyl-enzyme intermediate that is formed during reaction of gammaGTase with gammaGlu-AMC. Thus, the magnitude of this isotope effect suggests the formation of a catalytically important protonic bridge in the rate-limiting transition state for deacylation.     

Modulation of gamma-glutamyl transpeptidase activity by bile acids

The free bile acids (cholate, chenodeoxycholate, and deoxycholate) stimulate the hydrolysis and transpeptidation reactions catalyzed by gamma-glutamyl transpeptidase, while their glycine and taurine conjugates inhibit both reactions. Kinetic studies using D-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor indicate that the free bile acids decrease the Km for hydrolysis and increase the Vmax; transpeptidation is similarly activated. The conjugated bile acids increase the Km and Vmax of hydrolysis and decrease both of these for transpeptidation. This mixed type of modulation has also been shown to occur with hippurate and maleate (Thompson, G.A., and Meister, A. (1980) J. Biol. Chem. 255, 2109-2113). Glycine conjugates are substantially stronger inhibitors than the taurine conjugates. The results with free cholate indicate the presence of an activator binding domain on the enzyme with minimal overlap on the substrate binding sites. In contrast, the conjugated bile acids, like maleate and hippurate, may overlap on the substrate binding sites. The results suggest a potential feedback role for bile ductule gamma-glutamyl transpeptidase, in which free bile acids activate the enzyme to catabolize biliary glutathione and thus increase the pool of amino acid precursors required for conjugation (glycine directly and taurine through cysteine oxidation). Conjugated bile acids would have the reverse effect by inhibiting ductule gamma-glutamyl transpeptidase.     

Kinetics of the conversion of leukotriene C by gamma-glutamyl transpeptidase

γ-Glutamyl transpeptidase (EC 2.3.2.2) converts leukotriene C to leukotriene D by removal of a glutamyl residue. The Michaelis constant for leukotriene C₄ hydrolysis was found to be 5.6 μM. Under the same conditions the K_m value for hydrolysis of reduced glutathione was 5.7 μM. This suggests that leukotriene C₄ and glutathione may be competing substrates for γ-glutamyl transpeptidase under physiological conditions. The apparent K_I for inhibition of leukotriene C₄ hydrolysis by equimolar amounts of L-serine and sodium borate was 0.8 mM.     

Inhibition of the hydrolytic and transpeptidase activities of rat kidney gamma-glutamyl transpeptidase by specific monoclonal antibodies.

Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.     

Duplication of the bcr and gamma-glutamyl transpeptidase genes.

The Philadelphia (Ph') translocation involves rearrangement of the bcr gene located on chromosome 22. Hybridization experiments revealed the presence of multiple bcr gene-related loci within the human genome. Two of these were molecularly cloned and characterized. Both loci contain exons and introns corresponding to the 3' region of the bcr gene. Restriction enzyme and DNA sequence analysis indicate a very high degree of conservation between bcr and the two related genomic sequences. Both bcr-related loci are located on chromosome 22, one centromeric, the other telomeric, of the bcr gene. Within the two bcr related genomic sequences, fragments or the complete coding sequences of an unrelated gene were found to be present. This gene was identified; it encodes gamma-glutamyl transferase, an enzyme involved in the glutathione metabolism.     

Regulation of testicular and Sertoli cell gamma-glutamyl transpeptidase by follicle-stimulating hormone

Hormonal deprivation achieved by hypophysectomy or gonadotropin-releasing hormone (GnRH)-antagonist treatment of immature rats resulted in markedly lower testicular gamma-glutamyl transpeptidase (GGT) activity than in the testes of age-matched controls. When begun 15 days after hypophysectomy, follicle-stimulating hormone (FSH) treatment significantly increased testicular GGT above that in testes from hypophysectomized controls in a time- and dose-dependent manner. In contrast, testosterone propionate had only a small effect. Testicular GGT was higher in adult hypophysectomized rats treated with FSH from the time of surgery than in untreated hypophysectomized rats; testosterone propionate treatment had no effect. GGT activity in Sertoli cells isolated from GnRH antagonist-treated or hypophysectomized immature rats was also lower than in cells from control rats. FSH treatment from the day of hypophysectomy resulted in Sertoli cell GGT values equivalent to those from intact controls. These data indicate that FSH regulates GGT activity in rat testis and Sertoli cells.