Alton Meister’s research while affiliated with Osaka City University and other places

The serine residue required for catalysis of gamma-glutamyl transpeptidase was identified by site-specific mutagenesis of the conserved serine residues on the basis of sequence alignment of the light subunit of human, rat, pig and two bacterial enzymes. Recombinant human gamma-glutamyl transpeptidases with replacements of these serine residues by Ala were expressed using a baculovirus-insect cell system. Substitutions of Ala at Ser-385, -413 or -425 yielded almost fully active enzymes. However, substitutions of Ala at Ser-451 or -452 yielded enzymes that were only about 1% as active as the wild-type enzyme. Further, their double mutant is only 0.002% as active as the wild type. Kinetic analysis of transpeptidation using glycylglycine as acceptor indicates that the Vmax values of Ser-451 and -452 mutants are substantially decreased (to about 3% of the wild type); however, their Km values for L-gamma-glutamyl-p-nitroanilide as donor were only increased about 5 fold compared to that of the wild type. The double mutation of Ser-451 and -452 further decreased the Vmax value to only about 0.005% of the wild type, while this mutation produced only a minor effect (2-fold increase) on the Km value for the donor. The kinetic values for the hydrolysis reaction of L-gamma-glutamyl-p-nitroanilide in the mutants followed similar trends to those for transpeptidation. The rates of inactivation of Ser-451, -452 and their double mutant enzymes by acivicin, a potent inhibitor, were less than 1% that of the wild-type enzyme. The Ki value of the double mutant for L-serine as a competitive inhibitor of the gamma-glutamyl group is only 9 fold increased over that of the wild type, whereas the Ki for the serine-borate complex, which acts as an inhibitory transition-state analog, was more than 1,000 times higher than for the wild-type enzyme. These results suggest that both Ser-451 and -452 are located at the position able to interact with the gamma-glutamyl group and participate in catalysis, probably as nucleophiles or through stabilization of the transition state.

Glutathione deficiency produced by giving buthionine sulfoximine (an inhibitor of gamma-glutamylcysteine synthetase) to animals, leads to biphasic decline in cellular glutathione levels associated with sequestration of glutathione in mitochondria. Liver mitochondria lack the enzymes needed for glutathione synthesis. Mitochondrial glutathione arises from the cytosol. Rat liver mitochondria have a multicomponent system (with Kms of approx. 60 microM and 5.4 mM) that underlies their remarkable ability to transport and retain glutathione. Mitochondria produce substantial quantities of reactive oxygen species; this is opposed by reactions involving glutathione. Glutathione deficiency leads to widespread mitochondrial damage which is lethal in newborn rats and guinea pigs, animals that do not synthesize ascorbate. Glutathione esters and ascorbate protect against the lethal and other effects of glutathione deficiency. Ascorbate spares glutathione; it increases mitochondrial glutathione in glutathione-deficient animals. Glutathione esters delay onset of scurvy in ascorbate-deficient guinea pigs; thus, glutathione spares ascorbate. Glutathione and ascorbate function together in protecting mitochondria from oxidative damage.

Mutant human γ-glutamyl transpeptidases with amino acid substitutions on the light subunit at the Asp residues conserved among several species, and at the unique cysteine residue (Cys-454), were prepared and expressed in a baculovirus insect cell system. Replacement of Asp-423 by Ala or Glu led to major loss of enzyme activity, consistent with the conclusion that Asp-423 is essential for activity. A mutant in which Cys-454 was replaced by Ala was fully active, indicating that the unique light subunit thiol is not required for catalysis. Kinetic analysis of the hydrolysis reaction of L-γ-glutamyl-p-nitroanilide indicated that the decreased activity of Asp-423 mutants is the consequence of an extremely high substrate Km value, which is more than a 1000-fold greater than that for the wild-type enzyme, whereas the Vmax is decreased only less than 90-fold. The results suggest that Asp-423, and to a lesser extent Asp-422, interact electrostatically with the α-amino group of the γ-glutamyl donor substrate. Although further studies are required to evaluate the possibility that the reaction involves function of a charge (or proton) relay system, the present work suggests that the γ-glutamyl moiety of the substrate binds electrostatically to specific groups on the enzyme; this facilitates γ-glutamyl enzyme formation.

γ-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 γ-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 γ-glutamylhydroxamate. Stabilization of the γ-glutamyl-enzyme appears to be associated with acetylation of lysine residues (including Lys-99). These and other findings suggest that the α-amino group of the γ-glutamyl substrate is linked electrostatically to Asp-422 so as to facilitate reaction of the γ-carbonyl of the substrate with an enzyme hydroxyl group to form a γ-glutamyl-enzyme.

The efficiency of L-2-oxothiazolidine-4-carboxylate, a cysteine precursor, in stimulating glutathione synthesis and growth was evaluated in growing rats. Animals were fed a sulfur amino acid-deficient diet (0.25% L-methionine and no cysteine) supplemented with L-2-oxothiazolidine-4-carboxylate (0.35%) for 3 wk and compared with age-matched animals receiving the sulfur amino acid-deficient diet alone. Rats fed the sulfur amino acid-deficient diet had lower glutathione concentrations in bronchoalveolar lining fluid, lung, lymphocytes, and liver than rats fed a sulfur amino acid-deficient diet supplemented with L-2-oxothiazolidine-4-carboxylate. Rats fed the supplemented diet had normal tissue and bronchoalveolar lining fluid glutathione levels. Central venous plasma glutathione concentrations, mostly reflecting liver excretion, were less affected by L-2-oxothiazolidine-4-carboxylate supplementation. Rats fed L-2-oxothiazolidine-4-carboxylate supplementation had normal weight gain compared with a much lower weight gain in animals fed the sulfur amino acid-deficient diet alone. Thus, L-2-oxothiazolidine-4-carboxylate increased tissue glutathione concentrations and stimulated growth in rats. The lung glutathione status of the rats was reflected by glutathione concentrations in lymphocytes and the bronchoalveolar lining fluid, but not by the central venous plasma glutathione concentrations.

This chapter focuses on the Glutathione Biosynthesis and its inhibition. Glutathione (GSH) is synthesized in two steps that are catalyzed, respectively, by γ-glutamylcysteine synthetase (glutamate-cysteine ligase) and GSH synthetase. The reaction catalyzed by T-glutamylcysteine synthetase is feedback inhibited by GSH, and thus the enzyme functions normally at less than its maximal rate. Inhibition by GSH is not allosteric, but is competitive with respect to glutamate. Inhibition is associated with binding of the glutamyl moiety of GSH to the glutamate binding site of the enzyme, and there is evidence that the thiol moiety of GSH binds to another site on the enzyme, possibly to the cysteine binding site. Studies on the mapping of the active site of glutamine synthetase led to the conclusion that the S-methyl group of methionine sulfoximine binds to the ammonia binding site of the enzyme. Sulfoximines with larger S-alkyl moieties were found not to inhibit glutamine synthetase significantly because they are hindered from binding to the ammonia binding site of this enzyme. However, such compounds effectively bind to and inactivate γ-glutamylcysteine synthetase. Thus, Buthionine sulfoximine (BSO) does not inhibit glutamine synthetase significantly and thus decreases GSH levels without affecting the synthesis of glutamine (and does not produce convulsions).

gamma-Glutamyl transpeptidase, a highly glycosylated heterodimeric enzyme that is usually attached to the external surface of cell membranes, is of major importance in the metabolism of glutathione. The enzyme, which has been isolated from many animal sources, contains a large amount of carbohydrate, which is linked to both protein subunits. Previous work has not shown whether such carbohydrate is needed for enzyme activity nor indicated its functional role. Notably, gamma-glutamyl transpeptidase isolated from Escherichia coli, which exhibits about 80% amino acid sequence homology with the rat enzyme, has only about 0.1% of its specific enzymatic activity and is not glycosylated. Here we treated the highly glycosylated gamma-glutamyl transpeptidases isolated from rat and pig kidneys with a mixture of glycosidases and then separated two completely active gamma-glutamyl transpeptidase fractions from each species. One fraction was completely devoid of carbohydrate and was fully active as compared with the respective isolated enzymes, but differed in solubility and stability. The other fraction, which contained 10-20% of the initially bound carbohydrate, exhibited a marked increase in susceptibility to proteases. The oligosaccharide chains of gamma-glutamyl transpeptidase may protect against protease action (including self-destruction by the inherent protease activity of the light subunit) during synthesis of the active enzyme from its single chain precursor, as well as after enzyme synthesis.

This chapter discusses the preparation and use of glutathione monoesters. Glutathione monoesters are useful glutathione delivery agents because they are readily transported into many types of cells and split intracellularly to form glutathione. Treatment of cells (in suspension or culture) and of intact animals with glutathione itself may lead to some increase in cellular glutathione levels; such increases are usually caused by extracellular breakdown of glutathione into its constituent amino acids and into dipeptides and the transport of these into cells followed by intracellular glutathione synthesis. Studies in which glutathione synthesis is completely inhibited, for example, by treatment with buthionine sulfoximine, show that administration of glutathione leads to little increase in cellular glutathione levels. Glutathione monoesters are transported into many rodent tissues, including kidney, liver, pancreas, spleen, skeletal muscle, heart, lung, and, in neonatal animals, the lens and the brain. Monoesters of glutathione are also transported into human red blood cells, lymphocytes, and fibroblasts.

This chapter discusses the preparation and properties of glutathione diethyl ester and related derivatives. Glutathione diethyl ester, like glutathione mono(glycyl) esters, is an effective glutathione delivery compound. When mice are injected intraperitoneally with glutathione diethyl ester, the tissue levels of glutathione increase to about the same extent as found after injection of glutathione monoethyl ester. Essentially equivalent effects are found after administration of the monoester and the diester is explained by the finding that mouse blood plasma contains appreciable amounts of glutathione diester α-esterase, which rapidly converts the diester to the monoester. On the other hand, human plasma does not have this esterase; for this reason, glutathione diethyl ester is stable in human blood and serves as a cellular glutathione delivery agent for human cells. Studies indicate that glutathione diethyl ester is effectively transported into human cells. It is evident that neither rats nor mice are good experimental models for studies on glutathione diester. However, studies on hamsters, guinea pigs, rabbits, and sheep have shown that plasma from these species lacks glutathione diethyl ester α-esterase; thus, these animals are potentially useful as experimental models for studies on the in vivo disposition of glutathione diethyl ester.