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L-Methionine sulfoximine is a substrate of L-amino acid oxidase (Crotalus adamanteus), glutamine transaminase, and gamma-cystathionase. In the reaction catalyzed by L-amino acid oxidase, methionine sulfoximine is converted to aplph-imino-gamma-methylsulfoximinylbutyrate, which undergoes rapid gamma elimination yielding methane sulfinimide and 2-imino-3-butenoic acid. Methane sulfinimide is converted to methane sulfonamide, methane sulfinic acid, and methane sulfonic acid; 2-imino-3-butenoic acid is hydrolyzed to vinylglyoxylate, which polymerizes to an insoluble product. When the reaction is carried out in the presence of semicarbazide, the imine formed initially is quantitatively trapped as alpha-keto-gamma-methylsulfoximinylbutyrate semicarbazone, from which the free alpha-keto acid may be obtained. When the reaction is carried out in the presence of a mercaptan (RSH), a gamma exchange reaction occurs leading to formation of a new alpha-keto acid substituted in the gamma position by an SR-group; thus, alpha-keto-gamma-(beta-hydroxyethiol)butyric acid (S-(hydroxyethyl)-2-keto-4-mercaptobutyric acid) was obtained when L-methionine sulfoximine was oxidized in the presence of 2-mercaptoethanol, and enzymatic transamination of this alpha-keto acid with L-glutamine gave the new amino acid, L-omega-hydroxyethionine. The reaction of L-methionine sulfoximine with gamma-cystathionase yields 1 mol each of alpha-ketobutyrate and methane sulfinimide; the latter is hydrolyzed almost exclusively to methane sulfinic acid. Transamination of L-methionine sulfoximine yields the corresponding alpha-keto acid (alpha-keto-gamma-methylsulfoximinylbutyrate), which is stable. Some of these reactions may occur in vivo, and thus contribute to the toxicity of L-methionine sulfoximine.

Active-site ligand interactions with dodecameric glutamine synthetase from Escherichia coli have been studied by calorimetry and fluorometry using the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate (AMP-PNP), L-glutamate, L-Met-(S)-sulfoximine, and the transition-state analogue L-Met-(S)-sulfoximine phosphate. Measurements were made with the unadenylylated enzyme at pH 7.1 in the presence of 100 mM KCl and 1.0 mM MnCl2, under which conditions the two catalytically essential metal ion sites per subunit are occupied and the stoichiometry of active-site ligand binding is equal to 1.0 equiv/subunit. Thermodynamic linkage functions indicate that there is strong synergism between the binding of AMP-PNP and L-Met-(S)-sulfoximine (delta delta G' = -6.4 kJ/mol). In contrast, there is a small antagonistic effect between the binding of AMP-PNP and L-glutamate (delta delta G' = +1.4 kJ/mol). Proton effects were negligible (less than or equal to 0.2 equiv of H+ release or uptake/mol) for the different binding reactions. The binding of AMP-PNP (or ATP) to the enzyme is entropically controlled at 303 K with delta H = +5.4 kJ/mol and delta S = +150 J/(K.mol). At 303 K, the binding of L-glutamate (delta H = -22.2 kJ/mol) or L-Met-(S)-sulfoximine [delta H = -45.6 kJ/mol with delta Cp approximately equal to -670 +/- 420 J/(K.mol)] to the AMP-PNP.Mn.enzyme complex is enthalpically controlled with opposing delta S values of -29 or -46 J/(K.mol), respectively. The overall enthalpy change is negative and the overall entropy change is positive for the simultaneous binding of AMP-PNP and L-glutamate or of AMP-PNP and L-Met-(S)-sulfoximine to the enzyme. For the binding of the transition-state analogue L-Met-(S)-sulfoximine phosphate (which inactivates the enzyme by blocking active sites), both enthalpic and entropic contributions also are favorable at 303 K [delta G' approximately equal to -109 and delta H = -54.8 kJ/mol of subunit and delta S approximately equal to +180 J/(K.mol)].

Electron paramagnetic resonance (EPR) was used to study the environment of Mn2+ bound to the tight (n1) metal ion binding site of glutamine synthetase in the presence of analogues of the tetrahedral adduct, L-methionine (S)-sulfoximine [Met(O)(NH)-S] and L-methionine (R)-sulfoximine [Met(O)(NH)-R]. The Mn2+ EPR spectrum in the presence of Met(O)(NH)-S is identical with the previously published spectrum obtained from a mixture of isomers [Met(O)(NH)-RS] [Villafranca, J. J., Ash, D. E., & Wedler, F. C. (1976) Biochemistry 15, 544] and is characteristic of a highly octahedral metal ion environment with a small zero field splitting. The presence of Met(O)(NH)-R produces an EPR spectrum that appears characteristic of a more distorted metal ion environment, with a larger zero field splitting. These data demonstrate that the two isomers interact differently with the enzyme-bound Mn2+. Broadening of the Mn2+ EPR spectrum in the presence of Met(O)(NH) is observed in 17O-enriched water due to superhyperfine coupling of water to the metal ion. Deconvolution of the spectrum demonstrates the presence of at least a single water molecule in the inner coordination sphere of the metal ion. Superhyperfine coupling due to the 14N nucleus of the imine nitrogen of the sulfoximine moiety of Met(O)(NH)-S but not of Met(O)(NH)-R has been detected by electron spin-echo envelope modulation spectroscopy. Two intense peaks are evident in the presence of Met(O)(NH)-S with frequencies at 1.7 and 3.3 MHz. These peaks are absent when [15N]imine-labeled Met(O)(NH) is used, indicating the presence of the sulfoximine nitrogen of Met(O)(NH)-S in the inner coordination sphere of the metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)

The pKa values of enzyme groups of Escherichia coli glutamine synthetase which affect catalysis and/or substrate binding were determined by measuring the pH dependence of Vmax and V/K. Analysis of these data revealed that two enzyme groups are required for catalysis with apparent pKa values of approximately 7.1 and 8.2. The binding of ATP is essentially independent of pH in the range studied while the substrate ammonia must be deprotonated for the catalytic reaction. Using methylamine and hydroxylamine in place of ammonia, the pKa value of the deprotonated amine substrate as expressed in the V/K profiles was shifted to a lower pKa value for hydroxylamine and a higher pKa value for methylamine. These data indicate that the amine substrate must be deprotonated for binding. Hydroxylamine is at least as good a substrate as ammonia judged by the kinetic parameters whereas methylamine is a poor substrate as expressed in both the V and V/K values. Glutamate binding was determined by monitoring fluorescence changes of the enzyme and the data indicate that a protonated residue (pKa = 8.3 +/- 0.2) is required for glutamate binding. Chemical modification by reductive methylation with HCHO indicated that the group involved in glutamate binding most likely is a lysine residue. In addition, the Ki value for the transition state analog, L-3-amino-3-carboxy-propanesulfonamide was measured as a function of pH and the results indicate that an enzyme residue must be protonated (pKa = 8.2 +/- 0.1) to assist in binding. A mechanism for the reaction catalyzed by glutamine synthetase is proposed from the kinetic data acquired herein. A salt bridge is formed between the gamma-phosphate group of ATP and an enzyme group prior to attack by the gamma-carboxyl of glutamate on ATP to form gamma-glutamyl phosphate. The amine substrate subsequently attacks gamma-glutamyl phosphate resulting in formation of the tetrahedral adduct before phosphate release. A base on the enzyme assists in the deprotonation of ammonia during its attack on gamma-glutamyl phosphate or after the protonated carbinol amine is formed. Based on the kinetic data with the three amine substrates, catalysis is not rate-limiting through the pH range 6-9.

In rats, the glutathione content of the gastrointestinal mucosa amounted to 50-60% of that of the liver. The GSH-S-transferase activity towards an aryl substrate (CDNB) was low in the stomach, colon and rectum, i.e. 5% of hepatic activity. In the small intestine there was a typical decline of activity from proximal to distal segments. GSH-Peroxidase was much lower in the intestinal mucosa as compared to the stomach and liver, whereas the GSSG-reductase was 2-3 times higher in the gastrointestinal tract in comparison to the liver. Fasting for 24 h significantly decreased the GSH content, GSH-aryltransferase and GSSG-reductase activities in the liver but not in the intestine, where even higher GSH concentrations were found in the proximal segments. L-Buthionine-sulfoximine, an inhibitor of the GSH-synthesis, caused a marked decrease of the GSH levels in the liver, stomach, proximal small intestine, colon and rectum and a concomitant decline in GSSG-reductase activity. Among the GSH-depleting agents, paracetamol exerted the strongest effect, whereas 1,1-dichloroethylene and phorone only decreased the GSH content in the liver and stomach.

Active site ligand interactions with dodecameric glutamine synthetase from Escherichia coli were studied spectrally, using the resolved L-S- and L-R-diastereoisomers of the substrate analog L-methionine-SR-sulfoximine. direct measurements of the reversible binding of the S-isomer to unadenylylated manganese-enzyme show a stoichiometry of 1 eq/subunit and negative cooperativity with a Hill coefficient of 0.7. The affinity of this enzyme complex is greatest for the S-isomer alone ([S]0.5 = 35 microM), least with the R-isomer alone ([S]0.5 = 0.38 mM), and intermediate (but closer to that for the S-isomer) for an equimolar mixture of S- and R-isomers ([S]0.5 = 61 microM). The affinity for the S-isomer is enhanced greater than 35-fold by ADP and is decreased approximately 3-fold by adenylylation of the enzyme. Shrake, A., Whitley, E. J. Jr., and Ginsburg, A. ((1980) J. Biol. Chem. 255, 581-589) reported that UV spectral perturbations markedly differ for binding commercial L-methionine-SR-sulfoximine to unadenylylated and adenylylated manganese enzymes. However, essentially the same saturating protein difference spectrum is produced by binding the resolved S- and R-diastereoisomers, and equimolar mixture of S- and R-isomers, and the commercial S- and R-isomeric mixture to a particular enzyme complex. Since neither the subunit interactions that give rise to the observed negative cooperativity of binding nor the affinity differences in binding the S- and R-isomers are reflected in protein difference spectra, spectral perturbations derive from a conformational change that is solely a marked for the occupancy of the single subunit site by either isomer.

It was observed previously that hydroxyguanidine is formed in the reaction of canavanine(2-amino-4-guanidinooxybutanoate) with amino acid oxidases. The present work shows that hydroxyguanidine is formed by a nonenzymatic beta,gamma-elimination reaction following enzymatic oxidation at the alpha-C and that the abstraction of the beta-H is general-base catalyzed. The elimination reaction requires the presence in the alpha-position of an anion-stabilizing group--the protonated imino group (iminium ion group) or the carbonyl group. The iminium ion group is more activating than the carbonyl group. Elimination is further facilitated by protonation of the guanidinooxy group. The other product formed in the elimination reaction was identified as vinylglyoxylate (2-oxo-3-butenoate), a very highly electrophilic substance. The product resulting from hydrolysis following oxidation was identified as alpha-keto-gamma-guanidinooxybutyrate (ketocanavanine). The ratio of hydroxyguanidine to ketocanavanine depended upon the concentration and degree of basicity of the basic catalyst and on pH. In the presence of semicarbazide, the elimination reaction was prevented because the imino group in the semicarbazone derivative of ketocanavanine is not significantly protonated. Incubation of canavanine with 5'-deoxypyridoxal also yielded hydroxyguanidine. Since the elimination reactions take place under mild conditions, they may occur in vivo following oxidation at the alpha-C of L-canavanine (ingested or formed endogenously) or of other amino acids with a good leaving group in the gamma-position (e.g., S-adenosylmethionine, methionine sulfoximine, homocyst(e)ine, or cysteine-homocysteine mixed disulfide) by an L-amino acid oxidase, a transaminase, or a dehydrogenase. Therefore, vinylglyoxylate may be a normal metabolite in mammals which at elevated concentrations may contribute to the in vivo toxicity of canavanine and of some of the other above-mentioned amino acids.

Pseudomonas tabaci produces a toxin, tabtoxin, that causes wildfire disease in tobacco. The primary target of tabtoxin is presumed to be glutamine synthetase. Some effects of tabtoxin in tobacco can be mimicked by methionine sulfoximine (MSO), a compound that is known to inactivate glutamine synthetase. To understand how organisms can be made resistant to tabtoxin and MSO, we used Saccharomyces cerevisiae. We demonstrate that yeast strains carrying the glutamine synthetase gene, GLN1, on a multicopy plasmid overproduced glutamine synthetase and showed increased drug resistance. These and other data indicate that glutamine synthetase is the primary target of tabtoxin and MSO in S. cerevisiae. We also isolated three S. cerevisiae DNA inserts of 2.1, 2.3, and 2.8 kilobases that conferred tabtoxin and MSO resistance when the inserts were present on a multicopy plasmid. These plasmids conferred resistance to MSO by blocking intracellular transport of the drug. Transport appeared to occur by one or more methionine permeases. Resistance to tabtoxin could also occur by blockage of intracellular transport, but the drug was transported by some permease other than a methionine permease. These drug resistance plasmids did not block transport of citrulline, indicating that they did not affect the general amino acid permease.

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.

Previous data have demonstrated that methyl chloride (MeCl) is toxic to B6C3F1 mice under both acute and chronic exposure conditions, and that conjugation of MeCl with glutathione (GSH) is a key step in the metabolism of MeCl. This study examined the role of GSH in mediating the acute toxicity of MeCl to liver, kidney, and brain of male B6C3F1 mice. The lethal effects of a single 6-hr inhalation exposure of B6C3F1 males to 2500 ppm MeCl were completely prevented by pretreatment with the GSH synthesis inhibitor, L-buthionine-S,R-sulfoximine (4 mmol L-BSO/kg, ip 1.5 hr prior to MeCl exposure). GSH levels (measured as nonprotein sulfhydryl) in liver and kidney were depleted to 19 and 25% of control values, respectively, at the start of the exposure; the ratio of dead/exposed mice during the 18-hr postexposure declined from 14/15 mice to 0/10. Also, the LC50 for MeCl increased from 2200 to 3200 ppm in male mice pretreated with BSO. The hepatic toxicity of MeCl was detected by increased alanine aminotransferase (ALT) activities in serum 18 hr after a 6-hr exposure to 1500 ppm MeCl (2147 +/- 1327 IU/liter vs 46 +/- 6 in controls). Liver toxicity was inhibited when B6C3F1 males were depleted of GSH prior to MeCl exposure by BSO pretreatment (43 +/- 2), fasting (100 +/- 47), or injection of diethyl maleate (42 +/- 16). The effects of GSH depletion on MeCl toxicity to brain and kidney were determined in B6C3F1 males exposed to 1500 ppm MeCl 6 hr/day, 5 days/week for 2 weeks, with and without daily pretreatment with 2 mmol L-BSO/kg. This dose of BSO depleted hepatic and renal GSH by 28 and 60%, respectively, at the start of MeCl exposure. BSO-pretreated mice were protected from the central nervous system toxicity of MeCl, as assessed by microscopic examination of the granule cell layer of the cerebellum. BSO pretreatment also inhibited the renal toxicity of MeCl as measured by incorporation of [3H]thymidine ([3H]TdR) into renal DNA, an indicator of cell regeneration after cortical necrosis. [3H]TdR incorporation was 105 +/- 10,337 +/- 40, and 60 +/- 15 dpm/microgram DNA in nonexposed controls, MeCl, and MeCl + BSO treatment groups, respectively. These results indicate that GSH is an important component in the toxicity of MeCl to multiple organ systems in B6C3F1 mice. Reaction of MeCl with GSH appears to constitute a mechanism of toxication, contrary to the role usually proposed for GSH in detoxifying xenobiotics.

A mutant of Escherichia coli defective in thioredoxin reductase has been isolated and partially characterized. This mutant has no detectable thioredoxin reductase activity in vitro and yet it exhibits no in vivo defect in reduction of ribonucleotides. Evidence is presented that indicates that, in cells permeabilized via ether treatment, ribonucleoside diphosphate reduction can utilize glutathione as an alternate reducing system.