DL-threo-4-fluoroglutamic acid. A chain-terminating inhibitor of folylpolyglutamate synthesis

The effects of 4-fluoroglutamate on the reaction catalyzed by partially purified rat liver folylpolyglutamate synthetase have been investigated. DL-threo-4-Fluoroglutamate was an effective, concentration-dependent inhibitor of polyglutamylation of both tetrahydrofolate and methotrexate, while the erythro isomer was weakly inhibitory. 4-Fluoroglutamate acted as an alternate substrate; the DL-threo isomer was incorporated only slightly less effectively than L-glutamate, while the erythro isomer was poorly incorporated. The resulting product, a pteroylglutamyl-gamma-(4-fluoro)glutamate, was a very poor substrate for further glutamylation. Thus, when tetrahydrofolate and 4-fluoroglutamate were substrates, the sole Zn/HCl cleavage product co-chromatographed on high performance liquid chromatography with chemically synthesized p-aminobenzoylglutamyl-gamma-(4-fluoro)glutamate. When [3H]methotrexate (4-NH2-10-CH3PteGlu) and 4-fluoroglutamate were the substrates, one product was obtained which co-chromatographed on high performance liquid chromatography with chemically synthesized 4-NH2-CH3PteGlu-gamma-(4-fluoro)glutamate. Further evidence that the product from [3H]methotrexate was a dipeptide came from gamma-glutamyl hydrolase digestion experiments and quantitative amino acid analysis. The appearance of trace amounts of a product having properties consistent with the addition of a second 4-fluoroglutamate occurred only under forcing conditions. The chemically and enzymatically synthesized fluoroglutamate-containing products were at least 15 times poorer than the analogous diglutamyl compound as substrates for rat liver folylpolyglutamate synthetase. These results are consistent with inhibition of polyglutamate synthesis by 4-fluoroglutamate through a "leaky" chain termination mechanism.     

Synthesis and properties of 7-hydroxymethotrexate polyglutamyl derivatives in Ehrlich ascites tumor cells in vitro

The synthesis of poly-gamma-glutamyl derivatives of 7-hydroxymethotrexate (7-OH-4-NH2-10-CH3-pteroyl-glutamic acid (PteGlu1] was evaluated by direct hydroxylation of the tetraglutamyl derivative of methotrexate (4-NH2-10-CH3-PteGlu4) by a cell-free preparation of rabbit liver aldehyde oxidase and by polyglutamylation of 7-OH-methotrexate in Ehrlich ascites tumor cells in vitro. The polyglutamyl derivatives of 7-OH-methotrexate rapidly accumulate in cells to the 7-OH-4-NH2-10-CH3-PteGlu4. While 7-OH-methotrexate monoglutamate does not bind to dihydrofolate reductase, 7-OH-4-NH2-10-CH3-PteGlu4 does bind to the enzyme as established by gel filtration analysis of cell extracts and by use of purified dihydrofolate reductase from Ehrlich cells. Within cells, the rate of formation of 7-OH-methotrexate polyglutamyl derivatives exceeds that for methotrexate by a factor of 2.7 at comparable free monoglutamyl substrate levels, suggesting that 7-OH-methotrexate may be a better substrate than methotrexate for the folylpolyglutamate synthetase. 7-OH-methotrexate slows the rate of methotrexate polyglutamylation in cells, a consequence of the inhibition of methotrexate transport with reduced methotrexate substrate available for polyglutamylation. When 7-OH-methotrexate polyglutamyl derivatives were accumulated inside the cells following which extracellular 7-OH-methotrexate was removed, the monoglutamate, and to a lesser extent the diglutamate, exited the cells whereas the majority of the longer polyglutamyl derivatives were retained and continued to be metabolized to higher forms. These studies suggest that 7-OH-methotrexate and its polyglutamyl derivatives may play a role in modulating methotrexate action, either by their own inhibitory effects on folate-dependent enzymes or by their effects on methotrexate transport and metabolism within cells.     

alpha-Aminomethylglutarate, a beta-amino analog of glutamate that interacts with glutamine synthetase and the enzymes that catalyze glutathione synthesis.

The glutamate analog, alpha-aminomethylglutaric acid, was synthetized by Michael addition of ammonia to 2-methylene glutaronitrile followed by hydrolysis of the intermediate alpha-aminomethylglutaryl nitrile; the analog cyclizes readily on heating to 2-piperidone-5-carboxylic acid. Sheep brain glutamine synthetase utilizes one isomer of DL-alpha-aminomethylglutarate at about 10% of the rate with L-glutamate. gamma-Glutamylcysteine synthetase uses both isomers of DL-alpha-aminomethylglutarate, preferentially acting on the same isomer used by glutamine synthetase. gamma-(alpha-Aminomethyl)glutaryl-alpha-aminobutyrate, prepared enzymatically with gamma-glutamylcysteine synthetase, was found to be a substrate and an inhibitor of glutathione synthetase. alpha-Aminomethylglutarate does not inhibit gamma-glutamyl cyclotransferase and gamma-glutamyl transpeptidase appreciably. When alpha-aminomethylglutarate was administered to mice, there were substantial decreases in the levels of glutamine, glutathione, glutamate, and glycine in the kidney, and of glutamine and glutamate in the liver, indicating that this glutamate analog is effective as an inhibitor of glutamine and glutathione synthesis in vivo, and suggesting that it may also inhibit other enzymes.     

A new synthesis of adenosine 5'-([gamma(R)-17O,18O]-gamma-thiotriphosphate) and its use to determine the stereochemical course of the activation of glutamate by glutamine synthetase

A new synthetic route to adenosine 5'-([gamma(R)-17O,18O]-gamma-thiotriphosphate) is described which combines chemical methods for introducing the heavy oxygen isotopes and enzymic methods for achieving the enantiospecificity. This material was used as a substrate for the activation of glutamate catalyzed by glutamine synthetase from Salmonella typhimurium. Analysis of the chirality of the [16O,17O,18O]thiophosphate produced showed that the reaction proceeds with inversion of configuration on phosphorus. This result, taken together with the positional isotope exchange studies of Midelfort and Rose [Midelfort, C. F., & Rose, I.A. (1976) J. Biol. Chem. 251, 5881-5887], demonstrates that the activation of glutamate to form gamma-glutamyl phosphate proceeds by a direct "in-line" transfer of the phosphoryl group.     

Intermediates of the gamma-glutamyl cycle in mouse tissues. Influence of administration of amino acids on pyrrolidone carboxylate and gamma-glutamyl amino acids.

GAMMA-Glutamyl transpeptidase, gamma-glutamyl cyclotransferase, L-pyrrolidone carboxylate hydrolase, gamma-glutamylcysteine synthetase and glutathione synthetase, the enzymes of the gamma-glutamyl cycle, were found in mouse brain, liver and kidney. The activity of L-pyrrolidone carboxylate hydrolase was many times lower than the activities of the other enzymes, and thus the conversion of L-pyrrolidone carboxylate to L-glutamate is likely to be the rate-limiting step of the cycle. The specificity of gamma-glutamyl cyclotransferase from mouse tissues was similar to that from rat tissues. The concentration of pyrrolidone carboxylate and gamma-glutamyl amino acids, intermediates of the gamma-glutamyl cycle, was determined by a gas chromatographic procedure coupled with electron capture detection. Administration of L-2-aminobutyrate, an amino acid that is utilized as substrate in the reaction catalyzed by gamma-glutamylcysteine synthetase, led to a large accumulation of gamma-glutamyl-2-aminobutyrate and pyrrolidone carboxylate in mouse tissues. L-Methionine-RS-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, abolished the increase in concentration of pyrrolidone carboxylate. No accumulation of pyrrolidone carboxylate was observed after L-cysteine. The separate administration of several protein amino acids had little effect on the concentration of pyrrolidone carboxylate; however formation of small amounts of the corresponding gamma-glutamyl derivatives (e.g. gamma-glutamylmethionine and gamma-glutamylphenylalanine) was detected. These intermediates are probably formed by transpeptidation between glutathione and the corresponding amino acid, catalyzed by gamma-glutamyl transpeptidase. The concentration of pyrrolidone carboxylate increased significantly after administration of a mixture containing all protein amino acids, the highest increase occurring in the kidney. The results suggest that two separate pathways for the formation of gamma-glutamyl amino acids and pyrrolidone carboxylate exist in vivo. One of these results from the function of gamma-glutamylcysteine synthetase in glutathione synthesis. The other pathway involves the amino-acid-dependent degradation of glutathione, mediatedby gamma-glutamyl transpeptidase. Only very small amounts of free intermediates are apparently derived from the latter pathway, suggesting that the gamma-glutamyl amino acids formed in this pathway are either enzyme-bound or are directly hydrolyzed to glutamate and free amino acid.     

Developmental change of endogenous glutamate and gamma-glutamyl transferase in cultured cerebral cortical interneurons and cerebellar granule cells,and in mouse cerebral cortex and cerebellum in vivo

The developmental change of endogenous glutamate, as correlated to that of gamma-glutamyl transferase and other glutamate metabolizing enzymes such as phosphate activated glutaminase, glutamate dehydrogenase and aspartate, GABA and ornithine aminotransferases, has been investigated in cultured cerebral cortex interneurons and cerebellar granule cells. These cells are considered to be GABAergic and glutamatergic, respectively. Similar studies have also been performed in cerebral cortex and cerebellum in vivo. The developmental profiles of endogenous glutamate in cultured cerebral cortex interneurons and cerebellar granule cells corresponded rather closely with that of gamma-glutamyl transferase and not with other glutamate metabolizing enzymes. In cerebral cortex and cerebellum in vivo the developmental profiles of endogenous glutamate, gamma-glutamyl transferase and phosphate activated glutaminase corresponded with each other during the first 14 days in cerebellum, but this correspondence was less good in cerebral cortex. During the time period from 14 to 28 days post partum the endogenous glutamate concentration showed no close correspondence with any particular enzyme. It is suggested that gamma-glutamyltransferase regulates the endogenous glutamate concentration in culture neurons. The enzyme may also be important for regulation of endogenous glutamate in brain in vivo and particularly in cerebellum during the first 14 days post partum. Gamma-glutamyl transferase in cultured neurons and brain tissue in vivo appears to be devoid of maleate activated glutaminase.Abbreviations used Asp-T aspartate aminotransferase (EC 2.6.1.1) - GABA-T GABA aminotransferase (EC 2.6.1.19) - GAD glutamate decarboxylase (EC 4.1.1.15) - gamma-GT gamma-glutamyl transferase (gamma-glutamyl transpeptidase) (EC. 2.3.2.2) - Glu glutamate - GDH glutamate dehydrogenase (EC 1.4.1.3) - GS glutamine synthetase (EC 6.3.1.2) - MAG maleate activated glutaminase - Orn-T ornithine aminotransferase (EC 2.6.1.13) - PAG phosphate activated glutaminase (EC 3.5.1.1)     

In vitro synthesis of alpha-carboxyl-linked folylpolyglutamates by an enzyme preparation from Escherichia coli

Extracts of Escherichia coli contained an enzymatic activity which catalyzed the addition of L-glutamate to the alpha-carboxyl of various polyglutamate substrates, including folylpolyglutamates. Much of the enzyme activity was separated by DE52 chromatography and gel filtration from the enzyme which adds the first three glutamates in the biosynthesis of folylpolyglutamates, dihydrofolate synthetase-folylpolyglutamate synthetase. The two enzyme activities differed in many properties. Whereas dihydrofolate synthetase-folylpolyglutamate synthetase preferred pteroate or pteroylmonoglutamate substrates, the folylpoly-alpha-glutamate synthetase preparations effectively utilized tetrahydropteroylpolyglutamates, pteroylpolyglutamates, p-aminobenzoylpolyglutamates (pAB(Glu)n), and even a polyglutamate tripeptide. Several di- and triglutamyl peptides were inhibitory to folylpoly-alpha-glutamate synthetase activity, but not to dihydrofolate synthetase-folylpolyglutamate synthetase. Conversely, dihydropteroate noncompetitively inhibits the folylpolyglutamate synthetase reaction of the dihydrofolate synthetase-folylpolyglutamate synthetase protein, but did not inhibit the folylpoly-alpha-glutamate synthetase reaction. Potassium chloride was inhibitory to folylpoly-alpha-glutamate synthetase activity (as were other salts and several polyanions), in contrast to the absolute requirement of dihydrofolate synthetase-folylpolyglutamate synthetase activity for a monovalent cation such as K+. Incubation of a folylpoly-alpha-glutamate synthetase preparation with (6S)-tetrahydropteroyltri(gamma)glutamate generated products which after chemical cleavage to pAB(Glu)n were identical to those from growing E. coli, in high performance liquid chromatography retention times and in pattern of digestion by alpha-COOH bond-specific carboxypeptidase Y. High performance liquid chromatography and mass spectral analysis of the products of the in vitro reactions of folylpoly-alpha-glutamate synthetase with several substrates also demonstrated the addition of glutamate residues via alpha-COOH linkages. Thus, there appear to be two folylpolyglutamate synthetase activities in E. coli, dihydrofolate synthetase-folylpolyglutamate synthetase which adds the first three glutamate residues by gamma-COOH linkages and the folylpoly-alpha-glutamate synthetase activity which extends the folylpolyglutamate chain via gamma-COOH peptide bonds.     

Biosynthesis of Tetrapyrrole Pigment Precursors : Formation and Utilization of Glutamyl-tRNA for delta-Aminolevulinic Acid Synthesis by Isolated Enzyme Fractions from Chlorella Vulgaris

The universal tetrapyrrole precursor δ-aminolevulinic acid (ALA) is formed from glutamate (Glu) in algae and higher plants. In the postulated reaction sequence, Glu-tRNA is produced by a Glu-tRNA synthetase, and the product serves as a substrate for a reduction step catalyzed by a pyridine nucleotide-requiring Glu-tRNA dehydrogenase. The reduced intermediate is then converted into ALA by a transaminase. An RNA and three enzyme fractions required for ALA formation from Glu have been isolated from soluble Chlorella extracts. The recombined fractions catalyzed ALA production from Glu or Glu-tRNA. The fraction containing the synthetase produced Glu-tRNA from Glu and tRNA in the presence of ATP and Mg²⁺. The isolated product of this reaction served as substrate for ALA production by the partially reconstituted enzyme system lacking the synthetase fraction and incapable of producing ALA from Glu. The production of ALA from Glu-tRNA by this partially reconstituted system did not require free Glu or ATP, and was not affected by added ATP. These results show that (a) free Glu-tRNA is an intermediate in the formation of ALA from Glu, (b) ATP is required only in the first step of the reaction sequence, and NADPH only in a later step, (c) Glu-tRNA production is the essential reaction catalyzed by one of the enzyme fractions, (d) this enzyme fraction is active in the absence of the other enzymes and is not required for activity of the others. The specific Glu-tRNA synthetase required for ALA formation has an approximate molecular weight of 73,000 ± 5,000 as determined by Sephadex G-100 gel filtration and native polyacrylamide gel electrophoresis. Other Glu-tRNA synthetases were present in the cell extracts but were ineffective in the the ALA-forming process.     

Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.     

DNA replication and poly(ADP-ribosyl)ation of chromatin

The role of poly(ADP-ribosyl)ation in chromatin replication and the activity of poly(ADP-ribose) synthetase in the newly synthesized and old chromatin was studied. It was found that 3-aminobenzamide, which is an inhibitor of poly(ADP-ribose) synthetase, had no effect on the initiation of DNA synthesis and only a moderate effect on DNA chain elongation. However, poly(ADP-ribose) synthetase activity in the newly replicated chromatin was two to three times higher than that of the unreplicated chromatin.     

Fungitoxicity of 1,4-naphthoquinones to Candida albicans and Trichophyton mentagrophytes.

Twenty-one substituted 1,4-naphthoquinones and five 8-quinolinols and copper(II) chelates were tested for antifungal activity against Candida albicans and Trichophyton mentagrophytes. Compounds containing electron-releasing or weak electron-withdrawing groups in the 2 and 3 positions of the 1,4-naphthoquinone ring were the most active against C. albicans at pH 7.0 in the presence of beef serum in the following order: 2-CH3O = 2,3-(CH3O)2 greater than 2-CH3 greater than 2-CH3S greater than 2-NH2 greater than 2,6-(CH3)2. For T. mentagrophytes under the same conditions the inhibitory 1,4-naphthoquinones contained the substituents 2-CH3O greater than 2,3-(CH3O)2 greater than 2-CH2S greater than 2-CH3 greater than 2-CH3(NaHSO3) greater than 2-NH2 greater than 2-C2H5S, 3-CH3 greater than 2,6-(CH3)2 greater than 2,3-CL2 greater than 5,8-(OH)2.     

Proliferation-dependency of gamma-glutamyl hydrolase activity in various mouse cells.

The activity of gamma-glutamyl hydrolase, the enzyme which deglutamylates folyl and antifolyl polyglutamates, changed significantly in mouse cells during different phases of growth, being about two times lower in actively proliferating mice splenocytes and fibroblasts than in nondividing cells. In EAC cells growing in vivo the lowest activity was observed in cells in the logarythmic phase. Methotrexate treatment of mice in a dose of 500 mg/kg body weight increased the activity of the enzyme in EAC cells about 1.5 times. We suggest that gamma-glutamyl hydrolase is a proliferating dependent enzyme which together with folypolyglutamate synthetase ensures in cells an appropriate amount of folates in the form of polyglutamates necessary for optimizing folate-dependent biosynthetic activities.     

The synthesis of pteroylpolyglutamates by sheep liver enzymes in vitro

1. Sephadex G-15 was used to separate pteroylmonoglutamates from corresponding polyglutamate derivatives. 2. Pteroylpolyglutamates were formed when 5-formyltetrahydro[2-(14)C]pteroylglutamic acid, 5-[methyl-(14)C]tetrahydropteroylglutamic acid or tetrahydro[2-(14)C]pteroylglutamic acid was incubated at pH8.4 with ATP, MgCl(2), KCl, l-glutamic acid and sheep liver cytosol. The gamma-glutamyl side chain appeared to be lengthened by the stepwise addition of single glutamate moieties. 3. The subcellular distribution of pteroylpolyglutamates paralleled that of pteroylpolyglutamate synthetase activity, and followed the order cytosol>;nuclear' fraction>microsomal fraction>mitochondria.     

Studies of the mechanism of glutamine synthetase utilizing pH-dependent behavior in catalysis and binding

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.     

Purification and characterization of Chlamydomonas reinhardtii chloroplast glutamyl-tRNA synthetase, a natural misacylating enzyme

Glutamyl-tRNA synthetase from Chlamydomonas reinhardtii was purified by sequential column chromatography on DEAE-cellulose, phosphocellulose, Mono Q, and Mono S. The apparent molecular mass of the protein when analyzed under both denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 62,000 Da; this indicates that the active enzyme is a monomer. The purified glutamyl-tRNA synthetase was identified as the chloroplast enzyme by its tRNA charging specificity. Reversed-phase chromatography of unfractionated C. reinhardtii tRNA resolved four peaks of glutamate acceptor RNA when assayed with the purified enzyme. The enzyme can also glutamylate Escherichia coli tRNA(2Glu), but not cytoplasmic tRNA(Glu) from yeast or barley. In addition, the enzyme misacylates chloroplast tRNA(Gln) with glutamate. A similar mischarging phenomenon has been demonstrated for the barley chloroplast enzyme (Sch?n, A., Kannangara, C.G., Gough, S., and S?ll, D. (1988) Nature 331, 187-190) and for Bacillus subtilis glutamyl-tRNA synthetase (Proulx, M., Duplain, L., Lacoste, L., Yaguchi, M., and Lapointe, J. (1983) J. Biol. Chem. 258, 753-759).     

Structural bases of transfer RNA-dependent amino acid recognition and activation by glutamyl-tRNA synthetase

Glutamyl-tRNA synthetase (GluRS) is one of the aminoacyl-tRNA synthetases that require the cognate tRNA for specific amino acid recognition and activation. We analyzed the role of tRNA in amino acid recognition by crystallography. In the GluRS*tRNA(Glu)*Glu structure, GluRS and tRNA(Glu) collaborate to form a highly complementary L-glutamate-binding site. This collaborative site is functional, as it is formed in the same manner in pretransition-state mimic, GluRS*tRNA(Glu)*ATP*Eol (a glutamate analog), and posttransition-state mimic, GluRS*tRNA(Glu)*ESA (a glutamyl-adenylate analog) structures. In contrast, in the GluRS*Glu structure, only GluRS forms the amino acid-binding site, which is defective and accounts for the binding of incorrect amino acids, such as D-glutamate and L-glutamine. Therefore, tRNA(Glu) is essential for formation of the completely functional binding site for L-glutamate. These structures, together with our previously described structures, reveal that tRNA plays a crucial role in accurate positioning of both L-glutamate and ATP, thus driving the amino acid activation.     

Three-dimensional structure of human gamma -glutamyl hydrolase. A class I glatamine amidotransferase adapted for a complex substate

gamma-Glutamyl hydrolase catalyzes the cleavage of the gamma-glutamyl chain of folylpoly-gamma-glutamyl substrates and is a central enzyme in folyl and antifolyl poly-gamma-glutamate metabolism. The crystal structure of human gamma-glutamyl hydrolase, determined at 1.6-A resolution, reveals that the protein is a homodimer. The overall structure of human gamma-glutamyl hydrolase contains 11 alpha-helices and 14 beta-strands, with a fold in which a central eight-stranded beta-sheet is sandwiched by three and five alpha-helices on each side. The topology is very similar to that of the class I glutamine amidotransferase domains, with the only major differences consisting of extensions in four loops and at the C terminus. These insertions are important for defining the substrate binding cleft and/or the dimer interface. Two sequence motifs are found in common between human gamma-glutamyl hydrolase and the class I glutamine amidotransferase family and include the catalytically essential residues, Cys-110 and His-220. These residues are located in the center of a large l-shaped cleft that is closed at one end and open at the other. Several conserved residues, including Glu-114, His-171, Gln-218, and Lys-223, may be important for substrate binding. Modeling of a methotrexate thioester intermediate, based on the corresponding complex of the glutamate thioester intermediate of Escherichia coli carbamoyl-phosphate synthetase, indicates that the substrate binds in an orientation with the pteroyl group toward the open end of the cleft.     

The effect of methionine on methotrexate metabolism in rat hepatocytes in monolayer culture

The effect of methyl donors on the metabolism of methotrexate has been investigated in rat hepatocytes in monolayer culture. Pulse exposure to low concentrations of methotrexate (1 microM, 3h) in the absence of methionine results in the facile formation of the di- to pentaglutamates with the di- and triglutamate predominating. Further incubation after the removal of methotrexate (MTX) results in a shift to the tetra- and pentaglutamate at the expense of the shorter chain length derivatives. The same measurement in the presence of 1 mM methionine causes approx. an 80% inhibition in the formation of polyglutamates. This effect can be partially achieved when methionine is replaced by choline or betaine. No alteration in the formation of 7-hydroxymethotrexate could be detected by similar changes in methionine concentrations in the medium. The activity of the enzymes which synthesize and degrade methotrexate polyglutamates, folylpolyglutamate synthetase and gamma-glutamyl hydrolase, respectively, were the same in extracts of cells grown in the absence and in the presence of 1 mM methionine. Incubation of the hepatocytes with methionine causes a significant increase in 5,6,7,8-tetrahydrofolate (H4folate), 5,10-methylenehydrofolate and 10-formyltetrahydrofolate and a decrease in 5-methyltetrahydrofolate. These results suggest that the inhibition of glutamylation of methotrexate could be due in part to an elevation in reduced folates which can more effectively compete with methotrexate as a substrate for folylpolyglutamate synthetase. Inhibition in methotrexate glutamylation by methionine, betaine and choline in hepatocytes may contribute to the alleviation of hepatic toxicity by methyl donors.     

Catalytic cycle of the biosynthetic reaction catalyzed by adenylylated glutamine synthetase from Escherichia coli

The covalently attached AMP moiety of adenylylated glutamine synthetase from Escherichia coli has been replaced by its fluorescent analog, 2-aza-1,N6-etheno-AMP (aza-epsilon-AMP). The modified glutamine synthetase (aza-epsilon-GS) exhibits divalent cation requirement (Mn2+, rather than Mg2+), pH profile, Vmax, and Km similar to those of naturally adenylylated glutamine synthetase. Whereas naturally adenylylated glutamine synthetase exhibits only negligible fluorescence changes upon the binding of substrates, aza-epsilon-GS exhibits large fluorescence changes. The fluorescence changes have been used by means of a stopped flow technique to reveal the involvement of five fluorometrically distinct intermediates in the catalytic cycle for the biosynthesis of glutamine catalyzed by the adenylylated glutamine synthetase. The mechanism is very similar to that previously established for the unadenylylated enzyme, using intrinsic tryptophan fluorescence. Substrates bind via a rapid equilibrium random mechanism, but the reaction proceeds in a stepwise manner. The formation of an enzyme-bound intermediate (probably gamma-glutamyl phosphate + ADP) from ATP and L-glutamate is the rate-limiting step, with the subsequent reaction of the enzyme-bound intermediate occurring very rapidly. The success in elucidating this complex mechanism is due largely to the vastly different amplitudes of the fluorescence changes at the two excitation maxima (300 nm and 360 nm) of the aza-epsilon-AMP moiety which accompany the formation of the various intermediates.