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Query: CAS:6893-26-1 (glutamate)
 73,096 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat kidney 5-oxo-L-prolinase catalyzes the endergonic hydrolysis of 5-oxo-L-proline (L-pyroglutamate, L-2-pyrrolidone-5-carboxylate) to form L-glutamate; the reaction is driven by and dependent on the stoichiometric concomitant hydrolysis of ATP to ADP and inorganic phosphate. The present studies are concerned with the mechanism by which the free energy of ATP hydrolysis is conserved and made available for 5-oxoproline hydrolysis. Studies with 18O-labeled substrates showed that (a) all three oxygen atoms of 5-oxoproline are recovered in the product glutamate, and (b) the two water molecules consumed in the reaction contribute one oxygen atom to inorganic phosphate and one oxygen atom to the gamma-carboxyl group of glutamate. It was shown that the enzyme also catalyzes the intrinsically exergonic hydrolysis of alpha-hydroxyglutarate lactone, a reaction that is ATP-dependent. Intermediates in the 5-oxoprolinase reaction were not detected by exchange experiments with radioactive ADP and phosphate, nor were they trapped by adding hydroxylamine. In the presence of very high glutamate concentrations, a slow reversal of the 5-oxoprolinase reaction was demonstrated by measuring ATP formation. The findings are consistent with a mechanism in which 5-oxo-L-proline is phosphorylated by ATP on the amide carbonyl oxygen and the resulting intermediate is subsequently hydrolyzed to yield gamma-glutamyl phosphate; the latter is hydrolyzed to glutamate and inorganic phosphate.
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PMID:5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Studies of the chemical mechanism. 611 61

5-Oxoprolinase catalyzes the coupled hydrolysis of ATP and 5-oxoproline to yield glutamate, ADP, and Pi; the reaction may be partially or completely uncoupled by structural modification of either substrate. In the present work, we found slow 5-oxoproline-dependent changes in the rates of hydrolysis of ITP, GTP, and UTP. For example, in the absence of 5-oxoproline, the enzyme catalyzes the hydrolysis of UTP at a rapid and constant rate. Following addition of 5-oxo-L-proline, the rate of hydrolysis decreases slowly; after about 25 min, a much slower and constant rate of hydrolysis is attained. This change in rate is associated with a decrease in Vmax and an increase in the Km for UTP. In similar studies with ATP, both Vmax and Km increase over a much shorter time period (less than 10 s). The findings indicate that 5-oxoprolinase is a hysteretic enzyme, and are consistent with the hypothesis that in the normal catalytic reaction, the binding of both ATP and 5-oxo-proline to the enzyme induces a conformational change that brings the substrates into a juxtaposition that facilitates the reaction.
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PMID:Interaction of 5-oxo-L-prolinase with nucleoside triphosphates. Evidence suggesting substrate-dependent conformational change. 612 2

5-Oxo-L-prolinase catalyzes a reaction in which the endergonic cleavage of 5-oxo-L-proline to form L-glutamate is coupled to the exergonic cleavage of ATP to ADP and Pi. In the present research, the enzyme present in a strain of Pseudomonas putida isolated from soil by enrichment culture was found to be composed of two protein components. Neither component alone could catalyze the 5-oxoprolinase reaction, but the reaction was effectively catalyzed when they were mixed. One component (A) exhibited 5-oxo-L-proline-dependent ATPase activity indicating that Component A can interact with both ATP and 5-oxo-L-proline. The other component (coupling protein; B) does not exhibit ATPase activity nor is there evidence that it binds 5-oxo-L-proline. The findings are consistent with (but do not prove) the hypothesis that the Component A catalyzes an initial step in the reaction which involves 5-oxoproline and ATP, such as phosphorylation of 5-oxoproline. The coupling protein (B) may function as a catalyst that converts a phosphorylated form of 5-oxoproline to glutamate, or it might alter the conformation of Component A so as to facilitate the reaction.
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PMID:Resolution of 5-oxo-L-prolinase into a 5-oxo-L-proline-dependent ATPase and a coupling protein. 614 10

5-Oxo-L-prolinase, the enzyme that catalyzes the conversion of 5-oxo-L-proline to L-glutamate coupled to the cleavage of ATP to ADP and Pi, also acts on L-2-oxothiazolidine-4-carboxylate (an analog of 5-oxoproline in which the 4-methylene moiety is replaced by sulfur) and ATP to yield cysteine and ADP. The enzyme, which exhibits an affinity for the analog similar to that for the natural substrate, is inhibited by the analog in vitro and in vivo. L-2-oxothiazolidine-4-carboxylate thus serves as a potent inhibitor of the gamma-glutamyl cycle at the step of 5-oxoprolinase. Administration of L-2-oxothiazolidine-4-carboxylate to mice that had been depleted of hepatic glutathione led to restoration of normal hepatic glutathione levels. Since L-2-oxothiazolidine-4-carboxylate is an excellent substrate of the enzyme, it may serve as an intracellular delivery system for cysteine and thus has potential as a therapeutic agent for conditions in which there is depletion of hepatic glutathione.
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PMID:Stimulation of hepatic glutathione formation by administration of L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-prolinase substrate. 694 Jan 59

A screening test was undertaken to isolate a microorganism that produced 5-oxoprolinase (without ATP-hydrolyzing). The 5-oxoprolinase (without ATP-hydrolyzing) activity (decyclization activity toward L-pyroglutamate) was found in a cell-free extract of Alcaligenes faecalis N-38A, newly isolated from a soil sample. The enzyme was purified as a homogeneous preparation. The molecular weight of the enzyme was estimated to be 47,000. The decyclization activity was specific for L-pyroglutamate, and independent of ATP and metal ions. The reaction was a reversible one, i.e., cyclization reaction of L-glutamate to yield pyroglutamate was identified.
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PMID:Isolation and characterization of a novel 5-oxoprolinase (without ATP-hydrolyzing) from Alcaligenes faecalis N-38A. 853 1

5-Oxoprolinase (EC 3.5.2) catalyzes a reaction in which the endergonic cleavage of 5-oxo-L-proline to form L-glutamate is coupled to the exergonic hydrolysis of ATP to ADP and inorganic phosphate. Highly purified preparations of the enzyme have been obtained from rat kidney and Pseudomonas putida. The rat kidney enzyme is composed of two strongly interacting, apparently identical subunits (Mr = 142,000), whereas that from P. putida is composed of two functionally different protein components that can readily be dissociated. Here we report the cloning of rat kidney 5-oxoprolinase with preliminary expression studies. cDNA clones encoding the enzyme were isolated by screening a lambdagt11 cDNA library beginning with a degenerate oligonucleotide probe based on peptide sequence data obtained from the purified enzyme. The whole cDNA clone was completed by amplifying its 5' end from a premade library of rat kidney Marathon-ReadyTM cDNAs using polymerase chain reaction methodology. The composite cDNA (4,016 bases) revealed an uninterrupted open reading frame encoding 1,288 amino acid residues (Mr = 137,759). The deduced amino acid sequence contains all four of the peptide sequences that were independently found in peptide fragments derived from the enzyme. Expression of the full-length clone in Escherichia coli yielded a product of the same size as the rat kidney enzyme and which reacted with antibodies directed against the rat kidney enzyme. The predicted amino acid sequence is almost 50% identical throughout its entire length to that of a hypothetical yeast protein YKL215C. It is also 26% identical in half its length to the bacterial hydantoinase HyuA and 26% identical in the other half to the bacterial hydantoinase HyuB. The results suggest unexpected evolutionary relationships among the hydantoinases and rat kidney 5-oxoprolinase which share the common property of hydrolyzing the imide bond of 5-membered rings but which do not all require ATP.
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PMID:The amino acid sequence of rat kidney 5-oxo-L-prolinase determined by cDNA cloning. 894 90

The ability of astroglia-rich primary cultures derived from the brains of neonatal rats to take up and metabolize various sulfur containing compounds to cysteine was investigated using the content of intracellular glutathione as an indicator. Astroglial cells were partially depleted of glutathione by starvation for 24 h. Subsequent feeding for 4 h with glucose, glycine, and glutamate resulted in a restoration of the glutathione level, if cysteine was present. Substitution of cysteine by cystine during resynthesis of glutathione led to a glutathione content which exceeded that of cysteine-refed cells by 41%. Half-maximal content of glutathione was found at a concentration of about 12 microM cysteine and a maximal content at a concentration of at least 50 microM cysteine. In contrast, no plateau in the glutathione level was reached with increasing concentrations of cystine. The cystine effect could not be due to a contamination, since it was abolished after reduction of cystine by dithiothreitol. Since the cystine effect was not affected by inhibiting gamma-glutamyl transpeptidase, a promotion of cystine uptake by formation of gamma-glutamylcystine can also be excluded. Of the potential cysteine precursors tested, N-acetylcysteine was able to replace cysteine half-maximally at a concentration of 1 mM and fully at 5 mM. Feeding 2-oxothiazolidine-4-carboxylic acid at a concentration of 5 mM resulted in 64% of the glutathione level found in the presence of cysteine. A half-maximal glutathione content was attained at 50 microM 2-oxothiazolidine-4-carboxylic acid. While cystathionine could partially replace cysteine, methionine and homocysteine were not at all able to substitute for cysteine. These results demonstrate that astroglial cells prefer cystine from cysteine for glutathione synthesis and express uptake systems for N-acetylcysteine, 2-oxothiazolidine-4-carboxylic acid, and cystathionine, as well as the enzymes N-deacetylase, 5-oxoprolinase, and cystathionine gamma-lyase.
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PMID:Utilization of cysteine and cysteine precursors for the synthesis of glutathione in astroglial cultures: preference for cystine. 943 84

A novel type of 5-oxoprolinase was found in a cell extract of strain N-38A, which was later identified as Alcaligenes faecalis. The enzyme in the cell extract was purified to a homogeneous state with a yield of 16.6%. The molecular weight of the purified enzyme was estimated to be 47,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, suggesting that the enzyme is a monomeric protein. The enzyme specifically catalyzed a decyclization of L-pyroglutamate without hydrolyzing ATP and also without any requirements for metal ions such as Mg2+ and K+. The optimal pH for the decyclization was 7.4. The reaction was reversible. The equilibrium constant of the reaction, Keq = [L-glutamate]/[L-pyroglutamate], was evaluated to be approximately 0. 035, which indicates that the reaction tends to form L-pyroglutamate. The amino-terminal amino acid sequence of the enzyme was H-Glu-Pro-Arg-Leu-Asp-Thr-Ser-Gln-Leu-Tyr-Ala-Asp-Val-His-Phe-. No protein with a similar sequence was found in the DNASIS database. Based on these data, it was strongly suggested that the enzyme described here is a novel type of 5-oxoprolinase.
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PMID:Purification and characterization of a novel 5-oxoprolinase (without ATP-hydrolyzing activity) from Alcaligenes faecalis N-38A. 992 5

L-Glutamine and L-glutamate, which are important flavor components in soy sauce, are converted to L-pyroglutamate during brewing. Therefore, it is necessary that the L-glutamate and L-pyroglutamate contents can be measured accurately. We developed a simultaneous assay method for L-glutamate and L-pyroglutamate by using 5-oxoprolinase (without ATP hydrolyzing activity) and glutamate oxidase. By this method, the L-pyroglutamate could be measured accurately in a range of 0.05 to 1.0 mM in the presence of 1.0 mM L-glutamate. This system is effective for process and quality controls.
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PMID:A simultaneous assay method for L-glutamate and L-pyroglutamate contents in soy sauce using a 5-oxoprolinase (without ATP hydrolyzing activity). 1130 95

Glutathione is a tripeptide composed of glutamate, cysteine and glycine. Glutathione is present in millimolar concentrations in most mammalian cells and it is involved in several fundamental biological functions, including free radical scavenging, detoxification of xenobiotics and carcinogens, redox reactions, biosynthesis of DNA, proteins and leukotrienes, as well as neurotransmission/neuromodulation. Glutathione is metabolised via the gamma-glutamyl cycle, which is catalyzed by six enzymes. In man, hereditary deficiencies have been found in five of the six enzymes. Glutathione synthetase deficiency is the most frequently recognized disorder and, in its severe form, it is associated with hemolytic anemia, metabolic acidosis, 5-oxoprolinuria, central nervous system (CNS) damage and recurrent bacterial infections. Gamma-glutamylcysteine synthetase deficiency is also associated with hemolytic anemia, and some patients with this disorder show defects of neuromuscular function and generalized aminoaciduria. Gamma-glutamyl transpeptidase deficiency has been found in patients with CNS involvement and glutathionuria. 5-Oxoprolinase deficiency is associated with 5-oxoprolinuria but without a clear association with other symptoms. Dipeptidase deficiency has been described in one patient. All disorders are very rare and inherited in an autosomal recessive manner. Most of the mutations are leaky so that many patients have residual enzyme activity. Diagnosis is made by measuring the concentration of different metabolites in the gamma-glutamyl cycle, enzyme activity and in glutathione synthetase and gamma-glutamylcysteine synthetase deficiency, also by mutation analysis. Prenatal diagnosis has been preformed in glutathione synthetase deficiency. The prognosis is difficult to predict, as few patients are known, but seems to vary significantly between different patients. The aims of the treatment of glutathione synthesis defects are to avoid hemolytic crises and to increase the defense against reactive oxygen species. No treatment has been recommended for gamma-glutamyl transpeptidase, 5-oxoprolinase and dipeptidase deficiency.
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PMID:Inborn errors in the metabolism of glutathione. 1739 29


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