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1.
Nishimura A Itoh H Oyama H Murao S Oda K 《Bioscience, biotechnology, and biochemistry》2001,65(2):477-479
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. 相似文献
2.
Nishimura A Oyama H Hamada T Nobuoka K Shin T Murao S Oda K 《Applied and environmental microbiology》2000,66(8):3201-3205
The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited by p-chloromercuribenzoic acid, EDTA, o-phenanthroline, HgCl(2), and CuSO(4). The EDTA-inactivated enzyme was completely restored by the addition of Zn(2+) or Co(2+). In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme. 相似文献
3.
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. 相似文献
4.
A new assay for the determination of 5-oxoprolinase activity is described. The enzyme 5-oxoprolinase was purified from rat kidney 285-fold to apparent homogeneity, as judged by analytical disc electrophoresis and discontinous polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The specific activity of the preparation was 122 mU/mg of protein. A complete initial rate kinetic analysis of the forward reaction catalyzed by 5-oxoprolinase was carried out using 5-oxo-L-proline and MgATP2theta as substrates. The computer-fitted double reciprocal plots showed intersecting patterns indicating a sequential mechanism. The data were fitted by weighted linear regression analysis using the complete equation for bisubstrate reactions. The limiting Michaelis constants for 5-oxoproline and MgATP2theta were calculated to be 31.6 +/- 2.3 muM and 172.7 +/- 11.5muM, respectively. The maximum forward rate is 1.2 +/- 0.02 mumol X min-1; the turnover number 7.0 min-1. 相似文献
5.
Bacterial 5-oxoprolinase is composed of two protein components: Component A, which catalyzes 5-oxoproline-dependent ATP-hydrolysis and Component B, which couples the hydrolysis of ATP with the decyclization of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). Studies on this unusual enzyme system have led to evidence that an intermediate is formed by Component A. Application of the isotope-trapping method demonstrated an activated 5-oxoproline intermediate, whose formation requires ATP, Mg2+, and Component A. The amount of ATP-dependent trapping was close to the number of enzyme active sites. The intermediate formed by Component A was shown to be reducible by potassium borohydride to proline in low yield; when Component B was added, the formation of proline was abolished. Treatment of reaction mixtures containing Component A, 5-oxoproline, and [gamma-32P] ATP with diazomethane led to appearance of a 32P-labeled compound (found on thin layer chromatography), whose formation was significantly reduced when Component B was present. The new compound, which is labile, breaks down to form dimethyl[32P]phosphate. The total amount of dimethyl[32P]phosphate formed after breakdown is close to the number of active sites of Component A. The data are consistent with the conclusion that a phosphorylated form of 5-oxoproline is formed by Component A and suggest that Component B is required for conversion of this intermediate to glutamate. 相似文献
6.
Potentiometric and colorimetric methods for the assay of 2',3'-cyclic nucleotide 3'-phosphohydrolase 总被引:5,自引:3,他引:2
Abstract— A potentiometric titration method for the assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase is presented. Progress curves of the reaction were recorded automatically by pH-stat. 2-Mercaptoethanol was added to the reaction mixture to maintain a linear rate of reaction. The method is suitable for obtaining kinetic parameters and can be used for the rapid assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in nervous tissues. An improved colorimetric method for estimation of 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity at the optimum pH is described. This method employs the two-step procedure in which decyclization by 2′,3′-cyclic nucleotide 3′-phosphohydrolase and dephosphorylation by Escherichia coli alkaline phosphatase (EC 3.1.3.1) are carried out separately under the optimum conditions for each enzyme. The method is sensitive and most convenient for routine assays. 相似文献
7.
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. 相似文献
8.
Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal
proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the
disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile
cycle that is generated between two ATP-utilizing enzymes of the γ-glutamyl cycle leads to ATP depletion. The enzyme γ-glutamyl cysteine synthetase (γ-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, γ-glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in
cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without
production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion
observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model
of cystinosis, where renal failure is not observed. 相似文献
9.
The hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to 2-formylglutarate is a central step in the catabolism of nicotinate in several Clostridia and Proteobacteria. This reaction is catalyzed by the novel enzyme enamidase, a new member of the amidohydrolase superfamily as indicated by its unique reaction, sequence relationship, and the stoichiometric binding of iron and zinc. A hallmark of enamidase is its capability to catalyze a two-step reaction: the initial decyclization of 1,4,5,6-tetrahydro-6-oxonicotinate leading to 2-(enamine)glutarate followed by an additional hydrolysis step yielding (S)-2-formylglutarate. Here, we present the crystal structure of enamidase from Eubacterium barkeri at 1.9 Å resolution, providing a structural basis for catalysis and suggesting a mechanism for its exceptional activity and enantioselectivity. The enzyme forms a 222-symmetric tetramer built up by a dimer of dimers. Each enamidase monomer consists of a composite β-sandwich domain and an (α/β)8-TIM-barrel domain harboring the active site. With its catalytic binuclear metal center comprising both zinc and iron ions, enamidase represents a special case of subtype II amidohydrolases. 相似文献
10.
5-Oxo-L-prolinase from Pseudomonas putida is composed of two reversibly dissociable proteins: Component A catalyzes 5-oxoproline-dependent cleavage of ATP, but does not catalyze the decyclization of 5-oxoproline; Component B is required for the coupling of ATP cleavage to ring-opening of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). We describe here the purifications of Components A and B to apparent homogeneity and the interactions between these two proteins. The cellular content of Component B activity is significantly greater than that of Component A. By gel filtration, Component A is a hexamer; but in the presence of substrates, it is a dimer. Component B can exist as an aggregate, an octamer, or a tetramer, depending upon the conditions used. Gel filtration of a mixture of Components A and B in the presence of substrates gives a unique protein species that exhibits 5-oxoprolinase activity. The Mr of this Component A-Component B complex indicates that it probably has an A2-B2 structure. The molar ratio of Component A to Component B in the complex was determined to be 1:1 by the continuous variation method (Job). Titrations of each component by the other suggest that phosphorylated 5-oxoproline-bound Component A is the entity that interacts with Component B. These studies indicate that the binding of phosphorylated 5-oxoproline-bound Component A to Component B to form a complex proceeds by a cooperative type mechanism. This is supported by the observed shifts of the intersection points of the Job curves (see Appendix). 相似文献
11.
5-Oxoprolinase catalyzes the ATP-dependent decyclization of 5-oxo-L-proline to L-glutamate. Previous studies provided evidence for the intermediate formation of a phosphorylated form of 5-oxoproline (Seddon, A. P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11541) and of a tetrahedral intermediate (Li, L., Seddon, A. P., and Meister, A. (1987) J. Biol. Chem. 262, 11020-11025). A new approach to the study of the reaction mechanism using the 18O isotope effect on the 13C NMR signals for 5-oxoproline and glutamate is reported here. The 13C chemical shifts induced by 18O substitution for the carbonyl group of 5-oxoproline and the gamma-carboxyl group of glutamate are about 0.03 ppm with respect to the corresponding 16O-compounds. Using 5-[18O]oxo[5-13C]proline (97 and 79.5 atom % excess, 13C and 18O, respectively), the disappearance of the 18O-labeled and unlabeled 5-oxoproline and formation of the corresponding glutamate species were followed in the reactions catalyzed by purified preparations of 5-oxoprolinase isolated from Pseudomonas putida and from rat kidney. This procedure permits simultaneous determinations of the rates of 18O exchange and of the overall decyclization reaction. The ratios of 18O exchange rates to the overall reaction rates for the bacterial and kidney enzyme catalyzed-reactions were 0.28 and 0.14, respectively. The findings support the view that the coupling of ATP hydrolysis to 5-oxoproline decyclization involves formation of a phosphorylated tetrahedal intermediate. Although the exchange phenomena are consistent with the mechanistic interpretations, they seem not to be required for catalysis. 相似文献
12.
Takimoto A Takakura T Tani H Yagi S Mitsushima K 《Applied microbiology and biotechnology》2004,65(3):263-267
Bacillus subtilis SHS0133 cephalosporin-C deacetylase (CAH) overexpressed in Escherichia coli was immobilized on an anion-exchange resin, KA-890, using glutaraldehyde. The activity yield of immobilized enzyme was approximately 55% of the free enzyme. The pH range for stability of the immobilized enzyme (pH 5–10) was broader than that for free enzyme. The Kmapp value of immobilized enzyme for 7-aminocephalosporanic acid (7-ACA) was similar to that of the free enzyme. This immobilized enzyme obeyed Michaelis–Menten kinetics similar to those of the free enzyme. A batch-type reactor with a water jacket was employed for deacetylation of 7-ACA using CAH immobilized on KA-890. Ten kilograms of 7-ACA were completely converted to deacetyl 7-ACA at pH 8.0 within 90 min. The reaction kinetics agreed well with a computer simulation model. Moreover, the immobilized enzyme exhibited only a slight loss of the initial activity even after repeated use (52 times ) over a period of 70 days. This reaction will thus be useful for the production of cephalosporin-type antibiotics. 相似文献
13.
The degradation pathway of glutathione (GSH) in plants is not well understood. In mammals, GSH is predominantly metabolized through the γ-glutamyl cycle, where GSH is degraded by the sequential reaction of γ-glutamyl transpeptidase (GGT), γ-glutamyl cyclotransferase, and 5-oxoprolinase to yield glutamate (Glu) and dipeptides that are subject to peptidase action. In this study, we examined if GSH is degraded through the same pathway in Arabidopsis (Arabidopsis thaliana) as occurs in mammals. In Arabidopsis, the oxoprolinase knockout mutants (oxp1-1 and oxp1-2) accumulate more 5-oxoproline (5OP) and less Glu than wild-type plants, suggesting substantial metabolite flux though 5OP and that 5OP is a major contributor to Glu steady-state levels. In the ggt1-1/ggt4-1/oxp1-1 triple mutant with no GGT activity in any organs except young siliques, the 5OP concentration in leaves was not different from that in oxp1-1, suggesting that GGTs are not major contributors to 5OP production in Arabidopsis. 5OP formation strongly tracked the level of GSH in Arabidopsis plants, suggesting that GSH is the precursor of 5OP in a GGT-independent reaction. Kinetics analysis suggests that γ-glutamyl cyclotransferase is the major source of GSH degradation and 5OP formation in Arabidopsis. This discovery led us to propose a new pathway for GSH turnover in plants where GSH is converted to 5OP and then to Glu by the combined action of γ-glutamyl cyclotransferase and 5-oxoprolinase in the cytoplasm. 相似文献
14.
Glenwyn D. Kemp F. Mark Dickinson Colin Ratledge 《Applied microbiology and biotechnology》1991,34(4):441-445
Summary The alkane-induced, membrane-bound fatty alcohol oxidase of Candida tropicalis was functional with decanol as a model substrate in C8 to C12 alkanes and in cyclohexane. Optimal activity was with octane. Although some reaction took place without added water, 5–10% water gave optimal activity. A continuous spectrophotometric assay for following the enzyme activity in this system was developed. The enzyme, besides oxidizing primary long straight-chained alcohols, also oxidized long-chain diols, -hydroxy fatty acids, unsaturated fatty alcohols and branched-chained unsaturated fatty alcohols, although at diminished rates. Secondary alcohols or arylalkan-1-ols were not attacked. The K
m for dodecanol was some 5000-fold higher than the K
m for the same substrate when the reaction was carried out in water. 相似文献
15.
Summary A technique which allows for the continuous microdensitometric monitoring (at 3.3 s intervals) of an enzyme reaction has been applied to a study of 3-hydroxy-5-steroid dehydrogenase activity in regressing corpora lutea cells of unfixed tissue sections (5µm thick) of the pro-oestrous rat ovary. Initial reaction rates for NAD+ reduction by the activity of the enzyme were maintained for less than 3 min at all the concentrations (0-500µmol/l) of co-factor employed. The relationship between NAD+ concentration and enzyme activity under initial velocity rate conditions was hyperbolic and maximum enzyme activity was seen when the NAD+ concentration was greater than 250µmol/l. The apparent Michaelis—Menten constant (K
m) was 29µmol/l and Vmax 0.63µmol NAD+ reduced/min/cm3 corpora lutea. 相似文献
16.
Measurements have been made of the enzymes associated with γ-glutamyl cycleviz, γ-glutamyl transpeptidase, γ-glutamyl cysteine synthetase, and 5-oxoprolinase in human fetal brain, liver and kidney over
12–36 weeks of gestation. γ-Glutamyl transpeptidase activity increases gradually with age. γ-Glutamyl cysteine synthetase
and 5-oxoprolinase show biphasic pattern of development in human fetal brain. The data presented in this communication may
indicate a relationship between γ-glutamyl cycle and amino acid transport. 相似文献
17.
Gerry R. Boss Soha D. Idriss Randall C. Willis J. E. Seegmiller 《Analytical biochemistry》1983,130(2)
Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the transfer of the amide group of glutamine to 5-phospho-α-
-ribose-1-pyrophosphate. It is the first enzyme committed to the synthesis of purines by the de novo pathway. Previous assays of enzyme activity have either measured the phosphoribosylpyrophosphate-dependent disappearance of radioactive glutamine or have linked this reaction to subsequent steps in the purine pathway. A new assay for activity of the enzyme by directly measuring the synthesis of the product of the reaction, 5-β-phosphoribosyl-1-amine, using [1-14C]phosphoribosylpyrophosphate as substrate is described. Substrate and product are separated by thin-layer chromatography and identified by autoradiography. Glutamine or ammonia may be used as substrates; the apparent Km values of the human lymphoblast enzyme are 0.46 m
for glutamine and 0.71 m
for ammonia. GMP is a considerably more potent inhibitor of the human lymphoblast enzyme than is AMP; 6-diazo-5-oxo-
-norleucine inhibits only glutamine-dependent activity and has no effect on ammonia-dependent activity. 相似文献
18.
A simple method was developed for selective solubilization of membrane ATPase from etiolated corn seedlings using 0.01% Triton X100 and 0.01% deoxycholate containing 200 mM KI. An 81-fold enriched enzyme preparation, with specific activity of 133 μmol Pi/mg protein/hr, was obtained. The enzyme stored in 25 mM Tris-HCl buffer (pH 7.5) at 4° showed rapid loss of activity. The enzyme was stabilized by 1 mM EDTA with addition of 1.2 mM Mg2+°. Mg2+ and Ca2+ (1.2 mM) increased enzymatic activity by 12 and 10.8% respectively, whereas Na+ and K+ brought about a 20% increase in ATP-hydrolysis. The effect of combined mono- and di-valent ions was neither synergistic nor additive. Ouabain exerted no effect on enzyme activity. The enzyme showed two pH optima (6.0 and 7.5) in the presence of Na+ and K+, and one optimum at pH 6.5 in the absence of these ions. On polyacrylamide gel the enzyme was resolved into two protein bands, both exhibiting ATPase activity. It is suggested that the soluble enzyme from the microsomal fraction of corn seedlings contains two ATP-hydrolyzing enzymes, one of them being stimulated by Na+ and K+ ions. 相似文献
19.
The Wachstein and Meisel incubation medium was used to detect ATPase activity in epimastigote, spheromastigote (amastigote), and bloodstream trypomastigote forms of Trypanosoma cruzi. Reaction product, indicative of enzyme activity, was associated with the plasma membrane covering the cell body and the flagellum of the parasite. No reaction product was found in the portion of the plasma membrane lining the flagellar pocket. The plasma membrane-associated ATPase activity was not inhibited by ouabain or oligomycin, was detected in incubation medium without K+, was inhibited by prolonged glutaraldehyde fixation, and its activity was diminished when Mg2+ was omitted from the incubation medium. The Ernst medium was used to detect Na+-K+-ATPase activity in T. cruzi. No reaction product indicative of the presence of this enzyme was detected. Reaction product indicative of 5'-nucleotidase was not detected in T. cruzi. Acid phosphatase activity was detected in lysosomes. These results indicate that a Mg2+-activated ATPase is present in the plasma membrane of T. cruzi and that it can be used as an enzyme marker, provided that the mitochondrial and flagellar ATPases are inhibited, to assess the purity of plasma membrane fractions isolated from this parasite. 相似文献
20.
Feng Wang Yong Ma Yan-Hua Liu Xuan Zhang Fuming Zhang Robert J. Linhardt 《Biocatalysis and Biotransformation》2017,35(5):349-362
A β-glucosidase extracted from bitter almond (Prunus dulcis var. amara) was immobilized on polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH 6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobilization. In the co-solvent system using 10% t-butyl alcohol and 10% (v/v) water, the yield of OG was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on dynamic and Dixon plot analyses, the initial reaction velocity (V0) increased approximately three-fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dissociation constants for free and immobilized enzyme were 219?mM and 116?mM, respectively. A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336?h of reaction, the OG yield and the conversion rate of glucose reached 134?mM and 59.6%, respectively. Compared to the batch operation, the fed-bath operation increased the OG yield and the conversion rate of glucose by 340% and 381%, respectively. 相似文献