Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.4.3.11 (glutamate dehydrogenase)
4,437 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hysteresis in glutamate dehydrogenase is observed only in the reductive amination reaction and only with GTP present. The rate of reductive amination with NADH as coenzyme increases during the time course of the reaction. Premixing experiments, where glutamate dehydrogenase is preincubated with various combinations of substrates and GTP, suggest that the hysteresis phenomenon is not due to a time-dependent conformational change in the enzyme. Enzyme dilution experiments show (i) that the hysteresis is not due to enzyme association-dissociation effects and (ii) that the onset of the activation occurs after accumulation of about 25 microM NAD+. Addition of NAD+ to the initial reaction mixture prevents hysteresis from occurring. Although with NADPH as coenzyme hysteresis does not occur, addition of NADP+ to initial reaction mixtures containing NADH blocks hysteresis. A model based on reciprocating subunits is proposed whereby hysteresis results from product (NAD+) accumulation resulting in a half-of-the-sites activation of reductive amination.
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PMID:Mechanism of hysteresis in bovine glutamate dehydrogenase: role of subunit interactions. 707 37

The thionicotinamide analogues of NAD+ and NADP+ were shown to be good alternative coenzymes for bovine glutamate dehydrogenase, with similar affinity and approx. 40% of the maximum velocity obtained with the natural coenzymes. Both thionicotinamide analogues show non-linear Lineweaver-Burk plots, which with the natural coenzymes have been attributed to negative co-operativity. Since the reduced thionicotinamide analogues have an isosbestic point at 340nm and have an absorption maximum at 400nm, it is possible to monitor reduction of natural coenzyme and thionicotinamide analogue simultaneously by dual-wavelength spectroscopy. When glutamate dehydrogenase is presented with NADP+ and thio-NADP+ simultaneously, the enzyme oligomer senses saturation of its coenzyme-binding sites irrespective of the exact nature of the coenzyme and locks the oligomer into its highly saturated form even when low saturation of the monitored coenzyme is present. These experiments substantiate the suggestion that glutamate dehydrogenase shows negative co-operativity in its catalytically active form.
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PMID:Dual nucleotide specificity of bovine glutamate dehydrogenase. The role of negative co-operativity. 723 98

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.
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PMID:Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron. 736 28

1. The binding of NAD+ to glutamate dehydrogenase may be followed quantitatively by titration, using high-sensitivity circular dichroism (CD) difference spectroscopy. 2. The CD of the bound coenzyme in the binary complex E . NAD closely resembles that of bound ADP, although the affinity is much lower, being 350-fold less for NAD+ at 20 degrees C in 0.1 M phosphate, pH 7. 3. A family of CD spectra may be analysed by unconstrained linear regression assuming only three components: free enzyme, free coenzyme, and a single binary complex, E . NAD. 4. Taking the molar CD of bound ADP as representing the molar CD of the adenine chromophore of bound NAD+, the linear regression shows the formation of a simple 1 : 1 complex E . NAD with Kd = 0.72 mM in a simple binding process without positive or negative cooperativity. 5. NADP+ binding is more than 10-fold weaker than NAD+ binding. 6. From the similarity of the CD of bound ADP and bound NAD+ it is probable that NAD+, in forming a simple binary complex, binds preferentially at the regulatory (adenine nucleotide) binding site (site II). 7. Direct evidence has been obtained for the binding of a second molecule of NAD+ to the ternary complex E . NAD . glutarate. This process occurs with low affinity and is probably also located at the adenine regulatory site. 8. This second-site binding of NAD+ may contribute to the phenomena of non-Michaelis-Menten kinetics and apparent negative homotropic interactions in the binding of NAD+, previously attributed to subunit-subunit cooperative interactions.
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PMID:The binding of oxidised coenzyme to bovine-liver glutamate dehydrogenase studied by circular-difference spectroscopy. 746 Sep 36

A steady state kinetic study was carried out with the glutamate dehydrogenase from the thermophilic, archaebacterial isolate AN1. Initial velocity studies of the oxidative deamination reaction showed the mechanism is sequential and indicated that the order of substrate addition is random, while inhibition studies with products and substrate analogues suggested a strong preference for NADP+ to bind first. Initial velocity studies of the reductive amination reaction showed that the mechanism is sequential and indicated that the order of substrate addition is random, while product inhibition studies and the effect of substrate saturation on the initial velocity suggested that the preferred order of substrate addition is NADPH, 2-ketoglutarate, ammonia.
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PMID:Steady state kinetics of the glutamate dehydrogenase from an archaebacterial extreme thermophile, isolate AN1. 761 54

This review is an exhaustive description of the biochemistry and enzymology of all 17 known NAD(P)(+)-amino acid dehydrogenases. These enzymes catalyze the oxidative deamination of an amino acid to its keto acid and ammonia, with the concomitant reduction of either NAD+ or NADP+. These enzymes have many important applications in industrial and medical settings and have been the object of prodigious enzymological research. This article describes all that is known about the poorly characterized members of the family and contains detailed information on the better characterized enzymes, including valine, phenylalanine, leucine, alanine, and glutamate dehydrogenases. The latter three enzymes have been the subject of extensive enzymological experimentation, and, consequently, their chemical mechanisms are discussed. The three-dimensional structure of the Clostridium symbiosum glutamate dehydrogenase has been determined recently and remains the only structure known of any amino acid dehydrogenase. The three-dimensional structure and its implications to the chemical mechanisms and rate-limiting steps of the amino acid dehydrogenase family are discussed.
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PMID:The biochemistry and enzymology of amino acid dehydrogenases. 770 1

The effect of polyamines on glutamate dehydrogenase [L-glutamate: NAD(P) oxidoreductase (deaminating) [EC 1.4.1.3]) activity has been studied in both permeabilized kidney-cortex mitochondria and isolated renal tubules of rabbit. Spermidine was the most potent inhibitor of glutamate synthesis in permeabilized mitochondria resulting in about 80% decrease of the enzyme activity at 5 mM concentration. Putrescine, alpha-monofluoromethylputrescine (MFMP) and (R,R)-delta-methyl-alpha-acetylenic-putrescine (MAP) were more efficient than spermine. The inhibitory action of polyamines was potentiated by an elevated NADH content in the reaction mixture. Increasing concentrations of either NH4Cl, KCl or NaCl in the incubation medium resulted in a decrease of polyamine-induced inhibition of the enzyme activity, indicating that monovalent cations can compete with polyamines for the binding site at glutamate dehydrogenase. The inhibitory action of spermidine on glutamate synthesis was abolished by 2 mM ADP or 10 mM L-leucine, allosteric activators of the enzyme, as well as on the addition of either oxalate or sulphate at 20 mM concentrations. Spermidine did not affect glutamate formation when NADH was substituted by NADPH, suggesting an importance of the NADH binding to the inhibitory site of the enzyme for a decrease of reductive amination of 2-oxoglutarate by polyamine. Although spermidine did not influence glutamate deamination in the presence of NAD+, it stimulated this process by about 70% when NAD+ was substituted by NADP+. In the presence of ADP the stimulatory effect of polyamine was not significant. The data indicate that in permeabilized rabbit kidney-cortex mitochondria the effect of polyamines on both glutamate formation and glutamate deamination via the reaction catalysed by glutamate dehydrogenase is dependent upon the coenzyme utilized by the enzyme. In the presence of NADH their inhibitory effect on the glutamate formation may be alleviated by allosteric activators of the enzyme, and concentrations of potassium, sodium, sulphate and oxalate. In isolated rabbit renal tubules incubated with 5 mM methionine sulfoximine and aminooxyacetate, in order to inhibit glutamine synthetase and aminotransferases, respectively, 5 mM spermidine decreased glutamate formation by about 30%, while putrescine and spermine did not significantly diminish the enzyme activity. In the presence of octanoate glutamate formation was reduced by about 30% by naturally occurring polyamines as well as MFMP and MAP, indicating that under these conditions NADH rather than NADPH is utilized as the coenzyme. In view of these data it is possible to suggest that polyamines may be of importance to control glutamate dehydrogenase activity under physiological conditions.
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PMID:Effect of polyamines on glutamate dehydrogenase within permeabilized kidney-cortex mitochondria and isolated renal tubules of rabbit. 791 Apr 59

The effects of AMG-1 [6(5-hydroxy-2-methylpyridylamino)-9 ribofranosylpurine], an adenosine analogue, and adenosine on Ca2+-dependent and independent release of glutamate from rat synaptosomes induced by KCl 30 mmol.L-1 were studied with an enzyme-linked fluorometric assay. The synaptosomes were prepared and preincubated for 30 min at 37 degrees C. Two ml of incubation mixture containing synaptosomes (1.27 mg protein), NADP+ 1 mmol.L-1, L-glutamic dehydrogenase 50 U, CaCl2 1.3 mmol.L-1 (or EGTA 1.3 mmol.L-1) was transferred to the stirred cuvette in the fluorometer at 37 degrees C for 5 min. Then, AMG-1 (or adenosine) was added. Released glutamate (eg. fluorescent intensity) was monitored following the addition of 30 mmol.L-1 KCl. The results indicate that Ca2+-dependent glutamate release from depolarized synaptosomes is inhibited by AMG-1 (0.1 mmol.L-1) or adenosine (0.3 mmol.L-1). The action of AMG-1 seems to be similar to that of adenosine. However, no change was found on Ca2+-independent release of glutamate. This implies that the protective effect of AMG-1 against cerebral ischemia may be partially due to inhibiting glutamate release from nerve terminal via the activation of adenosine A1 receptor.
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PMID:[Effect of AMG-1 and adenosine on glutamate release from synaptosomes in rats]. 803 Apr 10

The NADP(+)-dependent hexameric glutamate dehydrogenase from Escherichia coli has been crystallized as the apo-enzyme and also in the presence of its substrates 2-oxoglutarate, glutamate or NADP+, using either pulsed equilibrium microdialysis, or the hanging drop method of vapour diffusion. Three non-isomorphous, but related, crystal forms have been obtained, all of which belong to the orthorhombic system and are most likely to be in space group P2(1)2(1)2(1). One crystal form is grown from ammonium sulphate, includes the apoenzyme and the binary complexes with 2-oxoglutarate or NADP+, and has cell dimensions a = 157.5 A, b = 212.5 A, c = 101.0 A with a hexamer in the asymmetric unit. Crystallizations using glutamate as the precipitant produced two further crystal forms, which show significant changes in the b and c cell dimensions with respect to the apo-enzyme crystals, with parameters a = 160.0 A, b = 217.5 A c = 92.4 A and a = 160.0 A, b = 223.0 A c = 92.4 A, respectively. X-ray diffraction photographs taken with synchrotron radiation show measurable reflections to beyond 3.0 A resolution.
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PMID:Crystallization of the NADP(+)-dependent glutamate dehydrogenase from Escherichia coli. 826 29

We describe an improved enzymatic ultraviolet absorbance method for assaying creatinine in serum, plasma, and urine. Creatinine is hydrolyzed by creatinine iminohydrolase (EC 3.5.4.21) to ammonia and N-methylhydantoin. The ammonia produced combines with 2-oxoglutarate and NADPH in the presence of glutamate dehydrogenase to yield glutamate and NADP+. The consumption of NADPH, measured by a two-point fixed-time assay, is proportional to the amount of creatinine in the sample. The assay is carried out in two steps: The first step eliminates background absorbance in hyperlipemic samples and endogenous ammonia through a "clearing system" and an isocitrate dehydrogenase-based "ammonia scavenger system"; the second step starts creatinine measurement. The method affords a simple, rapid, and sensitive assay with good precision and extended linearity; it employs working solutions stable at least 4 months. Test results compare closely with those of the isotope dilution-mass spectrometry Definitive Method, the HPLC procedure, and the fuller's earth method. The proposed method is not subject to interference from several metabolites or from the 72 drugs tested. Because it is easily automated, the method is suitable for routine work in clinical laboratories.
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PMID:A step forward in enzymatic measurement of creatinine. 828 20


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