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:1.4.1.2 (glutamate dehydrogenase)
4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A highly conserved lysine at position 128 of Escherichia coli glutamate dehydrogenase (GDH) has been altered by site-directed mutagenesis of the gdhA gene. Chemical modification studies have previously shown the importance of this residue for catalytic activity. We report the properties of mutants in which lysine-128 has been changed to histidine (K128H) or arginine (K128R). Both mutants have substantially reduced catalytic centre activities and raised pH optima for activity. K128H also has increased relative activity with amino acid substrates other than glutamate, especially L-norvaline. These differences, together with alterations in Km values, Kd values for NADPH and Ki values for D-glutamate, imply that lysine-128 is intimately involved in either direct or indirect interactions with all the substrates and also in catalysis. These multiple interactions of lysine-128 explain the diverse effects of chemical modifications of the corresponding lysine in homologous GHDs. In contrast, lysine-27, another highly reactive residue in bovine GDH, is not conserved in all of the sequenced NADP-specific GDHs and is therefore not likely to be involved in catalysis.
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PMID:Multiple interactions of lysine-128 of Escherichia coli glutamate dehydrogenase revealed by site-directed mutagenesis studies. 314 42

Two mechanisms have been postulated for the formation of bound alpha-iminoglutarate intermediate during the glutamate dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate; one involves the nucleophilic attack of ammonia on a covalently bound Schiff base in the enzyme-NADPH-alpha-ketoglutarate complex, and the other involves the reaction of ammonia with the carbonyl group of alpha-ketoglutarate in the ternary complex. We have measured the rates of carbonyl oxygen exchange in the complex to unambiguously distinguish between these two mechanisms. We find that the loss of label in the carbonyl oxygen-labeled ternary complex is at least 10(5) times slower than the rate of the reductive amination reaction. Therefore, the former mechanism cannot be operative. We also find that (i) the carbonyl oxygen exchange in free alpha-ketoglutarate proceeds without any significant catalysis by its gamma-carboxylate group; (ii) this exchange reaction has energy parameters which are comparable to those observed for the hydration of simple aliphatic ketones; and (iii) the carbonyl oxygen exchange in bound alpha-ketoglutarate is slower than that in the free keto acid over a wide pH range. We conclude that the oxygen exchange in the free and bound alpha-ketoglutarate must occur via a gem-diol intermediate. The observation that the enzyme inhibits the reaction of water with alpha-ketoglutarate while it catalyzes the reaction of ammonia with the same keto acid points to an extraordinary recognition of ammonia by the enzyme. We interpret this observation by assuming that the enzyme-NADPH-alpha-ketoglutarate complex exists in two forms, a predominant form which is produced rapidly upon mixing the components together and an unstable form which is produced in trace amounts from the predominant form via a gem-diol intermediate. These two forms are presumed to differ in the spatial relationship of the carbonyl group to the enzyme functional groups. The carbonyl group in the unstable form is assumed to be surrounded by the same enzyme groups as the iminium ion is in the bound iminoglutarate complex. We ascribe the remarkable catalysis of the ammonia reaction and the inhibition of the water reaction by the enzyme to the opposing interactions of the iminium and carbonyl groups with these surrounding enzyme groups.
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PMID:Mechanism of formation of bound alpha-iminoglutarate from alpha-ketoglutarate in the glutamate dehydrogenase reaction. A chemical basis for ammonia recognition. 333 11

There were significant changes in enzyme activities and concentrations of metabolites in the blood and liver of cows with fatty livers when compared to normal cows. Blood and liver samples were taken from cows at the abattoir immediately after slaughter. The liver was checked for pathological signs and the samples were divided according to the degree of fatty changes. Three groups were studied: controls showing no gross pathological signs, mild fatty infiltration and severe infiltration. In cows with fatty liver, there were significant increases in the serum activities of isocitric dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6PDH), glutamic dehydrogenase (GLDH), lactic dehydrogenase (LDH), malic dehydrogenase (MDH), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and acid phosphatase (ACP). In the fatty liver, the activities of the enzymes, ICDH, G6PDH, LDH, MDH, ALP and malic enzyme (ME) were significantly higher, while sorbitol dehydrogenase (SDH) was significantly lower. While serum total lipid decreased, the opposite was seen in the liver with higher lipid content, mainly due to triglycerides and cholesterol esters. The significant increases in the NADPH generating enzymes ME, ICDH, G6PDH and MDH, which are required for fatty acid synthesis, suggest that the lipids accumulated in the liver are not only of extrahepatic origin, mobilized into the liver, but also arise from increased lipid synthesis in the liver which is induced during the laying down of fat in the liver. Measurement of the serum NADPH generating enzymes may serve as a useful biochemical test specific for fatty liver in cows.
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PMID:Biochemical changes associated with the fatty liver syndrome in cows. 339 48

Direct transfer of NADPH between two NADP-dependent dehydrogenases, isocitrate dehydrogenase and glutamate dehydrogenase, has been investigated. These enzymes have opposite stereospecificity for hydrogen transfer to the coenzyme. In contrast with the general direct-transfer mechanism postulated for NAD-dependent dehydrogenases [Srivastava & Bernhard (1986) Science 234, 1081-1086], no evidence for direct transfer in either direction was found for these NADP-dependent dehydrogenases.
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PMID:Absence of direct coenzyme transfer in an A-B dehydrogenase system. 343 43

D-Glucose increased the cytosolic NADH/NAD+ ratio (but not the cytosolic NADPH/NADP+ ratio), augmented O2 uptake, raised the ATP/ADP ratio, decreased 86Rb outflow, and stimulated insulin release in tumoral insulin-producing cells of the RINm5F line. L-Leucine and 4-methyl-2-oxopentanoate also stimulated insulin secretion. In the RINm5F cells, as in normal islet cells, the nonmetabolized analogue of L-leucine, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), activated glutamate dehydrogenase, augmented L-[U-14C]glutamine oxidation, and induced a more reduced state of cytosolic redox couples. However, in sharp contrast to either its effect in normal islet cells or that of D-glucose in the tumoral cells, BCH severely decreased O2 uptake, lowered the ATP/ADP ratio, increased 86Rb outflow, and inhibited insulin release in the RINm5F cells. These findings are interpreted to support the concept that the rate of ATP generation represents an essential determinant of the secretory response of insulin-producing cells to nutrient secretagogues.
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PMID:Opposite effects of D-glucose and a nonmetabolized analogue of L-leucine on respiration and secretion in insulin-producing tumoral cells (RINm5F). 354 45

The nature of a general anion binding site that regulates NADPH binding to L-glutamate dehydrogenase has been explored. Dissociation constants for the enzyme-NADPH complex were measured by difference spectroscopy in the presence of phosphate, pyrophosphate, ADP and acetate ions. Whereas two molecules of phosphate, binding in a cooperative fashion, raise the Kd of the enzyme-NADPH complex 50-fold from 2.3 microM, a single pyrophosphate raises the Kd only 23-fold, disproving the notion that the anion binding site is simply the pyrophosphate binding site of NADPH. ADP raises the Kd of the enzyme-NADPH complex 2-fold for a given phosphate concentration, and formation of the enzyme-ADP complex is itself interfered with by phosphate and pyrophosphate, indicating that these anions interact with the same anion binding site. Acetate ion acts in a manner opposite to that of phosphate, pyrophosphate and ADP and reverses the weakening effect that these ions exert on NADPH binding, returning the Kd of the enzyme-NADPH complex to 2.3 microM. In the absence of these anions, however, acetate exerts no measurable effect on the Kd, suggesting an allosteric mechanism.
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PMID:The effects of an acetate-sensitive anion binding site on NADPH binding in glutamate dehydrogenase. 359 33

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.
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PMID:Properties of glutamate dehydrogenase purified from Bacteroides fragilis. 366 55

Functional group interactions involved in the formation of the glutamate dehydrogenase-NADPH binary complex have been studied by three independent but complementary approaches: the pH dependence of the overall dissociation constant measured by an improved differential spectroscopic technique; the pH dependence of the enthalpy of complex formation measured by flow calorimetry; and the pH dependence of the number of protons released to, or taken up from, the solvent in the complex formation reaction, measured by titration. We conclude that the coenzyme binds to the enzyme through three distinguishable interactions: a pH-independent process involving the binding of the reduced nicotinamide ring; a relatively weak "proton-stabilizing" process, occurring at low pH involving the shift at a pK of 6.3 in the free enzyme to 7.0 in the enzyme-NADPH complex; and a stronger "proton-destabilizing" process, occurring at a higher pH involving a shift of a pK of 8.5 in the enzyme down to 6.9 in the enzyme-NADPH complex. The proton ionization of the free enzyme involved in this third interaction exhibits some unusual thermodynamic parameters, having delta Go = +11.5 +/- 0.1 kcal mol-1, delta Ho = +19 +/- 1 kcal mol-1, and delta So = +23 eu. We show here that this proton ionization step is directly related to and indeed constitutes the "implicit" shift in enzyme macrostates which we have shown to be responsible for the existence of large highly nonlinear delta Cpo effects in the formation of this complex [Fisher, H. F., Colen, A. H., & Medary, R. T. (1981) Nature (London) 292, 271-272].
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PMID:NADPH binding induced proton ionization as a cause of nonlinear heat capacity changes in glutamate dehydrogenase. 371 28

The 2',3'-dialdehyde derivative of NADPH (oNADPH) acts as a coenzyme for the reaction catalyzed by bovine liver glutamate dehydrogenase. Incubation of 250 microM oNADPH with enzyme for 300 min at 30 degrees C and pH 8.0 yields covalent incorporation of 1.0 mol of oNADPH/mol of enzyme subunit. The modified enzyme has a functional catalytic site and is activated by ADP, but is no longer inhibited by high NADH concentrations and exhibits decreased sensitivity to GTP inhibition. Using the change in inhibition by 600 microM NADH or 1 microM GTP to monitor the reaction leads to rate constants of 44.0 and 41.5 min-1 M-1, respectively, suggesting that loss of inhibition by the two regulatory compounds results from reaction by oNADPH at a single location. The oNADPH incorporation is proportional to the decreased inhibition by 600 microM NADH or 1 microM GTP, extrapolating to less than 1 mol of oNADPH/mol of subunit when the maximum change in NADH or GTP inhibition has occurred. Modified enzyme is still 93% inhibited at saturating levels of GTP, although its K1 is increased 20-fold to 4.6 microM. The kinetic effects caused by oNADPH are not prevented by alpha-ketoglutarate, ADP, 5 mM NADH, or 200 microM GTP alone, but are prevented by 5 mM NADH with 200 microM GTP. Incorporation of oNADPH into enzyme at 255 min is 0.94 mol/mol of peptide chain in the absence of ligands but only 0.53 mol/mol of peptide chain in the presence of the protectants 5 mM NADH plus 200 microM GTP. These results indicate that oNADPH modifies specifically about 0.4-0.5 sites/enzyme subunit or about 3 sites/enzyme hexamer and that reaction occurs at a GTP-dependent inhibitory NADH site of glutamate dehydrogenase.
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PMID:Reaction of the 2',3'-dialdehyde derivative of NADPH at a nucleotide site of bovine liver glutamate dehydrogenase. 373 24

In porcine areolar placental epithelia, the following enzymes were demonstrated by histochemical methods after 30, 58, 80, 100, and 110 d of pregnancy, respectively: beta-N-acetyl-hexosaminidase, beta-galactosidase, beta-glucuronidase, alpha-mannosidase, acid phosphatase, alkaline phosphatase, nonspecific esterases, cytochrome oxidase, 5-nucleotidase, leucine aminopeptidase, adenosine triphosphatase, diaphorases (NADH, NADPH), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, succinate dehydrogenase, isocitrate dehydrogenase (NAD, NADP), beta-hydroxybutyrate dehydrogenase, glycero-3-phosphate dehydrogenase, NAD-glycero-3-phosphate dehydrogenase, glutamate dehydrogenase (NAD, NADP), lactate dehydrogenase. The results show that the enzyme activities remained almost unchanged during the period of investigation. Of the dehydrogenases, the diaphorases as well as succinate and lactate dehydrogenase demonstrated generally an intensive activity within the epithelia. The activity of the other dehydrogenases was only low. The activity of unspecific esterase was very intensive within the uterine epithelia but remarkably low within chorionic epithelia. Contrarily, the reaction of adenosine triphosphatase was more intensive within chorionic than uterine epithelia. All investigated glucosidases reacted distinctly positive within chorionic epithelia, but only beta-N-acetyl-hexosaminidase and beta-galactosidase in uterine epithelia. The high activity of acid phosphatase, especially within the chorionic epithelium, seems to be connected with uteroferrin, an iron-binding protein. The histochemical results are discussed in context with the function of the areolae in histiotrophic nutrition and iron transport.
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PMID:[Enzyme-histochemical studies of the pig placenta. II. Histotopics of enzymes in the areolar placenta epithelium]. 392 41


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