Spectra of glutamate dehydrogenase with diethylstilbestrol.

Glutamate dehydrogenase displays hyperchromicity at 256 nm and at 276 nm upon binding of diethylstilbestrol. Increase in absorbancy is linear at both regions up to 250 micrometer DES, and becomes parabolic at higher concentration of DES. ADP in the presence of DES causes decrease in absorbancy at 256 nm; absorbancy at 276 nm increased by DES is not affected by ADP. DES prevents spectral effects produced by GTP (decrease in absorbancy at 254 nm and at 276 nm). ADP still decreases absorbancy at 254 nm, leaving the 276 nm region unchanged. ADP enhances spectral effects produced by GTP. GTP, however, prevents changes produced by ADP.     

Spin-labeling studies of beef-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase was spin labeled with a nitroxide derivative of parachloromercuribenzoate. The ESR spectrum was of the immobilized type and the labeling yield 0.6 mole of spin label bound per mole of protomer under standard conditions. The specific activity of the labeled enzyme was not modified but the activation by ADP abolished. Inhibition by GTP was not altered but the ESR spectrum showed that the bound spin label was further immobilized in the presence of GTP and NADPH. In the presence of the coenzyme NADPH, the labeling yield decreased to half its initial value. Such a protection effect was observed neither with NADH nor with ADP.     

Folates as inhibitors of glutamate dehydrogenase.

Folates and tetrahydrofolates inhibit beef liver glutamate dehydrogenase (EC 1.4.1.2). Double reciprocal plats indicate a competitive inhibition for alpha-ketoglutarate-glutamate by folic acid and methotrexate and a complex or mixed type for NAD-NADH site. Pteroic acid is not inhibitory at the concentrations studied. The addition of up to four gamma-linked glutamyl residues to folic and tetrahydrofolic acids increases the inhibition. Further chain elongation of the gamma-peptide had no effect on the inhibitory activity. The p-aminobenzoate poly-gamma-glutamates were less inhibitory than the corresponding folyl polyglutamates.     

NAD-linked glutamate dehydrogenase in Trypanosoma cruzi.

1. Epimastigotes of Trypanosoma cruzi, Tulahuén strain, contained a NAD-linked glutamate dehydrogenase (EC 1.4.1.3), in addition to the already known NADP-linked enzyme enzyme (EC 1.4.1.4). 2. The partially purified NAD-linked enzyme had a higher molecular weight and was much more labile than the NADP-linked enzyme, and was inhibited by purine nucleotides. 3. These results further emphasize the difference in glutamate metabolism between the parasite and its mammalian host.     

Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition.

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000.     

Temperature-sensitive glutamate dehydrogenase mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium defective in glutamate dehydrogenase activity were isolated in parent strains lacking glutamate synthase activity by localizcd mutagenesis or by a general mutagenesis combined with a cycloserine enrichment for glutamate auxotrophs. Two mutants with temperature-sensitive phenotypes had glutamate dehydrogenase activities that were more thermolabile than that of an isogenic control strain. Eight other mutants had less than 10% of the wild-type glutamate dehydrogenase activity. All the mutations were cotransducible with a Tn10 element (zed-2:Tn10) located at approximately 23 U on the S. typhimurium linkage map. These data strongly indicate that this region contains the structural gene (gdhA) for glutamate dehydrogenase.     

Kinetic studies of dogfish liver glutamate dehydrogenase.

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.     

Regulation of glutamate dehydrogenase in Bacillus subtilis.

The activity of the nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase in Bacillus subtilis was influenced by the carbon source, but not the nitrogen source, in the growth medium. The highest specific activity for this enzyme was found when B. subtilis was grown in a minimal or rich medium that contained glutamate as the carbon source. It is proposed that glutamate dehydrogenase serves a catabolic function in the metabolism of glutamate, is induced by glutamate, and is subject to catabolite repression.     

Binding of ethidium to liver glutamate dehydrogenase.

Liver glutamate dehydrogenase forms a complex with ethidium bromide in solution; binding parameters of this complex depend on pH and enzyme concentration, but are independent of the ionic strength of solution. Binding of ethidium bromide occurs outside the coenzyme binding site, but appears to be closely related to the oligomer association sites of the enzyme.     

Heat denaturation of bovine liver glutamate dehydrogenase.

Heat denaturation of bovine liver glutamate dehydrogenase occurred at 47 degrees with loss of enzyme activity and formation of inactive, insoluble protein. Fractional loss of catalytic activity coincided with alteration in protein fluorescence and solubility for a corresponding percentage of protein molecules. Operationally, at 50% denaturation, one-half of the total population of enzyme molecules is fully active catalytically and soluble and the other half of the protein molecule population is completely inactive catalytically and insoluble.     

Phosphorylation of NAD-dependent glutamate dehydrogenase from yeast.

The NAD-dependent glutamate dehydrogenase from Candida utilis was isolated from 32P-labeled cells following enzyme inactivation promoted by glutamate starvation and found to exist in a phosphorylated form. Analysis of purified, fully active NAD-dependent glutamate dehydrogenase (a form) and inactive NAD-dependent glutamate dehydrogenase (b form) for alkalilabile phosphate revealed that the a form contained 0.09 +/- 0.06 mol of phosphate/mol of enzyme subunit and b form 1.25 +/- 0.06 mol of phosphate/mol of enzyme subunit. Phosphorylation caused a 10-fold reduction in enzyme specific activity. Dephosphorylation (release of 32P) and enzyme reactivation occurred on incubation with cell-free yeast extracts, indicating the presence of a phosphoprotein phosphatase in such preparations.     

Properties of glutamate dehydrogenase from Bacillus subtilis.

$\text{Bacillus subtilis}$ strain 168 possesses an NAD-dependent glutamate dehydrogenase. The level of this enzyme is influenced by the stage of growth, the source of nitrogen, and a high rate of tryptophan biosynthesis. The enzyme appears to serve an anabolic function and, therefore, must be considered as a possible route for the incorporation of inorganic nitrogen into an organic form.     

Reaction of formiminoglutamate with liver glutamate dehydrogenase.

1. Kinetic aspects of the reaction between crystalline bovine liver glutamate dehydrogenase and formiminoglutamate were investigated to establish the conditions under which the latter may interfere with the assay of glutamate by using glutamate dehydrogenase and to explain why formiminoglutamate accumulates in vivo after histidine loading, although it can react with glutamate dehydrogenase. The Km and Vmax. values were compared with those of the enzyme reacting with glutamate. At pH 7.4 Km for formiminoglutamate was much higher and Vmax. much lower than the values for glutamate. 2. The equilibrium constant at pH 7.0 was 0.017 micrometer with formiminoglutamate, i.e. about one two-hundredths that with glutamate. 3. In vivo the interaction between glutamate dehydrogenase and formiminoglutamate is minimal even when the concentration of the latter in the liver is greatly raised, as in cobalamine or folate deficiency after histidine loading. 4. At pH 9.3, i.e. under the conditions for the assay of glutamate by glutamate dehydrogenase, formiminoglutamate reacts readily with the enzyme.