.alpha.-Ketoglutaric acid: solution structure and the active form for reductive amination by bovine liver glutamate dehydrogenase

Biochemistry ◽  
1982 ◽  
Vol 21 (2) ◽  
pp. 339-345 ◽  
Author(s):  
Tenkasi S. Viswanathan ◽  
Robert E. Johnson ◽  
Harvey F. Fisher
Keyword(s):  
Acid Solution ◽  
Glutamate Dehydrogenase ◽  
Solution Structure ◽  
Reductive Amination ◽  
Active Form ◽  
Bovine Liver

scholarly journals Kinetic studies of dogfish liver glutamate dehydrogenase

1979 ◽  
Vol 177 (2) ◽  
pp. 449-459 ◽  
Author(s):  
A H Electricwala ◽  
F M Dickinson
Keyword(s):  
Glutamate Dehydrogenase ◽  
Kinetic Data ◽  
Reductive Amination ◽  
Initial Rate ◽  
Kinetic Studies ◽  
Bovine Liver ◽  
Binding Studies ◽  
Degree Of Protection ◽  
Binding Sequence ◽  
Kinetic Features

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.

scholarly journals Lysine and tyrosine in the NADH inhibitory site of bovine liver glutamate dehydrogenase.

1981 ◽  
Vol 256 (22) ◽  
pp. 11866-11872
Author(s):  
K.V. Saradambal ◽  
R.A. Bednar ◽  
R.F. Colman
Keyword(s):  
Glutamate Dehydrogenase ◽  
Bovine Liver ◽  
Inhibitory Site

scholarly journals Sequence of Bovine Liver Glutamate Dehydrogenase

1971 ◽  
Vol 246 (8) ◽  
pp. 2374-2399 ◽  
Author(s):  
Michael Landon ◽  
Dennis Piszkiewicz ◽  
Emil L. Smith
Keyword(s):  
Glutamate Dehydrogenase ◽  
Bovine Liver

scholarly journals Sequence of Bovine Liver Glutamate Dehydrogenase

1973 ◽  
Vol 248 (9) ◽  
pp. 3067-3081 ◽  
Author(s):  
Dennis Piszkiewicz ◽  
Michael Landon ◽  
Emil L. Smith
Keyword(s):  
Glutamate Dehydrogenase ◽  
Bovine Liver

scholarly journals Sequence of Bovine Liver Glutamate Dehydrogenase

1971 ◽  
Vol 246 (8) ◽  
pp. 2400-2418 ◽  
Author(s):  
William J. Brattin ◽  
Emil L. Smith
Keyword(s):  
Glutamate Dehydrogenase ◽  
Bovine Liver

Properties of NAD-dependent glutamate dehydrogenase from the tylosin producer Streptomyces fradiae

1997 ◽  
Vol 43 (11) ◽  
pp. 1005-1010 ◽  
Author(s):  
Kien Trung Nguyen ◽  
Lieu Thi Nguyen ◽  
Jan Kopecký ◽  
Vladislav Běhal
Keyword(s):  
Key Words ◽  
Glutamate Dehydrogenase ◽  
Reductive Amination ◽  
Divalent Cations ◽  
Ammonium Assimilation ◽  
Alkaline Ph ◽  
Streptomyces Fradiae ◽  
Oxidative Deamination

Glutamate dehydrogenase is an enzyme responsible for ammonium assimilation and glutamate catabolism in organisms. The tylosin producer Streptomyces fradiae possesses both NADP- and NAD-dependent glutamate dehydrogenases. The latter enzyme was purified 498-fold with a 7.5% recovery by a six-step protocol. The enzyme is composed of two subunits, each of Mr 47 000, and could form active aggregates of four or eight subunits. Its activity was inactivated by alkaline pH or temperatures of −20 °C or above 40 °C. Activities assayed in the direction of oxidative deamination and reductive amination were optimal at pH 9.2 and 8.8, respectively, and at temperatures of 30–35 °C. No activity was found when NAD(H) was replaced with NADP(H). The Km values were 32.2 mM for L-glutamate, 0.3 mM for NAD+, 3.4 mM for 2-ketoglutarate, 14.2 mM for NH4+, and 0.05 mM for NADH. Deamination activity was partially inhibited by adenyl nucleotides and several divalent cations; amination activity was not affected by the nucleotides but significantly inhibited by Cu2+ or Ni2+.Key words: Streptomyces fradiae, NAD-dependent glutamate dehydrogenase, purification, properties.

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