Glutamate Dehydrogenases of Thiobacillus novellus: KINETIC PROPERTIES AND A POSSIBLE CONTROL MECHANISM

Abstract

Kinetic analyses were conducted on two glutamate dehydrogenases isolated from the facultative chemoautotroph, Thiobacillus novellus. The enzymes were characterized as catalysts specific for the reversible oxidation and reduction of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate. The NAD-specific enzyme exhibited nonlinear kinetics in the assays of the reduction of NAD, but linear kinetics in the reverse direction. Addition of adenosine 5'-monophosphate to the assay system altered the kinetic parameters of this enzyme. The lines became linear and described the classical Michaelis-Menten kinetics. In addition, there was a dramatic increase in the Michaelis constants for ammonia, α-ketoglutarate, and NADH. The Michaelis constants for glutamate and NAD remained unchanged. The NADP-specific enzyme showed perfectly normal and linear kinetics. AMP had no effect on its kinetic properties. Both enzymes have been further characterized by their behavior on gel electrophoresis columns, molecular weight determinations by zone centrifugations in sucrose density gradients, sensitivity to inhibitors, substrate specificity, and interaction with macroions. The molecular weights are approximately the same, 120,000 ± 10,000.