Kinetic studies of glutamate dehydrogenase with glutamate and norvaline as substrates. Coenzyme activation and negative homotropic interactions in allosteric enzymes

Abstract

1. Kinetic studies of glutamate dehydrogenase were made with wide concentration ranges of the coenzymes NAD+ and NADP+ and the substrates glutamate and norvaline. Initial-rate parameters were evaluated. 2. Deviations from Michaelis–Menten behaviour towards higher activity were observed with increasing concentrations of either coenzyme with glutamate as substrate, but not with norvaline as substrate. 3. In phosphate buffer, pH7·0, Lineweaver–Burk plots with either coenzyme as variable and a constant, large glutamate concentration showed three or four linear regions of different slope with relatively sharp discontinuities. Maximum rates obtained by extrapolation and Michaelis constants for the coenzymes increased in steps with increase of coenzyme concentration. 4. In the absence of evidence of heterogeneity of the enzyme and coenzyme preparations, the results are interpreted in terms of negative homotropic interactions between the enzyme subunits. It is suggested that sharp discontinuities in Lineweaver–Burk plots or reciprocal binding plots may be characteristic of this new type of interaction, which can be explained in terms of an Adair–Koshland model, but not by the model of Monod, Wyman & Changeux.