Glutamic dehydrogenase. I. The effect of coenzyme on the sedimentation velocity and kinetic behavior.

It has been reported that high concentrations of reduced diphosphopyridine nucleotide as well as l,lO(ortho)-phenanthroline will cause dissociation of crystalline beef liver glutamic dehydrogenase as observed by the sedimentation behavior of the enzyme in the ultracentrifuge (1). Although this enzyme undergoes dissociation upon dilution (2), the effect of DPNH or l,lO-phenanthroline may be obtained at enzyme concentrations at which there should be essentially no dissociation at all. This result suggested that the extent of association or dissociation of the enzyme might play a role in the catalytic function of the enzyme. The purpose of the present investigation, then, was to study the effects of the different coenzymes for the reaction on the sedimentation and kinetic behavior and to determine the relationship between initial velocities of the enzymatic reaction obtained kinetically and the sedimentation behavior as observed in the ultracentrifuge. It will be shown in this paper that the coenzymes DPN, DPNH, TPN and TPNH, all of which are active with glutamic dehydrogenase, influence the sedimentation behavior of the enzyme at levels which are identical with the coenzyme concentrations used in the determination of the kinetic constants. Consequently, the rates of the enzymatic reaction are determined by the degree of association or dissociation of the enzyme. The data presented in this paper show that the enzyme, when dissociated, is inactive. The kinetic and sedimentation data also show that the active site for all the coenzymes is the same, but that in addition, DPN and DPNH are bound to a second, noncatalytically active, site.