Glutamic Dehydrogenase

Abstract

The previous paper has shown that for beef liver glutamic dehydrogenase, an enzyme which undergoes reversible dissociation, there is a direct correlation between the initial velocity and sedimentation behavior of the enzyme as a function of the coenzyme concentration(1).The kinetic studies carried out showed that the effects of the coenzyme on the initial velocity of the enzymatic reaction may be interpreted in terms of the association-dissociation behavior of the enzyme, a conclusion which should extend to the effects of other compounds on the reaction velocity.It is the purpose of the present investigation to study the effects of various compounds on the kinetic and molecular properties of the enzyme.It will be shown that compounds which have rather striking effects on the over-all reaction velocity exert these effects by influencing the degree of association or dissociation of the enzyme molecule.Furthermore, these effects are dependent on the coenzyme used, since the coenzymes themselves have different effects on the association-dissociation reaction.Most of the compounds studied bear some structural relation to portions of either the di-or triphosphopyridine nucleotide molecule, and those which exhibit the most unusual kinetic effects are the adenosine nucleotides.However, in addition, some experiments have been performed with l,lO(ortho)phenanthroline, a compound that causes dissociation of the enzyme into four subunits (2).The results support the conclusions of the previous paper concerning the presence of active and nonactive binding sites on the enzyme molecule (1). EXPERIMENTALThe kinetic and sedimentation experiments were carried out as described in the previous paper (1).All kinetic experiments were conducted at pH 8.0 in 0.01 M Tris-acetate' buffer.When DPN or TPN was used as coenzyme, the concentration of Lglutamate used was 0.05 M. When DPNH or TPNH was used as coenzyme, the reaction mixture was 0.05 M with respect to oc-ketoglutarate and 0.1 M with respect to NH&l.Reagents-Most of the nucleotides used were obtained from the Sigma Chemical Company, except that UDP, CDP, 5'-IMP and the deamino analogue of DPN were products of Pabst Laboratories, and 2'-AMP was obtained from Schwarz Laboratories.Reduced deamino DPN was prepared by chemical reduction with hydrosulfite.