Studies of Glutamate Dehydrogenase

Abstract

The detailed investigation of the binding of NAD(P)H to beef liver glutamate dehydrogenase in the presence of the effectors ADP and GTP shows that each oligomer (composed of six identical polypeptide chains) has a total of 18 nucleotide binding sites. They can be subdivided into three classes. A first set of six binding sites, the active sites, is occupied by NAD(P)H with nearly the same affinity for both coenzymes, and accompanied by negative cooperativity between the binding sites within the oligomer. The second set of six binding sites, the nonactive sites, binds the coenzyme less tightly than the first set and ADP competes with NAD(P)H for these sites. The third set of six binding sites, the GTP sites, is responsible for the GTP binding. ADP abolishes the negative cooperativity of the NAD(P)H binding to the active sites and causes a weaker binding of both reduced coenzymes to these sites ( K E · ADP, NAD(P)H = 70 μM for NADH and 64 μM for NADPH). Also the reduced coenzymes weaken the binding of ADP to the enzyme. The dissociation constant for the enzyme · ADP complex is found to be 17 μM in the presence and 3 μM in the absence of NAD(P)H. The results are interpreted on the basis of free energy conservation in internal equilibria. The coupling energy Δ F c was calculated to be about 1 kcal/mol. In contrast to ADP/NAD(P)H, GTP and NAD(P)H enhance each other's binding to glutamate dehydrogenase causing positive cooperativity which is stronger in the case of NADH than of NADPH. The Hill coefficient is found to be 2.1 for NADH and 1.8 for NADPH. The nonactive sites are occupied by NADH in the presence of GTP with a dissociation constant of 23 μM indicating a three‐fold enhancement of the affinity of the enzyme for NADH in the presence of GTP. This is not the case with NADPH, where the dissociation constant is about 600 μM. This value is similar to the dissociation constant of the binary complex.The influence of glutamate on the enzyme · NAD(P)H binding is similar to the effect obtained in the presence of GTP; glutamate and GTP, however, are most likely bound to Merent sites. Glutamate induces strong positive cooperativity of three of the active sites within the oligomer characterized by a Hill coefficient of 2.4 for both reduced coenzymes. The extrapolations yield dissociation constants of 2–3 μM for both reduced coenzymes. The nonactive sites are occupied with dissociation constants of 57 μM for NADH and 700 μM for NADPH. These data indicate that glutamate has only a small effect on the binding of the reduced coenzyme to the nonactive site.