Abstract

l-Glutamate dehydrogenase ( l-glutamate: NAD(P) + oxidoreductase (deaminating EC 1.4.1.3)), cooperatively binds NADP + and α-ketoglutarate in a highly stable dead-end complex exhibiting a near ultraviolet difference spectrum characteristic of red shifts of tryptophan and oxidized nicotinamide absorbance. The requirements of an intact amide on the micotinamide moiety and two carboxyl groups on the substrate for a 200-fold heterotropic cooperativity in binding are demonstrated by the use of coenzyme and substrate analogs. The agreement of the binary complex dissociation constants calculated from the concentration dependence of the formation of the abortive complex with those determined directly shows that both coenzyme and α-ketoglutarate are bound at the same site in the binary and ternary complexes. By analogy it is inferred that NADP + and l-glutamate bind to the enzyme to form the active complex with a cooperativity similar to that demonstrated for the dead-end complex. The ability of the enzyme to form the NADP + -α-ketoglutarate dead-end complex and other stable complexes is pertinent to the catalytic mechanisms proposed for glutamate dehydrogenase. This ability also provides a mechanism through which both the in vivo direction and rate of catalysis can be selectively and sensitively controlled by the cooperative binding of the reactants and products themselves.

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