THE MECHANISM OF SUBSTRATE AND COENZYME BINDING TO CLOSTRIDIAL GLUTAMATE-DEHYDROGENASE DURING REDUCTIVE AMINATION

The binding of NADH and 2-oxoglutarate to glutamate dehydrogenase (GDH ) from Clostridium symbiosum has been studied by fluorescence spectros copy. The K-d values for the binding of these ligands have been measur ed by titration of either the nucleotide or protein fluorescence. Duri ng ternary complex formation, the substrate and coenzyme binding sites interact in a positive cooperative manner, but steady-state studies r eveal a decrease in affinity of the catalytic complex indicative of ne gative cooperativity. It was possible to determine the kinetics of for mation of the glutamate-dehydrogenase - NADH complex by stopped-flow f luorescence spectroscopy but formation of the glutamate-dehydrogenase- 2-oxoglutarate complex was optically silent. Ternary complex formation was characterized by a large quench in protein fluorescence. The bind ing of NADH to the glutamate-dehydrogenase-2-oxoglutarate binary compl ex is characterised by a linear increase in the association rate const ant, consistent with a one-step binding process. However, the binding of 2-oxoglutarate to the glutamate-dehydrogenase-NADH binary complex i s characterised by a decrease in the rate for the observed transient. This suggests that 2-oxoglutarate binds to a different conformation of the enzyme to that stabilized by NADH, and that the transition betwee n these different conformational forms is rate limiting for ternary co mplex formation. NADH and 2-oxoglutarate can therefore stabilize diffe rent conformational states of the enzyme. Collectively, these studies are suggestive of a kinetic model for ternary complex formation that i nvolves the oscillation of the free, binary, and ternary glutamate deh ydrogenase complexes between two different conformational states, term ed E(1) and E(2). The equilibrium constants for ternary complex format ion via the predominant pathway have been determined. The cooperativit y betwen the substrate and coenzyme binding sites can be accounted for by the displacement of the equilibria between the E(1) and E(2) state s because of their difference in affinities for NADH and 2-oxoglutarat e.