The Mechanism of Substrate and Coenzyme Binding to Clostridial Glutamate Dehydrogenase During Reductive Amination

Abstract

The binding of NADH and 2-oxoglutarate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been studied by fluorescence spectroscopy. The Kd values for the binding of these ligands have been measured by titration of either the nucleotide or protein fluorescence. During ternary complex formation, the substrate and coenzyme binding sites interact in a positive cooperative manner, but steady-state studies reveal a decrease in affinity of the catalytic complex indicative of negative cooperativity. It was possible to determine the kinetics of formation of the glutamate-dehydrogenase-NADH complex by stopped-flow fluorescence 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 binding of NADH to the glutamate-dehydrogenase-2-oxoglutarate binary complex is characterised by a linear increase in the association rate constant, consistent with a one-step binding process. However, the binding of 2-oxoglutarate to the glutamate-dehydrogenase-NADH binary complex is 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 between these different conformational forms is rate limiting for ternary complex formation. NADH and 2-oxoglutarate can therefore stabilize different conformational states of the enzyme. Collectively, these studies are suggestive of a kinetic model for ternary complex formation that involves the oscillation of the free, binary, and ternary glutamate dehydrogenase complexes between two different conformational states, termed E1 and E2. The equilibrium constants for ternary complex formation via the predominant pathway have been determined. The cooperativity between the substrate and coenzyme binding sites can be accounted for by the displacement of the equilibria between the E1 and E2 states because of their difference in affinities for NADH and 2-oxoglutarate.