THE MECHANISM OF SUBSTRATE AND COENZYME BINDING TO CLOSTRIDIAL GLUTAMATE-DEHYDROGENASE DURING OXIDATIVE DEAMINATION

The binding of NAD+ and L-Glutamate to glutamate dehydrogenase (GDH) f rom Clostridium symbiosum has been investigated by stopped-flow fluore scence spectroscopy. The formation of the binary complexes produces li ttle change in the protein fluorescence but formation of the ternary c omplex results in quenching of its fluorescence with a maximum value o f 40%. This finding, coupled with the finding that a step prior to hyd ride transfer but subsequent to ternary complex formation is rate limi ting, has enabled us to monitor the kinetics of ternary complex format ion in detail. The ternary complex can be formed via the GDH-NAD+ or t he GDH-L-Glu binary complexes, but the route via the GDH-NAD+ binary c omplex is the preferred pathway. The equilibrium and rate constants fo r the formation of the two binary complexes and the ternary complex fo rmed via the two possible pathways have been determined. These studies have revealed an interaction between the coenzyme-binding site and th e substrate-binding site, which lead to a decrease in the binding cons tant for the second substrate binding to the enzyme. The free energy c oupling between the binary and ternary complexes is about 2.4 - 2.8 kJ . mol-1. We propose that there is a further isomerisation of the tern ary complex, which is rate limiting for the steady-state turnover of t he enzyme. Formation of this complex is characterised by an increased negative interaction, with a free energy coupling between these comple xes of 6.3 - 11.6 kJ . mol-1.