The mechanism of glutamate dehydrogenase reaction. IV. Evidence for random and rapid binding of substrate and coenzyme in the burst phase.

Stopped flow studies of the oxidative deamination of L-glutamate by TPN and beef liver glutamate dehydrogenase were performed at pH 6.5 and 7.6. At each pH value, the initial burst slopes obey an equation of the form: e/v = 40 + dd(TPN) + h/bglutamate) + &/(TPN)(L-glutamate). The agreement of dissociation constants for enzyme-TPN and enzyme-L-glutamate binary complexes obtained from the r#~ values with those obtained independently in equilibrium studies supports mechanisms which include random and rapid binding of substrate and coenzyme at the active site. The lack of effect of preincubation with either reactant on the transient state kinetics eliminates the possibility of any kinetically important slow isomerization steps in the formation of binary complexes. Comparison of the limiting Michaelis constants with the dissociation constants for TPN and L-glutamate shows that there is a cooperativity between coenzyme and substrate in the formation of an enzyme-TPN-L-glutamate ternary complex.