D-2-Hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp bulgaricus .1. Kinetic mechanism and pH dependence of kinetic parameters, coenzyme binding and substrate inhibition

The steady-state kinetics of D-2-hydroxy-4-methylvalerate dehydrogenase have been studied at pH 8.0 by initial velocity, product inhibition, and dead-end inhibition techniques. The mechanism is rapid-equilibrium ordered in the NAD(+) plus D-2-hydroxy-4-methylvalerate direction, and steady-state ordered in the other direction. In both cases coenzyme is the first substrate added and both the E-NADH-D-2-hydroxy-4-methylvalerate and E-NAD(+)-2-oxo-4-methylvalerate give rise to abortive complexes which cause excess substrate inhibition. Steady-state measurements show that the rate-limiting step in both directions at pH 8.0 is between formation of the enzyme-coenzyme-substrate ternary complex and the release of the first product of the reaction. Transient kinetics combined with primary kinetic deuterium isotope effects show that in the NADH --> NAD(+) direction there is a slow, rate-limiting rearrangement of the E-NADH-oxoacid complex while hydride transfer is very fast. The release of NAD(+) at pH 8.0 is 200-times faster than k(cat) (NADH --> NAD(+)) whereas the release of NADH is only 5-times faster than k(cat) (NAD(+) --> NADH). The pH dependence of NADH binding depends upon the presence of two ionizable residues with a pK(a) of about 5.9. The pH dependence of kinetic parameters is explained by a third ionizable residue with pK(a) values 7.2 (in the E-NADH complex) and less than or equal to 6.4 (in the E-NAD(+) complex) which may be the proton donor and acceptor for the chemical reaction. At pH 6.5 the mechanism changes in the NADH --> NAD(+) direction to be partly limited by the chemical step with a measured primary kinetic isotope effect of 5.7 and partly by an only slightly faster dissociation of NAD(+). In addition the inhibition by excess oxo-4-methylvalerate is more pronounced. The mechanism implies that removing the positive charges created by the the two groups which control coenzyme affinity could both enhance the catalytic rate at pH 6.5 and diminish excess substrate inhibition to provide an enzyme better suited to the bulk synthesis of D-2-hydroxyacids.