Effect of pH on sheep liver sorbitol dehydrogenase steady-state kinetics.

The variation with pH of the kinetic parameters for sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase has been studied over the pH 5-10 range. The reaction is compulsory ordered in both directions with the coenzyme as the leading substrate, and the rate-determining step in either direction is the enzyme-coenzyme product dissociation. Throughout the pH range, the lack of a primary kinetic isotope effect on Vm with (2H8) sorbitol confirms that the ternary complexes are not of rate-determining significance under maximum velocity conditions. The association rate constants for NAD and NADH increase and decrease, respectively, towards high pH. NAD binding to the enzyme is dependent upon pK values of 9.2 and 9.6. Whereas the dissociation rate constant for NAD release from the enzyme shows no pronounced variation with pH, NADH release is dependent upon pK values of 7.2 and 7.7. The kinetic constants that characterize the dependence on substrate concentration of the steady-state rate of catalysis vary with pH in accordance with a single pK of 7.1 for sorbitol oxidation and of 7.7 for fructose reduction. These pK values reflect the ionization properties of a catalytically essential group, which is tentatively considered to be either the H2O/OH- ligand binding to the catalytic zinc atom or a histidine residue. Catalysis by sorbitol dehydrogenase, due to the absence of a second ionization contribution, appears not to involve any obligatory step of proton transfer to solution at the ternary complex level. A mechanism for sorbitol dehydrogenase catalysis is proposed.