Isotopic Analysis of the Reaction Catalyzed by Glycerol Dehydrogenase

Glycerol dehydrogenase catalyzes the reversible NAD⁺-dependent oxidation of glycerol to form dihydroxyacetone. Initial velocity, product, and dead-end inhibition studies performed for the forward and reverse reactions support an ordered kinetic mechanism with NAD⁺ binding first and NADH released last. A monovalent cation is required for enzymatic activity and glycerol binding, with K⁺ having the highest activity as measured by V. The pH dependence of the kinetic parameters V and V/K_glycerol, as well as the temperature dependence of the V pH profile, suggested that an enzymic carboxylate group functions as a base in catalysis. The pH dependence of the primary deuterium kinetic isotope effect shows that ^DV/k_glycerol, increases from a pH-independent value of 1.15 at high pH values to a pH-independent value of 2.44 at low pH values. ^DV exhibits a similar pH dependence, increasing from a pH-independent value of 2.57 at high pH values to a pH independent value of 4.88 at low pH values. A chemical mechanism for enzymatic glycerol oxidation is proposed based on the data.