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/Kglycerol, 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/Kglycerol 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.
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Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Blanchard
J S
JS
D000192:Actinomycetales / Q000201:enzymology; D002384:Catalysis; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism*; D013696:Temperature
D003903:Deuterium; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase