Mitochondrial d-β-Hydroxybutyrate Dehydrogenase

A kinetic analysis of the reaction mechanism of d-β-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme from mitochondria, was made for both the membrane-bound enzyme in submitochondrial particles and for a purified soluble preparation. The results are consistent with an Ordered Bi Bi mechanism (nomenclature of Cleland) where NAD is the first substrate to add to the enzyme and NADH is the last product to leave. The mechanism is the same whether or not the enzyme is bound to the membrane. The dissociation constants for NADH and NAD of the soluble enzyme reactivated with mitochondrial phospholipid are the same as that for the membrane-bound enzyme. Replacing mitochondrial phospholipid with purified lecithin when reactivating the soluble enzyme results in tighter binding of these nucleotides to the enzyme. These studies (a) indicate that the enzyme is not appreciably altered by release from the membrane and by the purification procedure, (b) that the lipid environment of the enzyme in the membrane more closely approximates that of mitochondrial phospholipid than lecithin alone, and (c) provide insight into how phospholipid can modulate important parameters of enzymic catalysis.