Abstract
d-β-Hydroxybutyrate dehydrogenase ( d-3-hydroxybutyrate: NAD + oxidoreductase, EC 1.1.1.30) is a lipid-dependent enzyme which has an absolute and specific rquirement for phosphatidylcholine for function. Chemical derivatization studies using 1,2-cyclohexanedione, an arginine-specific reagent, have been carried out on the purified enzyme devoid of lipid as well as on the enzyme reactivated with phospholipid. Cyclohexanedione inactivated the active enzyme-phospholipid complex and the lipid-free enzyme was rendered inactivatable by phospholipid. From kinetic studies and by direct chemical derivatization studies with [1- 14C]cyclohexanedione, we find that incorporation of a single cyclohexanedione molecule per enzyme monomer resulted in complete loss of enzymic activity. The presence of NADH or NAD +, cofactors for the enzyme, offered no protection for the rate of inactivation. The substrates β-hydroxybutyrate and acetoacetate with or without coenzyme gave little or no protection. However, 2-methyl malonate, a competitive inhibitor for β-hydroxybutyrate, strongly protected against inactivation. These studies indicate that: (1) a single arginine serves a vital role and is essential for function; (2) the arginine is located in the proximity of the substrate binding site.
Published Version
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