Modification of arginines in D-beta-hydroxybutyrate dehydrogenase.
D-beta-Hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate: NAD+ oxidoreductase, EC 1.1.1.30) is a lipid-dependent enzyme which has an absolute and specific requirement 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 beta-hydroxybutyrate and acetoacetate with or without coenzyme gave little or no protection. However, 2-methyl malonate, a competitive inhibitor for beta-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.
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002417:Cattle; D003512:Cyclohexanones / Q000494:pharmacology; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008314:Malonates / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism
D003512:Cyclohexanones; D008314:Malonates; D010743:Phospholipids; D009243:NAD; C018031:1,2-cyclohexanedione; D001120:Arginine; D006884:Hydroxybutyrate Dehydrogenase; C005230:methyl malonate