Evidence for the presence of one carboxyl group in the catalytic center of D-beta-hydroxybutyrate dehydrogenase. Inactivation and binding studies with carbodiimide reagents.
D-beta-Hydroxybutyrate dehydrogenase D-3-hydroxybutyrate: NAD+ oxidoreductase, EC 1.1.1.30), a phosphatidylcholine-requiring enzyme, was irreversibly inactivated by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) or a hydrophobic carbodiimide, N,N'-dicyclohexylcarbodiimide (DCCD). The inactivation is pseudo-first-order with a kinetic stoichiometry of about 1. Phospholipid-free apoenzyme was more sensitive towards these reagents than reconstituted phospholipid-enzyme or membrane-bound enzyme forms. Reduced coenzyme (NADH) protected the enzyme against the inactivation, while oxidized coenzyme (NAD+) in presence of 2-methylmalonate (a pseudo-substrate) gave a better protection. It was found that the phospholipid-free apoenzyme bound about 1 mol [14C]DCCD. This incorporation was prevented by EDAC, indicating that both reagents react at the same site. [14C]Glycine ethyl ester, a nucleophilic compound which reacts specifically with the carboxylcarbodiimide derivative was incorporated to the enzyme (1 mol [14C]glycine ethyl ester per polypeptide chain), whatever its form, in the presence of DCCD or EDAC. These results indicate the presence of one carboxyl group probably located at or near the coenzyme-binding site and near the interacting domain of the enzyme with phospholipid.
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D002234:Carbodiimides / Q000378:metabolism / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D008764:Methylmalonic Acid / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000494:pharmacology; D051381:Rats
D001051:Apoenzymes; D002234:Carbodiimides; D010743:Phospholipids; D009243:NAD; C022010:glycine ethyl ester; D004024:Dicyclohexylcarbodiimide; D008764:Methylmalonic Acid; D006884:Hydroxybutyrate Dehydrogenase; D005022:Ethyldimethylaminopropyl Carbodiimide; D005998:Glycine