Possible occurrence for histidyl and cysteyl residues in the catalytic center of rat liver mitochondrial D (-)-beta-hydroxybutyrate dehydrogenase.
1. Rat liver mitochondrial D(-)-beta-hydroxybutyrate dehydrogenase (submitochondrial particles and partially purified preparation) is inhibited by some dicarboxylates, especially by malonate and succinate. The inhibition is reversible and competitive with beta-hydroxybutyrate while uncompetitive with acetoacetate, NAD and NADH: the inhibition is maximal at pH 6 and decrease with increasing pH. 2. Diethylpyrocarbonate (which reacts preferentially with histidyl residues at pH 6.6) inactivates the dehydrogenase at pH 6.1, beta-hydroxybutyrate protects against inactivation, this inactivation being almost completely released by hydroxylamine. The diethylpyrocarbonate-treated enzyme shows an absorbance increase at 242 nm which is characterisitic of reaction between diethylpyrocarbonate and histidyl residue. 3. The optimum pH of the enzyme for beta-hydroxybutyrate oxidation is around 8.2, while for acetoacetate reduction, the optimum pH is around 7. 4. All these results favour the existence of a histidyl residue in the catalytic center and taking into account previous results concerning the effect of thiol reagents on the same enzyme and especially, the protective effect of NAD+ and NADH against these reagents [11] we discuss the possible occurrence of, at least, one histidyl and one cysteyl residue on the catalytic center.
D000090:Acetoacetates / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003545:Cysteine; D003998:Dicarboxylic Acids / Q000494:pharmacology*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008314:Malonates / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats; D013386:Succinates / Q000494:pharmacology
D000090:Acetoacetates; D002729:Chloromercuribenzoates; D003998:Dicarboxylic Acids; D005561:Formates; D008314:Malonates; D013386:Succinates; D006639:Histidine; D006884:Hydroxybutyrate Dehydrogenase; D003545:Cysteine; D004047:Diethyl Pyrocarbonate