title: Essential histidine residue in 3-ketosteroid-delta 1-dehydrogenase.
abstract: The variation with pH of kinetic parameters was examined for 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina. The Vmax/Km profile for 4-androstenedione indicates that activity is lost upon protonation of a cationic acid-type group with a pK value of 7.7. The enzyme was inactivated by diethylpyrocarbonate at pH 7.4 and the inactivation was substantially prevented by androstadienedione. Analyses of reactivation with neutral hydroxylamine, pH variation, and spectral changes of the inactivated enzyme revealed that the inactivation arises from modification of a histidine residue. Studies with [14C]diethylpyrocarbonate provided support for the idea that the 1-2 essential histidine residues are essential for the catalytic activity of the enzyme. Dye-sensitized photooxidation led to 50% inactivation of the enzyme with the decomposition of two histidine residues. This inactivation was also prevented by androstadienedione. Dancyl chloride caused a loss of the enzyme activity. Modifiers of glutamic acid, aspartic acid, cysteine, and lysine did not affect the enzyme activity. Butanedione and phenylglyoxal in the presence of borate rapidly inactivated the enzyme, indicating that arginine residues also have a crucial function in the active site. The data described support the previously proposed mechanism of beta-oxidation of 3-ketosteroid.
authors.affiliation: Department of Chemistry, Faculty of Science, Kanazawa University, Ishikawa.
authors.forename: H
authors.initials: H
authors.lastname: Matsushita
authors.forename: E
authors.initials: E
authors.lastname: Itagaki
mesh_terms: D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009615:Nocardia / Q000201:enzymology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010777:Photochemistry
chemical_list: D006639:Histidine; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase; D004047:Diethyl Pyrocarbonate