Biosynthesis, isolation and properties of NAD-dependent formate dehydrogenase from the yeast Candida methylica.
NAD-dependent formate dehydrogenase (EC 1.2.1.2), was isolated from the methanol-utilizing yeast Candida methylica. Two purification techniques for the enzyme from the crude yeast extract have been developed: a two-step procedure, involving a sequential application of DEAE-cellulose ion-exchange chromatography and Sephadex G-200 gel filtration, and a single-step procedure, preparative isoelectric focusing in a granulated gel layer. The enzyme proved to be electrophoretically homogeneous. It consisted of two identical subunits with a relative molecular mass of 46 000, each containing one -SH group related to manifestation of the catalytic activity. The Michaelis constant was 1 X 10(-4) M for NAD and 1.3 X 10(-2) M for formate. Formate dehydrogenase was inhibited with p-chlormercuribenzoate, iodoacetamide, dithionitrobenzoate, cyanide and azide.
D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification*; D002175:Candida / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D005560:Formate Dehydrogenases / Q000096:biosynthesis / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009243:NAD / Q000502:physiology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis
D013438:Sulfhydryl Compounds; D009243:NAD; D005560:Formate Dehydrogenases; D000445:Aldehyde Oxidoreductases