title: Catalytic properties and stability of a Pseudomonas sp.101 formate dehydrogenase mutants containing Cys-255-Ser and Cys-255-Met replacements.
abstract: Two mutants of bacterial formate dehydrogenase from Pseudomonas sp.101 (EC 1.2.1.2, FDH)-C255S (FDH-S) and C255M (FDH-M), were obtained and its properties were studied. Both mutations provided the high resistance to inactivation by Hg2+. Slow inactivation of mutants by DTNB reveals the presence in FDH molecule of another essential cysteine residue. Specific activities of FDH, FDH-S and FDH-M were 16, 16 and 9.5 U/mg of protein, respectively. Km on formate was 7.5, 7.5 and 20 mM and Km on NAD(+)-0.1, 0.3 and 0.6 mM for FDH, FDH-S and FDH-M, respectively. Mutations of Cys255 on Ser or Met resulted in increasing of enzyme stability at 25 degrees C and decreasing of thermostability (above 45 degrees C). Data obtained show that Cys255 is unique residue for providing both enzyme thermostability and catalytically optimal binding of coenzyme.
authors: Department of Chemical Enzymology, Chemistry Faculty, Moscow State University, Russia.
authors: V I
authors: VI
authors: Tishkov
authors: A G
authors: AG
authors: Galkin
authors: G N
authors: GN
authors: Marchenko
authors: O A
authors: OA
authors: Egorova
authors: D V
authors: DV
authors: Sheluho
authors: L B
authors: LB
authors: Kulakova
authors: L A
authors: LA
authors: Dementieva
authors: A M
authors: AM
authors: Egorov
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D005560:Formate Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D008715:Methionine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides; D011549:Pseudomonas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism*
chemical_list: D009841:Oligonucleotides; D012694:Serine; D004228:Dithionitrobenzoic Acid; D008715:Methionine; D005560:Formate Dehydrogenases; D008628:Mercury; D003545:Cysteine