title: Effect of interactions between amino acid residues 43 and 61 on thermal stability of bacterial formate dehydrogenases.
abstract: NAD+-dependent formate dehydrogenases (EC 1.2.1.2, FDH) of methylotrophic bacteria Pseudomonas sp. 101 (PseFDH) and Mycobacterium vaccae N10 (MycFDH) exhibit high homology. They differ in two amino acid residues only among a total of 400, i.e., Ile35 and Glu61 in MycFDH substitute for Thr35 and Lys61 as in PseFDH. However, the rate constant for MycFDH thermal inactivation in the temperature range of 54-65 degrees C is 4-6-times higher than the corresponding rate constant for the enzyme from Pseudomonas sp. 101. To clarify the role of these residues in FDH stability the dependence of the apparent rate constant for enzyme inactivation on phosphate concentration was studied. Kinetic and thermodynamic parameters for thermal inactivation were obtained for both recombinant wild-type and mutant forms, i.e., MycFDH Glu61Gln, Glu61Pro, Glu61Lys and PseFDH Lys61Arg. It has been shown that the lower stability of MycFDH compared to that of PseFDH is caused mainly by electrostatic repulsion between Asp43 and Glu61 residues. Replacement of Lys61 with an Arg residue in the PseFDH molecule does not result in an increase in stability.
authors.affiliation: Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: V V
authors.initials: VV
authors.lastname: Fedorchuk
authors.forename: A G
authors.initials: AG
authors.lastname: Galkin
authors.forename: I E
authors.initials: IE
authors.lastname: Yasny
authors.forename: L B
authors.initials: LB
authors.lastname: Kulakova
authors.forename: A M
authors.initials: AM
authors.lastname: Rojkova
authors.forename: A A
authors.initials: AA
authors.lastname: Filippova
authors.forename: V I
authors.initials: VI
authors.lastname: Tishkov
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D016415:Sequence Alignment; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D005560:Formate Dehydrogenases