Catalytic properties and stability of a Pseudomonas sp.101 formate dehydrogenase mutants containing Cys-255-Ser and Cys-255-Met replacements

Biochem Biophys Res Commun. 1993 Apr 30;192(2):976-81. doi: 10.1006/bbrc.1993.1511.

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.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Catalysis
  • Cysteine / genetics
  • Cysteine / metabolism*
  • Dithionitrobenzoic Acid / pharmacology
  • Enzyme Stability
  • Formate Dehydrogenases / antagonists & inhibitors
  • Formate Dehydrogenases / genetics
  • Formate Dehydrogenases / metabolism*
  • Kinetics
  • Mercury / pharmacology
  • Methionine / genetics
  • Methionine / metabolism*
  • Molecular Sequence Data
  • Mutation*
  • Oligonucleotides
  • Pseudomonas / enzymology*
  • Sequence Homology, Amino Acid
  • Serine / genetics
  • Serine / metabolism*

Substances

  • Oligonucleotides
  • Serine
  • Dithionitrobenzoic Acid
  • Methionine
  • Formate Dehydrogenases
  • Mercury
  • Cysteine