Catalytic Properties and Stability of a Pseudomonas sp.101 Formate Dehydrogenase Mutants Containing Cys-255-Ser and Cys-255-Met Replacements

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 Hg 2+. 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. K m on formate was 7.5, 7.5 and 20 mM and K m 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°C and decreasing of thermostability (above 45°C). Data obtained show that Cys255 is unique residue for providing both enzyme thermostability and catalytically optimal binding of coenzyme.