Site-directed mutagenesis of the formate dehydrogenase active centre: role of the His 332-Gln 313 pair in enzyme catalysis

Abstract

Gln 313 and His 332 residues in the active centre of NAD +-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equivalent to the glutamate-histidine pair in active sites of d-specific 2-hydroxyacid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln 313Glu and His 332Phe, have been obtained and characterised. The Gln 313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild-type enzyme to 7.6 thus indicating that Gln 313 is essential for the broad pH affinity profile towards substrate. His 332Phe mutation leads to a complete loss of enzyme activity. The His 332Phe mutant is still able to bind coenzyme but not substrate or analogues. The role of histidine in the active centre of FDH is discussed. The protonation state of His 332 is not critical for catalysis but vital for substrate binding. A partial positive charge on the histidine imidazole, required for substrate binding, is provided via tight H-bond to the Gln 313 carboxamide.