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Site‐directed mutagenesis of the formate dehydrogenase active centre: role of the His 332 ‐Gln 313 pair in enzyme catalysis
Author(s) -
Tishkov Vladimir I.,
Matorin Andrey D.,
Rojkova Alexandra M.,
Fedorchuk Vladimir V.,
Savitsky Pavel A.,
Dementieva Larissa A.,
Lamzin Victor S.,
Mezentzev Alexander V.,
Popov Vladimir O.
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00641-2
Subject(s) - active site , formate , stereochemistry , histidine , formate dehydrogenase , chemistry , substrate (aquarium) , nad+ kinase , biochemistry , enzyme , dehydrogenase , site directed mutagenesis , binding site , cofactor , mutagenesis , mutant , biology , catalysis , ecology , gene
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 313 Glu and His 332 Phe, have been obtained and characterised. The Gln 313 Glu 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 332 Phe mutation leads to a complete loss of enzyme activity. The His 332 Phe 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.