Site-directed mutagenesis of the formate dehydrogenase active centre: role of the His332-Gln313 pair in enzyme catalysis.
Gln313 and His332 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, Gln313Glu and His332Phe, have been obtained and characterised. The Gln313Glu 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 Gln313 is essential for the broad pH affinity profile towards substrate. His332Phe mutation leads to a complete loss of enzyme activity. The His332Phe 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 His332 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 Gln313 carboxamide.
Tishkov
V I
VI
Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov, Moscow State University, Russian Federation. vit@enzyme.chem.msu.su
Matorin
A D
AD
Rojkova
A M
AM
Fedorchuk
V V
VV
Savitsky
P A
PA
Dementieva
L A
LA
Lamzin
V S
VS
Mezentzev
A V
AV
Popov
V O
VO
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D017931:DNA Primers; D005560:Formate Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D005973:Glutamine; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D005973:Glutamine; D006639:Histidine; D005560:Formate Dehydrogenases