Site-directed mutagenesis of the essential arginine of the formate dehydrogenase active centre.
Sequence alignment shows that residue Arg 284 (according to the numbering of the residues in formate dehydrogenase, FDH, from the methylotrophic bacterium Pseudomonas sp. 101) is conserved in NAD-dependent FDHs and D-specific 2-hydroxyacid dehydrogenases. Mutation of Arg 284 to glutamine and alanine results in a change of the catalytic, thermodynamic and spectral properties of FDH. In comparison to wild-type, the affinity of the mutants for the substrate (K(formate)m) or the transition state analogue (K(azide)i) decreases and correlates with the ability of the side chain of residue 284 to form H-bonds. In contrast, the affinity for the coenzyme (K(NAD)d or K(NAD)m) is either not affected or increases and correlates inversely with the partial positive charge of the side chain. The temperature dependence of circular dichroism (CD) spectra of the wild-type FDH and its Ala mutant has been studied over the 5-90 degrees C temperature range. Both proteins reveal regions of enhanced conformational mobility at the predenaturing temperatures (40-55 degrees C) associated with a change of enzyme kinetic parameters and a co-operative transition around 55-70 degrees C which is followed by the loss of enzyme activity. CD spectra of the wild-type and mutant proteins were deconvoluted and contributions from various types of secondary structure estimated. It is shown that the co-operative transition at 55-70 degrees C in the FDH protein globule is triggered by a loss of alpha-helical secondary structure. The results confirm the conclusion, from the crystal structures, that Arg 284 is directly involved in substrate binding. In addition this residue seems to exert a major structural role by supporting the catalytic conformation of the enzyme active centre.
Galkin
Andrey G
AG
Department of Chemical Enzymology, M.V. Lomonosov Moscow State University, Leninskie Gori, 119899 Moscow, Russia.
Kutsenko
Alexey S
AS
Bajulina
Nataliya P
NP
Esipova
Nataliya G
NG
Lamzin
Victor S
VS
Mesentsev
Alexander V
AV
Shelukho
Dmitriy V
DV
Tikhonova
Tamara V
TV
Tishkov
Vladimir I
VI
Ustinnikova
Tatyana B
TB
Popov
Vladimir O
VO
D001120:Arginine / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D002942:Circular Dichroism; D004795:Enzyme Stability; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D013696:Temperature
D001120:Arginine; D005560:Formate Dehydrogenases