Site-directed mutagenesis of the essential arginine of the formate dehydrogenase active centreстатья
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Аннотация: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-m(formate)) or the transition state analogue (K-i(azide)) 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-d(NAD) or K-m(NAD)) 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 5-90degreesC temperature range. Both proteins reveal regions of enhanced conformational mobility at the predenaturing temperatures (40-55degreesC) associated with a change of enzyme kinetic parameters and a co-operative transition around 55-70degreesC 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 55-70degreesC 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. (C) 2002 Elsevier Science B.V. All rights reserved.