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Site-directed mutagenesis of
the essential arginine of the formate dehydrogenase active
centre.
Galkin AG, Kutsenko AS, Bajulina NP,
Esipova NG, Lamzin VS, Mesentsev AV, Shelukho DV, Tikhonova TV, Tishkov
VI, Ustinnikova TB, Popov VO.
Department of Chemical
Enzymology, M.V. Lomonosov Moscow State University, Leninskie Gori,
119899 Moscow, Russia.
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.
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