Date Published: April , 2010
Publisher: A.I. Gordeyev
Author(s): S.S. Savin, V.I. Tishkov.
Abstract Kinetic studies on hydrogen peroxide–induced inactivation of mutant formate
dehydrogenase from Pseudomonas sp. 101 (PseFDH Cys255Ala) suggest a simple
bimolecular mechanism for enzyme reaction with the inactivation agent. In the excess of
hydrogen peroxide, the decrease in enzyme activity follows first–order kinetics.
Therefore, the first–order effective inactivation kinetic constants determined for
various FDH forms at a constant H2O2 concentration can be used as a
quantitative measure of the enzyme stability. It was shown that two cysteine residues located
in the active site formate– and coenzyme–binding domains (Cys145 and Cys255,
respectively) make similar contributions to the enzyme stability, while the contribution of
Cys354 is insignificant. The inactivation kinetics of wild–type PseFDH, mutant PseFDH
Cys145Ser/Cys255Ala, and FDH produced under stress conditions by bacterium
Staphylococcus aureus, higher plants Arabidopsis thaliana, and
soya Glycine max, was studied. It was found that the stress–induced
FDHs are at least 20 times more stable than the nonstress–induced PseFDH from
Pseudomonas sp. 101 grown on methanol.
Formate dehydrogenase (EC 184.108.40.206, FDH), a NAD+–dependent enzyme, catalyses
the oxidation of formate to carbon dioxide coupled to NAD+ reduction into NADH.
The preparations of recombinant formate dehydrogenase from bacterium
Pseudomonas sp.101 (PseFDH) and its mutant forms with a single substitution
Cys255Ala and double substitutions Cys145Ser/Cys255Ala and Cys255Ala/Cys354Ser, as well as
recombinant wild–type FDH from bacterium S. aureus (SauFDH), plants
A. thaliana (AraFDH) and soybeans G. max (SoyFDH), were
kindly provided by Innovations and High Technologies MSU Ltd.
(http://www.innotech–msu.com). All preparations were of 97–98% purity or higher as
judged by sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
Inactivation of mutant PseFDH Cys255Ala by hydrogen peroxide at various concentrations
The results of hydrogen peroxide inactivation of the wild–type and three mutant FDH
enzymes from Pseudomonas sp.101 with Cys substituted at various positions are
presented in Fig. 3. We had previously prepared a number
of mutant PseFDH enzymes with various substitutions for Cys 145, 255, and 354. For this study,
we selected only those mutants that had demonstrated the best kinetic behavior.
We studied the stability of FDH from various sources to inactivation by hydrogen peroxide. We
selected three FDH enzymes, the biosynthesis of which increases sharply under stress
conditions: SauFDH of bacterial origin and AraFDH and SoyFDH of higher plants origin (Fig. 4). The wild–type FDH from
Pseudomonas sp.101 and its most stable to hydrogen peroxide inactivation
Cys145Ser/Cys255Ala PseFDH mutant were used for reference. PseFDH is not a stress protein; its
biosynthesis is induced in bacterium Pseudomonas sp.101 under grown on
methanol. The stress–induced FDH of plant, as well as bacterial origin, shows very high
stability to inactivation induced by hydrogen peroxide (Fig.
4). They are much more stable than the wild–type PseFDH; only the best mutant,
PseFDH Cys145Ser/Cys255Ala, has a stability comparable to those of FDH of plant origin.
Although plant FDHs show almost identical stability to inactivation by hydrogen peroxide, they
differ in their thermal stability by more than 5,000 times . FDH from pathogenic bacterium S. aureus was the most
stable to inactivation by hydrogen peroxide. As shown in Fig.
4, the residual activity of this enzyme after 4 hours of incubation in the presence of
0.15М H2O2 was more than 90%. SauFDH also possesses high thermal
stability, being second only to PseFDH among all known formate dehydrogenases.