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Tatyana S. Iurchenko, Seseg B. Bolotova, Anastasia A. Loginova, Evgeny V. Pometun, Svyatoslav S. Savin, Anastasia A. Pometun, Vladimir I. Tishkov
Influence of the
components of buffer solution on the catalytic activity of the nad+-dependent
formate dehydrogenase from the bacterium Staphylococcus aureus
Abstract
Abstract. NAD+-dependent formate dehydrogenase
(FDH, EC 1.2.1.2) from bacterium Staphylococcus aureus (SauFDH) is the
most active enzyme among FDHs of this group, but high values of KM
of the enzyme with NAD+ and formate in standard 0.1 M phosphate
buffer result in lower catalytic efficiency kcat/KM
compared to ones for other FDHs. We have studied influence of different buffers
on catalytic properties of SauFDH. Sodium phosphate (NaPB) was used as the base
buffer component and Tris, Gly and citrate (Cit) were added to NaPB to prepare
double, ternary and quaternary buffer systems with different concentrations. It
was found that KM for formate does not depend on buffer composition
and concentration, while values of kcatи KMNAD+ increased and decreased
significantly. The highest positive effect achieved in the case of quaternary
buffer NaPB-Cit-Tris-Gly. At 0.05 M concentration of each component kcat
increased by 70% compared to one in standard 0.1 M NaPB. At 0.1 M of each
component improvement of both parameters, kcat and KMNAD+.
was observed. Thermal inactivation studies in NaPB and complex
NaPB-Cit-Tris-Gly buffer showed that at component concentrations 0.1 M and more
SauFDH thermal stability increased. Value of stabilization effect depends on
ion strength but not on type of buffer. Comparison of X-ray structures of
holo-forms of SauFDH and FDH from bacterium Pseudomonas sp.101 shows
that active site of PseFDH in complex with substrate is totally closed, while in
holo-SauFDH amino acid residues in active site can be accessed by water
molecules and buffer components. It could be the reason of kcatи KMNAD+ changes in in buffers of different compositions.
Key words: NAD+-dependent formate
dehydrogenase, complex buffer systems, catalytic activity, thermal stability
Copyright (C) Chemistry Dept., Moscow State University, 2002
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