Effect of the Components of a Buffer Solution on the Catalytic Activity of the NAD+-Dependent Formate Dehydrogenase from the Bacterium Staphylococcus aureus

AbstractNAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from bacteriumStaphylococcus aureus (SauFDH) is the most active enzyme among FDHs of this group; however, the high values ofKM of the enzyme with NAD+ and formate in the standard 0.1 M phosphate buffer result in lower catalytic efficiencykcat/KM than for other FDHs. Here, we study the effect of different buffers on the catalytic properties of SauFDH. Sodium phosphate (NaPB) is used as the base buffer component and Tris, Gly and citrate (Cit) are added to NaPB to prepare double, ternary, and quaternary buffer systems with different concentrations. It is found that KM for formate does not depend on the buffer composition and concentration, while the values ofkcat and\(K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}\) increase and decrease significantly. The highest positive effect is achieved in the case of quaternary buffer NaPB-Cit-Tris-Gly. At a 0.05 M concentration of each component,kcat increases by 70% compared to one in the standard 0.1 M NaPB. At a 0.1 M of each component, improvement in both parameters,kcat and\(K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}\), is observed. Thermal inactivation studies in NaPB and the complex NaPB-Cit-Tris-Gly buffer showed that at component concentrations of 0.1 M and more, SauFDH ’s thermal stability increased. The value of the stabilization effect depends on the ion strength but not on the type of buffer. A comparison of the X-ray structures of...
Source: European Journal of Applied Physiology - Category: Physiology Source Type: research