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

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; however, the high values of K_M of the enzyme with NAD⁺ and formate in the standard 0.1 M phosphate buffer result in lower catalytic efficiency k_cat/K_M 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 K_M for formate does not depend on the buffer composition and concentration, while the values of k_cat 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, k_cat 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, k_cat 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 holo-forms of SauFDH and FDH from bacterium Pseudomonas sp.101 shows that the active site of PseFDH in complex with the substrate is totally closed, while in holo-SauFDH, amino acid residues in the active site can be accessed by water molecules and buffer components. This could be the reason of the k_cat and \(K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}\) changes in buffers of different compositions.