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

• Published in: Moscow University chemistry bulletin Vol. 77; no. 6; pp. 330 - 339
• Main Authors: Iurchenko, T. S., Bolotova, S. B., Loginova, A. A., Pometun, E. V., Savin, S. S., Pometun, A. A., Tishkov, V. I.
• Format: Journal Article
• Language: English
• Published: Moscow Pleiades Publishing 01.12.2022; Springer Nature B.V
• Subjects: Amino acids; Bacteria; Buffer solutions; Catalytic activity; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Dehydrogenases; Enzymes; Formate dehydrogenase; Sodium phosphate; Substrates; Thermal stability; NAD; complex buffer systems; dependent formate dehydrogenase; thermal stability; catalytic activity
• ISSN: 0027-1314; 1935-0260
• DOI: 10.3103/S0027131422060074

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 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 , 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 changes in buffers of different compositions.