Activity modulation of the oligopeptidase B from Serratia proteamaculans by site-directed mutagenesis of amino acid residues surrounding catalytic triad histidine

Reversible Cyclic Thermal Inactivation of Oligopeptidase B from Serratia proteamaculans

“…As we have repeatedly demonstrated earlier, activity of wild-type PSP samples [ 15 , 17 , 18 ] and its mutant variants [ 20 ] in 0.1 Tris-HCl buffer, pH 8.0, at T ≤ 25 °C remains virtually unchanged for an appreciably long time (up to 10–14 days) irrespective of whether Ca 2+ ions are absent or present (50 mM). In this study, activity of the initial PSP samples stored at both 4°C and 25°C also remained constant during the entire experiment.…”

“…In order to elucidate the mechanism of action of PSP, we simulated the crystal structure of the protein in its closed and open forms [ 20 ].…”

“…Depending on the substrate used, substitution of a.a.r. 647 and 660 resulted in either a 2- to 3-fold reduction or a 1.5–2 increase in enzyme activity [ 20 ].…”

“…This hypothesis relies on the results of X-ray diffraction and NMR studies of the nature of reversible transitions between different forms of enzymes of the prolyl oligopeptidase family [ 12 ] and our earlier findings about the correlation between the thermal inactivation of PSP at 37°C and unfolding of its protein molecule (according to the intrinsic fluorescence spectra) [ 17 ]. Figure 5 shows the closed and open forms of PSP corresponding to an earlier obtained model of the enzyme [ 20 ]. It is clear that the inactive open form is an unfolded molecule (i.e., a denatured enzyme).…”

“…Wild-type PSP and point mutants expressed in E. coli BL21(DE3) (Novagen) were obtained and purified according to the procedure described in [ 17 , 20 ].…”