Effects of site-directed mutagenesis in the N-terminal domain of thermolysin on its stabilization

摘要

The thermolysin variant G8C/N60C/S65P in which the triple mutation in the N-terminal domain, Gly8→Cys/Asn60→Cys/Ser65→Pro, is undertaken increases stability [Yasukawa, K. and Inouye, K. (2007) Improving the activity and stability of thermolysin by site-directed mutagenesis. Biochim. Biophys. Acta 1774, 1281–1288] and its mechanism is examined in this study. The apparent denaturing temperatures based on ellipticity at 222 nm of the wild-type thermolysin (WT), G8C/N60C, S65P and G8C/N60C/S65P were 85, >95, 88 and >95°C, respectively. The first-order rate constants, kobs, of the thermal inactivation of WT and variants at 10 mM CaCl2 increased with increasing thermal treatment temperatures (70–95°C), and those at 80°C decreased with increasing CaCl2 concentrations (1–100 mM). The kobs values were in the order of WT > S65P > G8C/N60C≒G8C/N60C/S65P at all temperatures and CaCl2 concentrations. These results indicate that the mutational combination, Gly8→Cys/Asn60→Cys and Ser65→Pro, increases stability only as high as Gly8→Cys/Asn60→Cys does. Assuming that irreversible inactivation of thermolysin occurs only in the absence of calcium ions, the dissociation constants, Kd, to the calcium ions of WT, G8C/N60C, S65P and G8C/N60C/S65P were 47, 8.9, 17 and 7.2 mM, respectively, suggesting that Gly8→Cys/Asn60→Cys and Ser65→Pro stabilize thermolysin by improving its affinity to calcium ions, most probably the one at the Ca²⁺-binding site III in the N-terminal domain.