Publication Date:
2012-12-22
Description:
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, k obs , of the thermal inactivation of WT and variants at 10 mM CaCl 2 increased with increasing thermal treatment temperatures (70–95°C), and those at 80°C decreased with increasing CaCl 2 concentrations (1–100 mM). The k obs values were in the order of WT 〉 S65P 〉 G8C/N60CG8C/N60C/S65P at all temperatures and CaCl 2 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, K d , 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 Ca 2+ -binding site III in the N-terminal domain.
Print ISSN:
0021-924X
Electronic ISSN:
1756-2651
Topics:
Biology
,
Chemistry and Pharmacology
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