title: Effects of site-directed mutagenesis in the N-terminal domain of thermolysin on its stabilization.
abstract: 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 CaCl₂ increased with increasing thermal treatment temperatures (70-95°C), and those at 80°C decreased with increasing CaCl₂ concentrations (1-100 mM). The k(obs) 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, 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 Ca²⁺-binding site III in the N-terminal domain.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Kawasaki
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002122:Calcium Chloride / Q000737:chemistry; D002942:Circular Dichroism; D003553:Cystine / Q000032:analysis; D004795:Enzyme Stability; D006358:Hot Temperature / Q000009:adverse effects; D007202:Indicators and Reagents / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013820:Thermolysin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007202:Indicators and Reagents; D050505:Mutant Proteins; D011994:Recombinant Proteins; D003553:Cystine; D013820:Thermolysin; D002122:Calcium Chloride