Mechanism of thermostability in thermolysin – analysis of subsite S2 mutant enzymes of thermolysin |
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| Authors: | M. Kubo K. Itoh K. Nishikawa F. Hasumi K. Inouye |
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| Affiliation: | Department of Bio Science and Technology, Ritsumeikan University, Kusatsu,;Department of Chemistry and Biochemistry, Numazu College of Technology, Numazu, and;Department of Food Science and Technology, Kyoto University, Japan |
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| Abstract: | An aromatic amino acid at position 115 (tryptophan residue; subsite S2) in thermolysin is known to be essential for proteolytic activity of thermolysin. Mutant enzymes substituted by phenylalanine (W115F) and tyrosine (W115Y) at position 115 were expressed at similar levels as the wild type (WT) enzyme in Bacillus subtilis . The thermostability of the W115Y mutant enzyme was equal to that of the WT. However, that of the W115F mutant enzyme was significantly lower than the WT. Enzymatic kcat/Km values of W115F increased to about twice those of the WT, but W115F also seemed to promote increased autodegradation compared with the WT and W115Y enzymes. |
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