Effect of a charged residue at the 213th site of thermolysin on the enzymatic activity

Considering the electrostatic potential of active site, four mutants of thermolysin (EC 3.4.24.4) are designed in an attempt to change the optimum pH of the hydrolytic activity toward acidic regions. On the basis of the numerical calculation of the electrostatic potential in the thermolysin molecule, Asp213 is targeted to be replaced by a basic residue, His, Lys, Arg or a neutral one, Asn. The mutant enzymes are produced in Bacillus subtilis as a host using the method of site-directed mutagenesis and their optimum pH values for hydrolyzing a synthetic substrate furylacryloyl-Gly- l-Leu-NH 2 are found to be lowered by 0.2–0.4 pH units with reference to the wild type enzyme. The pl shifts of the mutants are evaluated. Neither optimum pH nor pl shift can be explained by the contribution of the p K change only at the mutation site. We find a clear negative correlation between the activities at pH 7.0 and the pI values among the four mutants and wild-type enzyme. It suggests that the contribution of p K shift of other residues must be taken into account in order to explain the activity change. Little change of thermal stability is observed among the mutants and wild type enzymes.