EFFECT OF A CHARGED RESIDUE AT THE 213TH SITE OF THERMOLYSIN ON THE ENZYMATIC-ACTIVITY

Considering the electrostatic potential of active site, four mutants o f thermolysin (EC 3.4.24.4) are designed in an attempt to change the o ptimum pH of the hydrolytic activity toward acidic regions. On the bas is of the numerical calculation of the electrostatic potential in the thermolysin molecule, Asp213 is targeted to be replaced by a basic res idue, His, Lys, Arg or a neutral one, Asn. The mutant enzymes are prod uced in Bacillus subtilis as a host using the method of site-directed mutagenesis and their optimum pH values for hydrolyzing a synthetic su bstrate furylacryloyl-Gly-L-Leu-NH2 are found to be lowered by 0.2-0.4 pH units with reference to the wild type enzyme. The pI shifts of the mutants are evaluated. Neither optimum pH nor pi shift can be explain ed by the contribution of the pK 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 suggest s that the contribution of pK shift of other residues must be taken in to account in order to explain the activity change. Little change of t hermal stability is observed among the mutants and wild type enzymes.