Site-directed mutagenesis reveals functional contribution of Thr218, Lys220 and Asp304 in chymosin.

The functional contributions of amino acid residues Thr218 and Asp304 of chymosin, both of which are highly conserved in the aspartic proteinases, are analysed by means of site-directed mutagenesis. The optimum pH values, milk-clotting (C) and proteolytic (P) activities and kinetic parameters for synthetic oligopeptides as substrates were examined for the mutant enzymes. The mutation Thr218Ser caused a marked increase in the C/P ratio, which seemed to be due to a change in substrate recognition. Although the negative charge of Asp304 had been expected to play a role in lowering the optimum pH values in the aspartic proteinases, this turned out not to be the case in chymosin because both the mutations Asp304Ala and Asp304Glu caused a similar shift of the optimum pH towards the acidic side. In addition, the mutation Lys220Leu, which we generated previously, was found to cause a decrease in the C/P ratio, mainly due to the increase in the proteolytic activity.