MUTAGENESIS, BIOCHEMICAL-CHARACTERIZATION AND X-RAY STRUCTURAL-ANALYSIS OF POINT MUTANTS OF BOVINE CHYMOSIN

Chymosin B point mutants, A115T and G243D (chymosin A), were expressed in Escherichia coli and Trichoderma reesei respectively, characterize d biochemically, crystallized and studied by X-ray analysis at 2.3 and 2.8 Angstrom resolutions respectively, The three-dimensional structur es showed that the mutations gave rise to local conformational changes only when compared with that of chymosin B, Kinetic analysis of the A 115T mutant with a six residue synthetic peptide revealed a reduction in K-m with respect to the wild type, possibly caused by the small loc al changes in the vicinity of S1 and S3, Although, kinetic analyses of the G243D mutant using the short substrate showed reduced catalytic a ctivity, use of a 15 residue substrate based on residues 98-112 of K-c asein, the natural substrate, revealed an increase in the k(cat) compa red with chymosin B, probably a consequence of the charge introduced t hat may interact with the substrate between P4 and P8.