Chymosin B point mutants, A115T and G243D (chymosin A), were expressed in Escherichia coli and Trichoderma reesei respectively, characterized biochemically, crystallized and studied by X-ray analysis at 2.3 and 2.8 Å resolutions respectively. The three-dimensional structures showed that the mutations gave rise to local conformational changes only when compared with that of chymosin B. Kinetic analysis of the A115T mutant with a six residue synthetic peptide revealed a reduction in K(m) with respect to the wild type, possibly caused by the small local changes in the vicinity of S1 and S3. Although, kinetic analyses of the G243D mutant using the short substrate showed reduced catalytic activity, use of a 15 residue substrate based on residues 98-112 of κ-casein, the natural substrate, revealed an increase in the k(cat) compared with chymosin B, probably a consequence of the charge introduced that may interact with the substrate between P4 and P8.
|Number of pages||7|
|Publication status||Published - 1 Dec 1997|
|MoE publication type||A1 Journal article-refereed|
- Aspartic proteinase
- Crystal structure