Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin.

The loop exchange mutant chymosin 155-164 rhizopuspepsin was expressed in Trichoderma reesei and exported into the medium to yield a correctly folded and active product. The biochemical characterization and crystal structure determination at 2.5 A resolution confirm that the mutant enzyme adopts a native fold. However, the conformation of the mutated loop is unlike that in native rhizopuspepsin and involves the chelation of a water molecule in the loop. Kinetic analysis using two synthetic peptide substrates (six and 15 residues long) and the natural substrate, milk, revealed a reduction in the activity of the mutant enzyme with respect to the native when acting on both the long peptide substrate and milk. This may be a consequence of the different charge distribution of the mutated loop, its increased size and/or its different conformation.