Design of Biotechnologically Applicable Yeast Carboxypeptidase Y Mutants with Increased Preference for Charged P1 Residues
In the next phase the crystal structure of CPD-Y had also been determined (Endrizzi et al., 1994). Comparing this to that of CPD-WII revealed that structural differences exist between the two enzymes giving rise to errors in the alignment of primary structures used to predict the constitution of the S1 subsite ofCPD-Y. Thus, the S1 pocketofCPD-Yturned out not to be constituted of residues Tyrl47, Leu178, Glu215, Arg216, lle340, and Cys341 as predicted but rather of the residues '!yrl47, Leu178, Tyr185, Tyrl88, Trp312, Ile340, and Cys341 (Figure 1). Thus, using the two structures as a guide a new and improved alignment of 30 primary sequences of serine carboxypeptidases was constructed (Olesen and Bred dam, 1995). Comparing this to the substrate preferences of kinetically characterised enzymes it was found
Yagure 1 Structure of the S1 binding pocket and catalytic triad (Serl46-His397-Asp338) of CPD-Y seen from the direction of the catalytic essential Serl46
that all enzymes with low activity with basic P1 residues possessed a Trp at position 312 while those with high activity toward such substrates possessed smaller residues like Asn, lle, and Gly. Furthermore, most of these enzymes possessed substitutions of Tyr185, e.g. to Glu as in CPD-S1 (Figure 2).