Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis.

The residue Tyr 248 of carboxypeptidase A (CPA) is thought to play a role in catalysis by contributing a proton to the incipient amine anion generated during cleavage of peptide substrates. To test this hypothesis we have modified the rat CPA cDNA by site-directed mutagenesis so that the codon for Tyr 248 is replaced by that for Phe. Here, we report the expression of the cDNAs for proCPA and its Tyr-to-Phe variant in yeast via the alpha-factor system. Following zymogen activation by trypsin, wild-type CPA (CPA-WT) and variant CPA (CPA-Phe 248) were purified to homogeneity and characterized enzymatically. CPA-Phe 248 displays essentially undiminished values for the catalytic constant (kcat) towards various peptide and ester substrates. However, the Michaelis constants (Km values) of peptide substrates and the inhibition constant (Ki) of the potato carboxypeptidase inhibitor are increased 6-fold and 70-fold, respectively. These data suggest that the phenolic hydroxyl of Tyr 248 does not act as the requisite general acid catalyst but participates in ligand binding.