Contribution of C-tail residues of potato carboxypeptidase inhibitor to the binding to carboxypeptidase A - A mutagenesis analysis

The role of each residue of the potato carboxypeptidase inhibitor (PCI) C-t erminal tail, in the interaction with carboxypeptidase A (CPA), has been st udied by the analysis of two main kinds of site-directed mutants: the point substitution of each C-terminal residue by glycine and the sequential dele tions of the C-terminal residues. The mutant PCI-CPA interactions have been characterized by the measurement of their inhibition constant, K-i, in sev eral cases, by their kinetic association and dissociation constants determi ned by presteady-state analysis, and by computational approaches. The role of Pro36 appears to be mainly the restriction of the mobility of the PCI C- tail. In addition, and unexpectedly, both Gly35 and Pro36 have been found t o be important for folding of the protein core. Val38 has the greatest enth alpic contribution to the PCI-CPA interaction. Although Tyr37 has a minor c ontribution to the binding energy of the whole inhibitor, it has been found to be essential for the interaction with the enzyme following the cleavage of the C-terminal Gly39 by CPA. The energetic contribution of the PCI seco ndary binding site has been evaluated to be about half of the total free en ergy of dissociation of the PCI-CPA complex.