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

Marino‐Buslje, Cristina, Venhudová, Gabriela, Molina, Miguel A., Oliva, Baldomero, Jorba, Xavier, Canals, Francesc, Avilés, Francesc X., Querol, Enrique
European journal of biochemistry 2000 v.267 no.5 pp. 1502-1509
binding sites, carboxypeptidase A, dissociation, energy, mutagenesis, mutants, potatoes, protein folding
The role of each residue of the potato carboxypeptidase inhibitor (PCI) C‐terminal tail, in the interaction with carboxypeptidase A (CPA), has been studied by the analysis of two main kinds of site‐directed mutants: the point substitution of each C‐terminal residue by glycine and the sequential deletions of the C‐terminal residues. The mutant PCI–CPA interactions have been characterized by the measurement of their inhibition constant, Ki, in several cases, by their kinetic association and dissociation constants determined 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 to be important for folding of the protein core. Val38 has the greatest enthalpic contribution to the PCI–CPA interaction. Although Tyr37 has a minor contribution 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 secondary binding site has been evaluated to be about half of the total free energy of dissociation of the PCI–CPA complex.