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Contribution of Câtail residues of potato carboxypeptidase inhibitor to the binding to carboxypeptidaseâA: A mutagenesis analysis
- MarinoâBuslje, Cristina, VenhudovÃ¡, Gabriela, Molina, Miguel A., Oliva, Baldomero, Jorba, Xavier, Canals, Francesc, AvilÃ©s, 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) Câterminal tail, in the interaction with carboxypeptidaseâA (CPA), has been studied by the analysis of two main kinds of siteâdirected mutants: the point substitution of each Câterminal residue by glycine and the sequential deletions of the Câterminal residues. The mutant PCIâCPA interactions have been characterized by the measurement of their inhibition constant, Ki, in several cases, by their kinetic association and dissociation constants determined by presteadyâstate analysis, and by computational approaches. The role of Pro36 appears to be mainly the restriction of the mobility of the PCI Câtail. 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 PCIâCPA 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 Câterminal 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 PCIâCPA complex.