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Comparative Study
. 1994 Aug 26;269(34):21467-72.

C-tail valine is a key residue for stabilization of complex between potato inhibitor and carboxypeptidase A

M A Molina  1 , C Marino, B Oliva, F X Avilés, E Querol
Affiliations
  • PMID: 8063780
Free article
Comparative Study

C-tail valine is a key residue for stabilization of complex between potato inhibitor and carboxypeptidase A

M A Molina et al. J Biol Chem. .
Free article

Abstract

Although the carboxypeptidase A-potato carboxypeptidase inhibitor (CPA-PCI) complex is a well known example of protein-protein interaction, little was known about the basis of its thermodynamic stability. Site-directed mutagenesis has been used to identify key residues in the PCI tail and estimate the contribution of their chemical groups to the binding to CPA. Two deletion mutants were created, one lacking the C-terminal residue of the tail (Gly39) and another one lacking the two C-terminal residues (Val38, Gly39). The last mutant had an inhibition constant for CPA 10(4)-fold higher than that of wild-type PCI, indicating that Val38 is a key residue. The interactions of Val38 with CPA residues contribute 5.4-5.7 kcal mol-1 to the overall stability of the CPA-PCI complex (11.9-12.1 kcal mol-1). A series of PCI point mutants at valine 38 were created, and their inhibition constant for CPA was measured. Two of these mutants with smaller side chains, V38G and V38A, allowed us to estimate that the contribution of the three side chain aliphatic groups of valine 38 to the overall stability of the complex is 3.4-4 kcal mol-1. Another two mutants with larger side chains, V38L and V38I, were constructed, the first being a significantly worse inhibitor than the wild type. These results suggest that only aliphatic groups in positions beta and gamma of residue 38 in PCI (but not those in delta) can establish van der Waals interactions with atoms of the active center of CPA and participate in binding. The energetic contribution of each methyl/methylene group in those positions can be estimated as 1-1.5 kcal mol-1. Our hypothesis is supported by computer simulation analysis.

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