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-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.
Marino-Buslje
C
C
Institut de Biologia Fonamental and Department de Bioquimica i Biologica Molecular, Universitat Autònoma de Barcelona, Spain.
Venhudová
G
G
Molina
M A
MA
Oliva
B
B
Jorba
X
X
Canals
F
F
Avilés
F X
FX
Querol
E
E
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D017931:DNA Primers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000596:Amino Acids; D017931:DNA Primers; D010940:Plant Proteins; D011480:Protease Inhibitors; D002268:Carboxypeptidases; D043422:Carboxypeptidases A