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Requirements for peptidyl‐prolyl isomerization activity: A comprehensive mutational analysis of the substrate‐binding cavity of FK506‐binding protein 12
Author(s) -
Ikura Teikichi,
Ito Nobutoshi
Publication year - 2007
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.073203707
Subject(s) - fkbp , isomerase , amino acid , active site , biochemistry , chemistry , stereochemistry , binding site , peptide sequence , peptide , chymotrypsin , enzyme , trypsin , gene
Peptidyl‐prolyl isomerase (PPIase) activity is exhibited by many proteins belonging to the PPIase family. However, the catalytic mechanism of this activity remains to be completely elucidated. Here, we selected human FK506‐binding protein 12 (FKBP12) as the model PPIase and investigated the nature of amino acid residues essential for the activity. The crystal structures of several complexes of PPIase with short peptides revealed that the residues Asp37, Arg42, Phe46, Val55, Trp59, and Tyr82 in the substrate‐binding cavity of FKBP12 appear to play key roles in the PPIase activity. Each of these six residues was substituted by 20 common amino acid residues. The activity of each mutant protein was measured using a peptide analog by the chymotrypsin digestion assay and then compared with wild‐type FKBP12. It was found that site‐specific interactions by the side chains of amino acid residues constituting the substrate‐binding cavity were not essential for the PPIase activity, although the 37th, 55th, and 82nd amino acid residues significantly contributed to the activity. This suggests that the PPIase activity requires only the hydrophobic cavity that captures the Pro‐containing peptide.