PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism.
FK506-binding protein (FKBP) catalyzes the cis-trans isomerization of the peptidyl-prolyl amide bond (the PPIase reaction) and is the major intracellular receptor for the immunosuppressive drugs FK506 and rapamycin. One mechanism proposed for catalysis of the PPIase reaction requires attack of an enzyme nucleophile on the carbonyl carbon of the isomerized peptide bond. An alternative mechanism requires conformational distortion of the peptide bond with or without assistance by an enzyme hydrogen bond donor. We have determined the kinetic parameters of the human FKBP-catalyzed PPIase reaction. At 5 degrees C, the isomerization of Suc-Ala-Leu-Pro-Phe-pNA proceeds in 2.5% trifluorethanol with kcat = 600 s-1, Km = 0.5 mM and kcat/Km = 1.2 x 10(6) M-1s-1. The kcat/Km shows little pH dependence between 5 and 10. A normal secondary deuterium isotope effect is observed on both kcat and kcat/Km. To investigate dependence on enzyme nucleophiles and proton donors, we have replaced eight potential catalytic residues with alanine by site-directed mutagenesis. Each FKBP variant efficiently catalyzes the PPIase reaction. Taken together, these data support an unassisted conformational twist mechanism with rate enhancement due in part to desolvation of the peptide bond at the active site. Fluorescence quenching of the buried tryptophan 59 residue by peptide substrate suggests that isomerization occurs in a hydrophobic environment.
Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4211.
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Park
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D000591:Amino Acid Isomerases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D019696:Peptidylprolyl Isomerase; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D016559:Tacrolimus / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins
D002352:Carrier Proteins; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D011994:Recombinant Proteins; D000591:Amino Acid Isomerases; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus