title: Kinetic isotope effects support the twisted amide mechanism of Pin1 peptidyl-prolyl isomerase.
abstract: The Pin1 peptidyl-prolyl isomerase catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac-Phe-Phe-phosphoSer-Pro-Arg-p-nitroaniline, were synthesized in unlabeled form, and with deuterium-labeled Ser-d3 and Pro-d7 amino acids. Kinetic data were collected as a function of Pin1 concentration to measure kinetic isotope effects (KIEs) on catalytic efficiency (kcat/Km). The normal secondary (2°) KIE value measured for the Ser-d3 substrate (kH/kD = 1.6 ± 0.2) indicates that the serine carbonyl does not rehybridize from sp(2) to sp(3) in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2° KIE can be explained by hyperconjugation between Ser α-C-H/D and C═O and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2° KIE value (kH/kD = 0.86 ± 0.08) measured for the Pro-d7 substrate indicates rehybridization of the prolyl nitrogen from sp(2) to sp(3) during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (kH2O/kD2O = 0.92 ± 0.12), indicating little or no involvement of exchangeable protons in the mechanism. These results support the formation of a simple twisted amide transition state as the mechanism for peptidyl prolyl isomerization catalyzed by Pin1. A model of the reaction mechanism is presented using crystal structures of Pin1 with ground state analogues and an inhibitor that resembles a twisted amide transition state.
authors.affiliation: Department of Chemistry, Virginia Tech , Blacksburg, Virginia 24061, United States.
authors.forename: Ana Y
authors.initials: AY
authors.lastname: Mercedes-Camacho
authors.forename: Ashley B
authors.initials: AB
authors.lastname: Mullins
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Mason
authors.forename: Guoyan G
authors.initials: GG
authors.lastname: Xu
authors.forename: Brendan J
authors.initials: BJ
authors.lastname: Mahoney
authors.forename: Xingsheng
authors.initials: X
authors.lastname: Wang
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Peng
authors.forename: Felicia A
authors.initials: FA
authors.lastname: Etzkorn
mesh_terms: D000577:Amides / Q000737:chemistry*; D056445:Cytoplasmic Dyneins / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D007536:Isomerism; D007553:Isotope Labeling; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000577:Amides; D010455:Peptides; D003903:Deuterium; C427758:DYNLL1 protein, human; D056445:Cytoplasmic Dyneins