Kinetic isotope effects support the twisted amide mechanism of Pin1 peptidyl-prolyl isomerase.
Biochemistry
; 52(44): 7707-13, 2013 Nov 05.
Article
en En
| MEDLINE
| ID: mdl-24116866
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 CâO 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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Dineínas Citoplasmáticas
/
Amidas
Idioma:
En
Revista:
Biochemistry
Año:
2013
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos