Kinetic Isotope Effects Support the Twisted Amide Mechanism of Pin1 Peptidyl-Prolyl Isomerase

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• 作者：Ana Y. Mercedes-Camacho ; Ashley B. Mullins ; Matthew D. Mason ; Guoyan G. Xu ; Brendan J. Mahoney ; Xingsheng Wang ; Jeffrey W. Peng ; Felicia A. Etzkorn
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：November 5, 2013
• 年：2013
• 卷：52
• 期：44
• 页码：7707-7713
• 全文大小：386K
• 年卷期：v.52,no.44(November 5, 2013)
• ISSN：1520-4995

文摘

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 (k_cat/K_m). The normal secondary (2掳) KIE value measured for the Ser-d₃ substrate (k_H/k_D = 1.6 卤 0.2) indicates that the serine carbonyl does not rehybridize from sp² to sp³ in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2掳 KIE can be explained by hyperconjugation between Ser 伪-C-H/D and C鈺怬 and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2掳 KIE value (k_H/k_D = 0.86 卤 0.08) measured for the Pro-d₇ substrate indicates rehybridization of the prolyl nitrogen from sp² to sp³ during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (k_H2O/k_D2O = 0.92 卤 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.