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标题：Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8
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作者：Joshua A. Linscott ; Kanishk Kapilashrami ; Zhen Wang 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：52
页码：E8369-E8378
DOI：10.1073/pnas.1609032114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceWe developed an MS-based method to determine kinetic isotope effects and binding isotope effects on protein lysine methyltransferase SET8-catalyzed monomethylation. These parameters, coupled with steady-state kinetics and molecular modeling, outlined the reaction path of SET8-catalyzed methylation. Upon the formation of the S-adenosyl-L-methionine-SET8-histone 4 lysine 20 intermediate complex followed by lysine deprotonation, the reaction goes through an early, asymmetrical transition state (TS) with the small engagement of the C-N bond and the partial dissociation of the C-S bond. This TS structure is distinct from the known TS structures of other protein lysine methyltransferases (PKMTs) and thus presents the feasibility to design selective TS analog inhibitors against PKMTs. The developed techniques can also be generally applicable to examining other protein methylation and posttranslational modifications. Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-L-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13C KIE of 1.04, an inverse intrinsic -secondary CD3 KIE of 0.90, and a small but statistically significant inverse CD3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35-2.40 [IMG]f1.gif" ALT="A" BORDER="0"> and 2.00-2.05 [IMG]f1.gif" ALT="A" BORDER="0">, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analog Se-adenosyl-L-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.
关键词：PMT ; PKMT ; KIE ; BIE ; methylation