Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8 Journal Article


Authors: Linscott, J. A.; Kapilashrami, K.; Wang, Z.; Senevirathne, C.; Bothwell, I. R.; Blum, G.; Luo, M.
Article Title: Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8
Abstract: 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 MSbased 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 α-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 A and 2.00-2.05 A, respectively. This transition state is further supported by the KIEs, BIEs, and steadystate 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.
Keywords: methylation; pkmt; bie; kie; pmt
Journal Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 113
Issue: 52
ISSN: 0027-8424
Publisher: National Academy of Sciences  
Date Published: 2016-12-27
Start Page: E8369
End Page: E8378
Language: English
DOI: 10.1073/pnas.1609032114
PROVIDER: scopus
PMCID: PMC5206543
PUBMED: 27940912
DOI/URL:
Notes: Article -- Export Date: 2 February 2017 -- Source: Scopus
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  1. Minkui Luo
    70 Luo
  2. Gil Blum
    15 Blum