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 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 intrinsic13C KIE of 1.04, an inverse intrinsic α-secondary CD3KIE of 0.90, and a small but statistically significant inverse CD3BIE 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 Å and 2.00-2.05 Å, 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.
authors.affiliation: Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.|Program of Pharmacology, Weill Graduate School of Medical Science, Cornell University, New York, NY 10021.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Linscott
authors.affiliation: Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
authors.forename: Kanishk
authors.initials: K
authors.lastname: Kapilashrami
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
authors.forename: Chamara
authors.initials: C
authors.lastname: Senevirathne
authors.affiliation: Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.|Tri-Institutional PhD Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Bothwell
authors.affiliation: Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.|Tri-Institutional PhD Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
authors.forename: Gil
authors.initials: G
authors.lastname: Blum
authors.affiliation: Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065; luom@mskcc.org.|Program of Pharmacology, Weill Graduate School of Medical Science, Cornell University, New York, NY 10021.
authors.forename: Minkui
authors.initials: M
authors.lastname: Luo
mesh_terms: D001667:Binding, Competitive; D002384:Catalysis; D002453:Cell Cycle; D003198:Computer Simulation; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry*; D006657:Histones / Q000737:chemistry; D006801:Humans; D007554:Isotopes / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D008958:Models, Molecular; D008962:Models, Theoretical; D009362:Neoplasm Metastasis; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D012436:S-Adenosylmethionine / Q000737:chemistry; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D006657:Histones; D007554:Isotopes; D010455:Peptides; D012436:S-Adenosylmethionine; D011495:Histone-Lysine N-Methyltransferase; C470730:KMT5A protein, human; D008239:Lysine
keywords: BIE; KIE; PKMT; PMT; methylation