title: Enzyme-dependent lysine deprotonation in EZH2 catalysis.
abstract: Protein lysine methyltransferases (PKMTs) are key players in epigenetic regulation and have been associated with a variety of diseases, including cancers. The catalytic subunit of Polycomb Repressive Complex 2, EZH2 (EC 2.1.1.43), is a PKMT and a member of a family of SET domain lysine methyltransferases that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to lysine 27 of histone 3 (H3K27). Wild-type (WT) EZH2 primarily catalyzes the mono- and dimethylation of H3K27; however, a clinically relevant active site mutation (Y641F) has been shown to alter the reaction specificity, dominantly catalyzing trimethylation of H3K27, and has been linked to tumor genesis and maintenance. Herein, we explore the chemical mechanism of methyl transfer by EZH2 and its Y641F mutant with pH-rate profiles and solvent kinetic isotope effects (sKIEs) using a short peptide derived from histone H3 [H3(21-44)]. A key component of the chemical reaction is the essential deprotonation of the ε-NH3(+) group of lysine to accommodate subsequent methylation. This deprotonation has been suggested by independent studies (1) to occur prior to binding to the enzyme (by bulk solvent) or (2) to be facilitated within the active site following binding, either (a) by the enzyme itself or (b) by a water molecule with access to the binding pocket. Our pH-rate and sKIE data best support a model in which lysine deprotonation is enzyme-dependent and at least partially rate-limiting. Furthermore, our experimental data are in agreement with prior computational models involving enzyme-dependent solvent deprotonation through a channel providing bulk solvent access to the active site. The mechanism of deprotonation and the rate-limiting catalytic steps appear to be unchanged between the WT and Y641F mutant enzymes, despite their activities being highly dependent on different substrate methylation states, suggesting determinants of substrate and product specificity in EZH2 are independent of catalytic events limiting the steady-state rate.
authors.affiliation: Novartis Institutes for Biomedical Research , 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: D Randal
authors.initials: DR
authors.lastname: Kipp
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Quinn
authors.forename: Pascal D
authors.initials: PD
authors.lastname: Fortin
mesh_terms: D055162:Biocatalysis; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D063151:Polycomb Repressive Complex 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011522:Protons
chemical_list: D011522:Protons; D063151:Polycomb Repressive Complex 2; D008239:Lysine