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 (S N 2) 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 13 C KIE of 1.04, an inverse intrinsic α-secondary CD 3 KIE of 0.90, and a small but statistically significant inverse CD 3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical S N 2 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 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.S tepwise progression of an enzyme-catalyzed chemical reaction is accompanied by changes of bond orders and vibrational modes involved with specific atoms of the reactant(s) (1, 2). Such changes can be traced experimentally by measuring the ratios of turnover rates [kinetic isotope effects (KIEs)] or binding affinities [binding isotope effects (BIEs)] of the reactant(s) when the relevant atoms are replaced by heavy isotopes (3, 4). KIEs and BIEs are thus useful parameters for elucidating transition-state (TS) structures and catalytic mechanisms, which sometimes cannot be elucidated readily through sole measurement of steady-state kinetics (5-9). A sufficient set of KIEs and BIEs at the positions involved with bond motions can afford electrostatic and geometric constraints, when combined with computational modeling, to define an enzymatic TS (10-12). This information provides not only the atomic resolution of the transient structure at the highest energy summit along the reaction path, but also structural guidance for designing tight-binding TS analog inhibitors (13...

Ability of IR and NMR Spectral Data to Distinguish between a Tetrel Bond and a Hydrogen Bond

“…There are also a number of proposals that it is just this sort of TB that is part and parcel of enzymatic activity [45][46][47][48][49] . However, structural studies are unable to deduce whether there is a truly attractive noncovalent bond present, or whether the two groups adopt their relative positions due to structural restraints in other parts of the system, with little or no attractive force between them.…”

Crystallographic and Computational Characterization of Methyl Tetrel Bonding in S-Adenosylmethionine-Dependent Methyltransferases

“…Correlatively, structural and biochemical characterization of the protein lysine N-methyltransferase (KMT) SET7/9 and the reactivation domain of methionine synthase demonstrated that these hydrogen bonds promote high affinity binding to AdoMet compared to the methyl transfer product S-adenosylhomocysteine (AdoHcy), thus mitigating product inhibition [ 18 , 21 ]. Moreover, CH∙∙∙O and CH∙∙∙N interactions with the AdoMet methyl group have been proposed to contribute to transition state stabilization in several methyltransferases, including SET7/9, SET8, NSD2, and glycine N-methyltransferase [ 18 , 22 , 23 , 24 ].…”

The transition to magic bullets – transition state analogue drug design

“…39 Kinetic isotope effects (KIEs) inform on bond vibrational changes between the ground state and TS of a chemical reaction and are one of the most powerful experimental techniques for interrogating TS structure. 40,41 Primary KIEs occur when a bond is formed or broken to the isotopic atom during the reaction while secondary KIEs are observed when the number of bonds to the isotopic atom remains unchanged. 42 The direct comparison, equilibrium perturbation and the internal competition method are the three main methods for the measurement of KIEs.…”