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_N2) 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 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 S_N2 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.

Keywords: BIE; KIE; PKMT; PMT; methylation.

Fig. 1.

A hypothetical reaction path of PKMT-catalyzed lysine monomethylation and relevant biochemical methods to examine each aspect of the process.

Fig. S1.

Steady-state kinetics and reaction mechanism of SET8-catalyzed H4K20 monomethylation. The kinetic parameters of SET8 methylation were examined using 1 µM SET8 by systematically varying the amount of the H4K20 substrate peptide (3–50 µM) and SAM (3–100 µM). The product of the methylation reaction was quantified with the enzyme-coupled luciferase. (A) Representative initial-rate kinetics for SET8-catalyzed H4K20 methylation with a fixed amount of peptide H4 substrate (8 µM) and varied amounts of SAM cofactor: (■, 3 μM; ▲, 6 μM; ▼, 10 μM; ◆, 15 μM; , 25 μM; , 50 μM; and ∆, 100 μM). The initial linear range varies with cofactor and substrate concentrations. (B and C) Lineweaver–Burk analysis of the initial velocities vs. varied concentrations of the H4K20 substrate (●, 30 μM; ■, 20 μM; ▲, 15 μM; ▼, 10 μM; and ◆, 8 μM) and SAM (●, 6 μM; ▼,15 μM; ◆, 25 μM; , 50 μM; and , 100 μM). All of the initial rates converge in the secondary quadrant. (D) Global fit of the initial velocities to a general, bisubstrate kinetic mechanism against SAM concentration to afford k_cat, K_m,SAM, K_m,H4K20 and α according to Eqs. S1 and S2 with k_cat = 7.0 ± 0.8 min⁻¹, K_m,peptide = 40 ± 8 µM, K_m,cofactor = 16 ± 6 µM, and α = 1.4 ± 0.8 (45).

Fig. 2.

Overview of KIE measurement under competitive conditions. (A) Schematic description of a competition reaction with a pair of isotopic cofactors and the resultant overlapping MS data with CH₃/CD₃ and CH₃/¹³CD₃ cofactor pairs. The reaction sample was split into two portions for partial consumption and full consumption of the pair of isotopic cofactors, respectively. Their light-to-heavy isotope ratios (blue vs. red) are quantified by MS as described in Materials and Methods. (B) Schematic description of BIE measurement under competitive conditions. Here a pair of prebound or SET8-bound CH₃/CD₃ isotopic cofactors were converted to label H4K20 peptide substrate under competitive conditions. The resultant methylated H4K20 peptide was subjected to MS analysis to quantify the isotopic ratios as described in Materials and Methods.

Fig. 3.

Overview of mathematical matrix to deconvolute the MS data and calculate light-to-heavy isotope ratios according to Eqs. S3–S7. (A) -CH₃ vs. -CD₃ (blue vs. red) (B) -CH₃ vs. ^-13CD₃ (blue vs. orange).

Fig. 4.

(A and B) Representative MALDI-TOF-MS data for partial consumption (Top) and 100% consumption (Bottom) of (A) [S-CH₃]-SAM and [S-CD₃]-SAM and (B) [S-CH₃]-SeAM and [S-¹³CD₃]-SeAM as a pair of isotopic cofactors. The ratios of [CH₃] vs. [CD₃] before initializing the reaction (R₀) and after reaching partial completion (R_f) were determined by the MS-deconvolution method (Fig. 3 and Materials and Methods). Their ratio R₀/R_f was used to determine the inverse KIEs according to Eq. 1.

Fig. 5.

TS structure of SET8-catalyzed H4K20 monomethylation with SAM as the cofactors. α-2°-CD₃ KIE and 1-¹³C KIE (intrinsic KIEs, Left) were used as computational constraints to solve the TS structures. The TS (Center) shows an early, asymmetrical character with the long C-N and short C-S distances of 2.35–2.40 Å and 2.00–2.05 Å, respectively. The structures of reactants (R, Left) and products (P, Right) are shown for comparison. The numbers in R, TS, and P are NBO charges for S, -CH₃, and -NH₂ with red for electron rich and blue for electron deficient in ESPS maps.

Fig. S2.

Noncanonical CH–O interaction of SET8-catalyzed H4K20 monomethylation. (A) Static distances of the amide oxygens of Cys270 and Arg295 and the phenolic oxygen of Tyr336 to the sulfonium–methyl moiety of SAM upon the formation of the SAM–SET8 intermediate complex. The structure was extracted from PDB file 4IJ8. Hydrogen atoms were added to the PDB file using MolProbity online software. Their bond distances were measured with the measurement wizard in PyMol. (B) Two-dimensional representative interactions between SAM and Cys270/Arg295/Tyr336 upon the formation of the SAM–SET8–peptide intermediate complex.

Fig. S3.

TS geometries matched with KIEs of SeAM. Constrained TS geometries with varied distances of C-Se and C-N were optimized for the frequency calculation by Gaussian09. KIEs (${}^{{}^{13}C{H}_3}k$ and ${}^{C{D}_3}k$) were calculated using the ISOEFF program and then scaled by 0.967 to match experimental vibrational frequencies. The matched TS geometrical landscapes were highlighted for ${}^{{}^{13}C{H}_3}k$ and ${}^{C{D}_3}k.$ The combined experimental KIEs (${}^{{}^{13}C{H}_3}k$ and ${}^{C{D}_3}k$) of the SeAM-dependent H4K20 methylation match a suite of TS geometries with a relatively fixed Se-N distance around 4.9 Å but the altered position of the methyl group between the methyl donor and acceptor.

Fig. 6.

Reaction path of SET8-catalyzed H4K20 monomethylation. (A) Reaction mechanism of SET8-catalyzed H4K20 monomethylation with the SAM cofactor. After the formation of a ternary SAM–SET8–H4K20 intermediate complex and the subsequent deprotonation, the rate-determining step of the catalysis is the formation of an early, asymmetrical S_N2 transition state for methyl transferring, reflected by an inverse CD₃ BIE of 0.96, an inverse intrinsic α-2°-CD₃ KIE of 0.90, and a normal 1°-¹³C KIE of 1.04. (B) Reaction mechanism of SET8-catalyzed H4K20 monomethylation with the SeAM cofactor. The reaction path for the SeAM cofactor is similar to that for SAM with CD₃ BIE of 0.96, α-2°-CD₃ KIE of 1.02, and 1-¹³C KIE of 1.06. (C) Relative energy barriers in SET8-catalyzed H4K20 monomethylation with SAM and SeAM as cofactors. SAM and SeAM are similar in terms of their affinity to SET8 and CD₃ BIE and show only 1.94-fold difference of their k_cat values along with the reaction path.