The Burden Borne by Protein Methyltransferases: Rates and Equilibria of Non-enzymatic Methylation of Amino Acid Side Chains by SAM in Water

Abstract

SAM is a powerful methylating agent, with a methyl group transfer potential matching the phosphoryl group transfer potential of ATP. SAM-dependent N-methyltransferases have evolved to catalyze the modification of specific lysine residues in histones and transcription factors, in addition to generating epinephrine, N-methylnicotinamide, and a quaternary amine (betaine) that is used to maintain osmotic pressure in plants and halophilic bacteria. To assess the catalytic power of these enzymes and their potential susceptibility to transition state and multisubstrate analogue inhibitors, we determined the rates and positions of the equilibrium of methyl transfer from the trimethylsulfonium ion to model amines in the absence of a catalyst. Unlike the methyl group transfer potential of SAM, which becomes more negative with an increase in pH throughout the normal pH range, equilibrium constants for the hydrolytic demethylation of secondary, tertiary, and quaternary amines are found to be insensitive to a change in pH and resemble each other in magnitude, with an average ΔG value of approximately −0.7 kcal/mol at pH 7. Thus, each of the three steps in the mono-, di-, and trimethylation of lysine by SAM is accompanied by a change in free energy of −7.5 kcal/mol in a neutral solution. Arrhenius analysis of the uncatalyzed reactions shows that the unprotonated form of glycine attacks the trimethylsulfonium ion (TMS+) with second-order rates constant of 1.8 × 10–7 M–1 s–1 at 25 °C (ΔH ⧧ = 22 kcal/mol, and TΔS ⧧ = −6 kcal/mol). Comparable values are observed for the methylation of secondary and tertiary amines, with k 25 values of 1.1 × 10–7 M–1 s–1 for sarcosine and 4.3 × 10–8 M–1 s–1 for dimethylglycine. The non-enzymatic methylations of imidazole and methionine by TMS+, benchmarks for the methylation of histidine and methionine residues by SETD3, exhibit k 25 values of 3.3 × 10–9 and 1.2 × 10–9 M–1 s–1, respectively. Lysine methylation by SAM, although slow under physiological conditions (t 1/2 = 7 weeks at 25 °C), is accelerated 1.1 × 1012 -fold at the active site of a SET domain methyltransferase.

Enzymology and significance of protein histidine methylation

“…This free electron pair can perform nucleophilic attacks on electrophiles such as the polarized methyl group of AdoMet ( Fig. 1 B ), resulting in methyl transfer from AdoMet to His ( 18 ). In cells, AdoMet-dependent PHMT enzymes catalyze the generation of N1-methylhistidine (1MeH) and N3-methylhistidine (3MeH) in specific protein substrates ( Fig.…”

“…For Hme, nonenzymatic reactions may not be negligible. It is well established that the major cellular methyl donor AdoMet can partake in nonenzymatic methylation ( 72 ) and the nucleophilic nature of the His side chain may engage in reactions with AdoMet in the absence of enzymes at the physiological pH ( 18 ). For SETD3, a recent study suggests that nonenzymatic methylation is slow compared with enzyme-catalyzed modification ( 18 ).…”

“…It is well established that the major cellular methyl donor AdoMet can partake in nonenzymatic methylation ( 72 ) and the nucleophilic nature of the His side chain may engage in reactions with AdoMet in the absence of enzymes at the physiological pH ( 18 ). For SETD3, a recent study suggests that nonenzymatic methylation is slow compared with enzyme-catalyzed modification ( 18 ). However, the relative contribution of nonenzymatic Hme to the Hme-ome, and its potential function, remains to be investigated in detail.…”

Structure, Activity and Function of the Dual Protein Lysine and Protein N-Terminal Methyltransferase METTL13

“…The protein Nt α-amino group and the ε-amino group of lysine are both chemical bases and exist in both possible protonation states; a neutral state and a positively charged state. The neutral, i.e., unprotonated state is characterized by a free electron pair capable of acting as a nucleophile in nucleophilic substitution reactions [8]. In cells, the differentially protonated forms are in equilibrium and their relative abundance is determined by the acid dissociation constant (pKa) and pH.…”