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

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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 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 increasing pH throughout the normal pH range, equilibrium constants for the hydrolytic demethylation of secondary, tertiary and quaternary amines are found to be insensitive to changing pH and resemble to each other in magnitude, with an average ΔG value of ∼ -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 free energy change of -7.5 kcal/mol in neutral solution. Arrhenius analysis of the uncatalyzed reactions shows that the unprotonated form of glycine attacks the trimethylsulfonium ion (TMS+ + ) with a 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 = 1.1 × 10 −7 M -1 s -1 for sarcosine and 4.3 × 10 −8 M -1 s -1 for dimethylglycine. The nonenzymatic methylation 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 × 10 12 -fold at the active site of a SET domain methyltransferase.

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