Department of Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, North Carolina 27514, United States.
Biochemistry. 2021 Mar 23;60(11):854-858. doi: 10.1021/acs.biochem.1c00028. Epub 2021 Mar 5.
SAM is a powerful methylating agent, with a methyl group transfer potential matching the phosphoryl group transfer potential of ATP. SAM-dependent -methyltransferases have evolved to catalyze the modification of specific lysine residues in histones and transcription factors, in addition to generating epinephrine, -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 Δ 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 M s at 25 °C (Δ = 22 kcal/mol, and Δ = -6 kcal/mol). Comparable values are observed for the methylation of secondary and tertiary amines, with values of 1.1 × 10 M s for sarcosine and 4.3 × 10 M s for dimethylglycine. The non-enzymatic methylations of imidazole and methionine by TMS, benchmarks for the methylation of histidine and methionine residues by SETD3, exhibit values of 3.3 × 10 and 1.2 × 10 M s, respectively. Lysine methylation by SAM, although slow under physiological conditions ( = 7 weeks at 25 °C), is accelerated 1.1 × 10 -fold at the active site of a SET domain methyltransferase.
SAM 是一种强大的甲基供体，其甲基转移潜力与 ATP 的磷酸化转移潜力相当。SAM 依赖性 - 甲基转移酶已进化为催化组蛋白和转录因子中特定赖氨酸残基的修饰，除了产生肾上腺素、-甲基烟酰胺和季铵盐（甜菜碱）外，还用于维持植物和嗜盐菌中的渗透压。为了评估这些酶的催化能力及其对过渡态和多底物类似物抑制剂的潜在敏感性，我们在没有催化剂的情况下，测定了从三甲基锍离子向模型胺转移甲基的平衡速率和位置。与 SAM 的甲基转移潜力不同，其在整个正常 pH 范围内随 pH 的增加而变得更负，发现二级、三级和季铵盐的水解脱甲基平衡常数对 pH 的变化不敏感，并且彼此相似，在 pH 7 时平均Δ值约为-0.7 kcal/mol。因此，SAM 对赖氨酸的单、二和三甲基化的三个步骤都伴随着在中性溶液中自由能变化-7.5 kcal/mol。未催化反应的 Arrhenius 分析表明，甘氨酸的未质子化形式以 2.8 × 10 M s 的二级速率常数攻击三甲基锍离子（TMS）（Δ = 22 kcal/mol，Δ =-6 kcal/mol）。对于二级和三级胺的甲基化，观察到类似的值，肌氨酸的 值为 1.1 × 10 M s，二甲基甘氨酸的 值为 4.3 × 10 M s。TMS 对咪唑和蛋氨酸的非酶甲基化，是 SETD3 对组氨酸和蛋氨酸残基甲基化的基准，分别表现出 3.3 × 10 和 1.2 × 10 M s 的 值。SAM 对赖氨酸的甲基化在生理条件下很慢（25°C 时为 7 周），但在 SET 结构域甲基转移酶的活性部位被加速 1.1 × 10 -倍。
