Linscott Joshua A, Kapilashrami Kanishk, Wang Zhen, Senevirathne Chamara, Bothwell Ian R, Blum Gil, Luo Minkui
Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
Program of Pharmacology, Weill Graduate School of Medical Science, Cornell University, New York, NY 10021.
Proc Natl Acad Sci U S A. 2016 Dec 27;113(52):E8369-E8378. doi: 10.1073/pnas.1609032114. Epub 2016 Dec 9.
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 (S2) 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 C KIE of 1.04, an inverse intrinsic α-secondary CD KIE of 0.90, and a small but statistically significant inverse CD BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical S2 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.
蛋白质赖氨酸甲基转移酶（PKMTs）催化蛋白质底物的甲基化，其失调与包括癌症在内的多种疾病有关。越来越多的证据表明，PKMT催化甲基化的反应路径包括辅因子（共底物）-PKMT-底物复合物的形成、通过动态水通道进行的赖氨酸去质子化以及转甲基化的亲核取代（S2）过渡态。然而，所提出过程的分子特征仍有待通过实验阐明。在此，我们开发了一种基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）方法及相应的数学矩阵，以精确测定同位素甲基化肽段的比例。这种方法可能普遍适用于研究翻译后修饰酶的动力学同位素效应（KIEs）。蛋白质赖氨酸甲基转移酶SET8是唯一能使组蛋白4赖氨酸20（H4K20）发生单甲基化的PKMT，其功能与正常细胞周期进程和癌症转移有关。因此，我们采用基于质谱的方法来测量SET8催化H4K20单甲基化时辅因子S-腺苷-L-甲硫氨酸（SAM）的KIEs和结合同位素效应（BIEs）。一个主要的内在C KIE为1.04，一个反向内在α-二级CD KIE为0.90，以及一个虽小但具有统计学意义的反向CD BIE为0.96，结合计算模型，表明SET8催化的甲基化通过一个早期的、不对称的S2过渡态进行，C-N和C-S距离分别为2.35 - 2.40 Å和2.00 - 2.05 Å。以SAM类似物硒代腺苷-L-硒代甲硫氨酸（SeAM）作为辅因子替代物的KIEs、BIEs和稳态动力学进一步支持了这种过渡态。蛋白质甲基转移酶之间不同的过渡态为设计选择性过渡态类似物抑制剂提供了机会。
