MECHANISM-BASED INHIBITION OF THYMIDYLATE SYNTHASE BY 5-(TRIFLUOROMETHYL)-2'-DEOXYURIDINE 5'-MONOPHOSPHATE
MECHANISM-BASED INHIBITION OF THYMIDYLATE SYNTHASE BY 5-(TRIFLUOROMETHYL)-2'-DEOXYURIDINE 5'-MONOPHOSPHATE
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DOI：
10.1021/bi00254a018
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发表时间：
1994-12-20
期刊：
影响因子：
2.9
通讯作者：
SANTI, DV
中科院分区：
文献类型：
--
作者：
ECKSTEIN, JW;FOSTER, PG;SANTI, DV
干酪乳杆菌胸苷酸合成酶（TS）被5-（三氟甲基）-2 '-脱氧尿苷5'-单磷酸（CF（3）dUMP）抑制。CF（3）dUMP在没有5，10-亚甲基四氢叶酸存在下与TS的活性位点结合，催化亲核试剂半胱氨酸198攻击嘧啶的C-6位，导致三氟甲基基团的活化和氟离子的释放。随后，活化的杂环与酶的亲核试剂反应以形成适度稳定的共价复合物。用[2 '-H-3]CF（3）dUMP处理TS，然后对标记肽进行蛋白水解消化，结果显示酪氨酸146和半胱氨酸198与酶-抑制剂复合物中的抑制剂共价结合。二硫苏糖醇（DTT）或β-巯基乙醇的存在下导致的共价复合物的分解，并从复合物的分解产物进行分离和表征。通过X射线晶体学确定酶抑制剂复合物的三维结构，清楚地表明核苷酸与酪氨酸146的共价连接。CF（3）dUMP抑制TS的化学反应机理与动力学，生化和结构的结果是一致的。
Thymidylate synthase (TS) from Lactobacillus casei is inhibited by 5- (trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF(3)dUMP). CF(3)dUMP binds to the active site of TS in the absence of 5,10-methylenetetrahydrofolate, and attack of the catalytic nucleophile cysteine 198 at C-6 Of the pyrimidine leads to activation of the trifluoromethyl group and release of fluoride ion. Subsequently, the activated heterocycle reacts with a nucleophile of the enzyme to form a moderately stable covalent complex. Proteolytic digestion of TS treated with [2'-H-3]CF(3)dUMP, followed by sequencing of the labeled peptides, revealed that tyrosine 146 and cysteine 198 are covalently bound to the inhibitor in the enzyme-inhibitor complex. The presence of dithiothreitol (DTT) or beta-mercaptoethanol resulted in the breakdown of the covalent complex, and products from the breakdown of the complex were isolated and characterized. The three-dimensional structure of the enzyme-inhibitor complex was determined by X-ray crystallography, clearly demonstrating covalent attachment of the nucleotide to tyrosine 146. A chemical reaction mechanism for the inhibition of TS by CF(3)dUMP is presented that is consistent with the kinetic, biochemical, and structural results.