EVIDENCE FROM O-18 EXCHANGE STUDIES FOR AN EXOCYCLIC METHYLENE INTERMEDIATE IN THE REACTION CATALYZED BY T4 DEOXYCYTIDYLATE HYDROXYMETHYLASE
EVIDENCE FROM O-18 EXCHANGE STUDIES FOR AN EXOCYCLIC METHYLENE INTERMEDIATE IN THE REACTION CATALYZED BY T4 DEOXYCYTIDYLATE HYDROXYMETHYLASE
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DOI：
10.1021/bi00200a038
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发表时间：
1994-08-30
期刊：
影响因子：
2.9
通讯作者：
HARDY, LW
中科院分区：
文献类型：
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作者：
BUTLER, MM;GRAVES, KL;HARDY, LW
O-18交换实验旨在鉴定噬菌体T4脱氧胞苷酸（dCMP）羟甲基化酶（CH）催化机制中的最终中间体。CH催化由dCMP和亚甲基四氢叶酸（CH 2-THF）形成5-（羟甲基）-dCMP（HmdCMP）。CH在氨基酸序列和催化的反应方面都类似于胸苷酸合成酶（TS），一种已知三维结构的酶。TS或CH催化反应的最终中间体被认为是环外5-亚甲基与酶共价连接的核苷酸。然后将该中间体水合为HmdCMP（通过CH）或还原为脱氧胸苷酸（通过TS）。我们在这里报告，CH催化纳入O-18从溶剂水到产品，HmdCMP，在四氢叶酸（THF）的存在下。这种交换的原因是一个逆反应，然后再合成。CH还催化交换O-18从溶剂水HmdCMP在不存在外源性THF和在THF类似物的存在下，缺乏N-5。N-5是在转移到dCMP时可能与亚甲基结合的氮原子。缺乏亲核Cys 148的CH变体不能促进这些O-18交换反应。缺乏N-5的THF类似物不促进CH催化的逆反应。相反，我们认为这些THF类似物促进的CH催化的O-18交换反应是通过Cys 148与酶连接的5-亚甲基-dCMP发生的。我们的结论是，酶结合的5-亚甲基-dCMP是CH催化过程中的最终中间体，也已经提出TS和dUMP。
O-18 exchange experiments were designed to identify the final intermediate in the catalytic mechanism of bacteriophage T4 deoxycytidylate (dCMP) hydroxymethylase (CH). CH catalyzes the formation of 5-(hydroxymethyl)-dCMP (HmdCMP) from dCMP and methylenetetrahydrofolate (CH2-THF). CH resembles thymidylate synthase (TS), an enzyme of known three-dimensional structure, in both amino acid sequence and the reaction catalyzed. The final intermediate in the reaction catalyzed by TS or CH has been proposed to be the nucleotide with an exocyclic 5-methylene group covalently linked to the enzyme. This intermediate is then hydrated to HmdCMP (by CH) or reduced to deoxythymidylate (by TS). We report here that CH catalyzes the incorporation of O-18 from solvent water into the product, HmdCMP, in the presence of tetrahydrofolate (THF). The cause of this exchange is a reverse reaction followed by a resynthesis. CH also catalyzes the exchange of O-18 from solvent water into HmdCMP in the absence of exogenous THF and in the presence of THF analogues that lack N-5. N-5 is the nitiogen that is likely to be bound to the methylene as it is transferred to dCMP. A CH variant that lacks the nucleophilic Cys 148 is incapable of promoting these O-18 exchange reactions. The THF analogues lacking N-5 do not promote a CH-catalyzed reverse reaction. Rather, we propose that the CH-catalyzed O-18 exchange reaction promoted by these THF analogues occurs via 5-methylene-dCMP linked to the enzyme through Cys 148. We conclude here that enzyme-bound 5-methylene-dCMP is the final intermediate during catalysis by CH, as has also een proposed for TS and dUMP.