Catalytic strategy of S-adenosyl-L-homocysteine hydrolase:: Transition-state stabilization and the avoidance of abortive reactions
Catalytic strategy of S-adenosyl-L-homocysteine hydrolase:: Transition-state stabilization and the avoidance of abortive reactions
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DOI：
10.1021/bi0262350
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发表时间：
2003-02-25
期刊：
影响因子：
2.9
通讯作者：
Schowen, RL
中科院分区：
文献类型：
--
作者：
Yang, XD;Hu, YB;Schowen, RL
S-腺苷高半胱氨酸水解酶（NAHcy水解酶）从含有中间体类似物neplanocin A的溶液中结晶，其中该类似物在其所有四个亚基的活性位点以其3 '-酮形式结合，四个紧密结合的辅因子以其还原（NADH）状态结合。酶处于闭合构象，这对应于催化化学发生的结构。根据现有的非常详细的动力学研究[Porter，D. J.，Boyd，F. L.等人（1991）J.Biol.Chem.266，21616-21625。波特，D. J.，Boyd，F. L.等人（1992）J.Biol.Chem.267，3205-3213。波特，D. J.（1998）J.Biol.Chem.268，66-73]提出了用于加速α-Hcy向腺苷（Ado）和高半胱氨酸（Hcy）的可逆转化的α-Hcy水解酶的催化策略的要素。该酶的每个亚基都具有底物结合结构域，在没有底物的情况下，该结构域相对于酶的四聚体核心快速运动，首先结合底物并停止运动。可能与底物氧化成其3 '-酮形式同时，封闭的活性位点与环境“密封”，如配体离开速率的大的（10（8-9）倍）降低所示，这是防止不稳定的3'-酮中间体暴露于水性环境的特征。5 '-取代基的消除（水解方向上的Hey，合成方向上的水）产生中心中间体4'，5 '-二氨基-5'-脱氧-3 '-酮腺苷。在中心中间体存在期间，通过暂时中止酶的全部或部分氧化还原催化能力来防止中心中间体的不成功的3 '-还原。在催化循环结束时，失败的还原比生产性还原慢10（4）倍，并且具有与非酶分子内模型反应相似的速率常数。暂停氧化还原催化能力的机制似乎是构象诱导的距离增加，在该距离上，氢化物转移必须发生在辅因子和底物之间，负责的构象变化再次被“密封”的活性位点。晶体结构揭示了一个明确定义的链的三个水分子导致从活性位点的亚基表面，这可能是作为一个中继溶剂和活性位点之间的质子交换在封闭形式的酶，允许维护的活性位点的功能基团在催化合适的质子化状态。
S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase) crystallizes from solutions containing the intermediate analogue neplanocin A with the analogue bound in its 3'-keto form at the active sites of all of its four subunits and the four tightly bound cofactors in their reduced (NADH) state. The enzyme is in the closed conformation, which corresponds to the structure in which the catalytic chemistry occurs. Examination of the structure in the light of available, very detailed kinetic studies [Porter, D. J., Boyd, F. L. (1991) J. Biol. Chem. 266, 21616-21625. Porter, D. J., Boyd, F. L. (1992) J. Biol. Chem. 267, 3205-3213. Porter, D. J. (1998) J. Biol. Chem. 268, 66-73] suggests elements of the catalytic strategy of AdoHcy hydrolase for acceleration of the reversible conversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy). The enzyme, each subunit of which possesses a substrate-binding domain that in the absence of substrate is in rapid motion relative to the tetrameric core of the enzyme, first binds substrate and ceases motion. Probably concurrently with oxidation of the substrate to its 3'-keto form, the closed active site is "sealed off" from the environment, as indicated by a large (10(8-9)-fold) reduction in the rate of departure of ligands, a feature that prevents exposure of the labile 3'-keto intermediates to the aqueous environment. Elimination of the 5'-substituent (Hey in the hydrolytic direction, water in the synthetic direction) generates the central intermediate 4',5'-didehydro-5'-deoxy-3'-ketoadenosine. Abortive 3'-reduction of the central intermediate is prevented by a temporary suspension of all or part of the redox catalytic power of the enzyme during the existence of the central intermediate. The abortive reduction is 10(4)-fold slower than the productive reductions at the ends of the catalytic cycle and has a rate constant similar to those of nonenzymic intramolecular model reactions. The mechanism for suspending the redox catalytic power appears to be a conformationally induced increase in the distance across which hydride transfer must occur between cofactor and substrate, the responsible conformational change again being that which "seals" the active site. The crystal structure reveals a well-defined chain of three water molecules leading from the active site to the subunit surface, which may serve as a relay for proton exchange between solvent and active site in the closed form of the enzyme, permitting maintenance of active-site functional groups in catalytically suitable protonation states.