Catalytic roles of substrate-binding residues in coenzyme B12-dependent ethanolamine ammonia-lyase
Catalytic roles of substrate-binding residues in coenzyme B12-dependent ethanolamine ammonia-lyase
复制标题
辅酶 B12 依赖性乙醇胺氨裂解酶中底物结合残基的催化作用
DOI：
10.1021/bi500223k
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发表时间：
2014
期刊：
影响因子：
2.9
通讯作者：
Toraya T
中科院分区：
文献类型：
--
作者：
Mori K;Oiwa T;Kawaguchi S;Kondo K;Takahashi Y;Toraya T
乙醇胺氨裂解酶（EAL）催化腺苷钴胺素依赖性的乙醇胺转化为乙醛和氨。底物的1-OH与Gluα287、Argα160和Asnα193形成氢键，2-NH 2与Gluα287、Glnα162和Aspα362形成氢键。活性部位有点类似于二醇脱氢酶的活性部位。所有五个残基是重要的高亲和力结合的基板和催化。Glu α287残基上的-COO-基团是活性和辅酶Co-C键断裂所必需的，并且存在一个空间最佳位置，表明Gluα287有助于Co-C键的均裂，通过旁观者OH的部分去质子化稳定NH 2从C2向C1迁移的过渡态，并在氨的消除中起着碱的作用。在α160位的正电荷和/或氢键以及在α162和α193位与底物的氢键对于催化和防止自由基中间体发生副反应是重要的。Argα160通过静电催化和与旁观者OH的氢键作用稳定NH 2迁移的三角过渡态。Asnα193也有助于保持Argα160胍基的适当位置和方向。在α162位上，氢键受体是必需的，而氢键供体则是有害的。Glnα162可能通过接受迁移的NH3+的氢键来稳定三角过渡态。活性对α362位-COO-的位置非常敏感。Aspα362通过接受迁移的NH3+和静电相互作用形成的氢键，间接地协助Co-C键的均裂，稳定三角过渡态。
Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. 1-OH of the substrate is hydrogen-bonded with Gluα287, Argα160, and Asnα193 and 2-NH2with Gluα287, Glnα162, and Aspα362. The active site somewhat resembles that of diol dehydratase. All five residues were important for the high-affinity binding of the substrate and for catalysis. The -COO–group at residue α287 was absolutely required for activity and coenzyme Co–C bond cleavage, and there was a spatially optimal position for it, suggesting that Gluα287 contributes to Co–C bond homolysis, stabilizes the transition state for the migration of NH2from C2 to C1 through partial deprotonation of spectator OH, and functions as a base in the elimination of ammonia. A positive charge and/or the hydrogen bond at position α160 and the hydrogen bonds at positions α162 and α193 with the substrate are important for catalysis and for preventing a radical intermediate from undergoing side reactions. Argα160 would stabilize the trigonal transition state in NH2migration by electrostatic catalysis and hydrogen bonding with spectator OH. Asnα193 would contribute to maintaining the appropriate position and direction of the guanidinium group of Argα160, as well. Hydrogen bond acceptors were necessary at position α162, but hydrogen bond donors were rather harmful. Glnα162 might stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3+. The activity was very sensitive to the position of -COO–at α362. Aspα362 would assist Co–C bond homolysis indirectly and stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3+and electrostatic interaction.