Coupling of cobalt-carbon bond homolysis and hydrogen atom abstraction in adenosylcobalamin-dependent glutamate mutase
Coupling of cobalt-carbon bond homolysis and hydrogen atom abstraction in adenosylcobalamin-dependent glutamate mutase
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DOI：
10.1021/bi980512e
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发表时间：
1998-08-25
期刊：
影响因子：
2.9
通讯作者：
Ballou, DP
中科院分区：
文献类型：
--
作者：
Marsh, ENG;Ballou, DP
腺苷钴胺素依赖性谷氨酸盐催化一种不寻常的碳骨架重排，该重排通过底物诱导的辅酶钴-碳键裂解产生的自由基中间体的形成而进行。在10 ° C下用不同浓度的L-谷氨酸和L-苏型-3-甲基天冬氨酸研究反应，并使用停流光谱法跟踪cob（II）alamin的形成。任一基板诱导快速形成的辅酶（II）的丙氨酸，积累占约25%的总酶物种在稳态时，基板饱和。对钴（II）丙氨素形成速率常数的测量表明，该酶将钴-碳键的均裂速率加速了至少10（12）倍。非常大的同位素效应对辅酶（II）的丙氨酸形成，28和35，观察到与氘代L-谷氨酸和氘代L-苏型-3-甲基天冬氨酸，分别。这意味着一种机制，其中Co-C键的均裂是动力学耦合到基板的氢提取。因此，腺苷自由基只能在非常低的酶浓度下作为高能中间体形成。同位素效应的大小表明氢隧穿可能在催化中起重要作用。
Adenosylcobalamin-dependent glutamate mutase catalyzes an unusual carbon skeleton rearrangement that proceeds through the formation of free radical intermediates generated by the substrate-induced cleavage of the coenzyme cobalt-carbon bond. The reaction was studied at 10 degrees C with various concentrations of L-glutamate and L-threo-3-methylaspartate and with use of stopped-flow spectroscopy to follow the formation of cob(II)alamin. Either substrate induces rapid formation of cob(II)alamin, which accumulates to account for about 25% of the total enzyme species in the steady state when substrate is saturating. Measurements of the rate constant for the formation of cob(II)alamin demonstrate that the enzyme accelerates the rate of homolysis of the cobalt-carbon bond by at least 10(12)-fold. Very large isotope effects on cob(II)alamin formation, of 28 and 35, are observed with deuterated L-glutamate and deuterated L-threo-3-methylaspartate, respectively. This implies a mechanism in which Co-C bond homolysis is kinetically coupled to substrate hydrogen abstraction. Therefore, adenosyl radical can only be formed as a high-energy intermediate only at very low concentrations on the enzyme. The magnitude of the isotope effects suggests that hydrogen tunneling may play an important role catalysis.