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首页> 外文期刊>Biochemistry >Photolysis of Adenosylcobalamin and Radical Pair Recombination in Ethanolamine Ammonia-Lyase Probed on the Micro- to Millisecond Time Scale by Using Time-Resolved Optical Absorption Spectroscopy
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Photolysis of Adenosylcobalamin and Radical Pair Recombination in Ethanolamine Ammonia-Lyase Probed on the Micro- to Millisecond Time Scale by Using Time-Resolved Optical Absorption Spectroscopy

机译：使用时间分辨光学吸收光谱法在微秒至毫秒级的乙醇胺氨水解酶中腺苷钴胺的光解和自由基对的重组

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The quantum yield and kinetics of decay of cob(II)alamin formed by pulsed-laser photolysis of adenosylcobalamin (AdoCbl; coenzyme B12) in AdoCbl-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied on the 10−7−10−1 s time scale at 295 K by using transient ultraviolet−visible absorption spectroscopy. The aim is to probe the mechanism of formation and stabilization of the cob(II)alamin−5′-deoxyadenosyl radical pair, which is a key intermediate in EAL catalysis, and the influence of substrate binding on this process. Substrate binding is required for cobalt−carbon bond cleavage in the native system. Photolysis of AdoCbl in EAL leads to a quantum yield at 10−7 s for cob(II)alamin of 0.08 ± 0.01, which is 3-fold smaller than for AdoCbl in aqueous solution (0.23 ± 0.01). The protein binding site therefore suppresses photoproduct radical pair formation. Three photoproduct states, Pf, Ps, and Pc, are identified in holo-EAL by the different cob(II)alamin decay kinetics (subscripts denote fast, slow, and constant, respectively). These states have the following first-order decay rate constants and quantum yields: 2.2 × 103 s−1 and 0.02 for Pf, 4.2 × 102 s−1 and 0.01 for Ps, and constant amplitude, with no recombination, and 0.05 for Pc, respectively. Binding of the substrate analogue (S)-1-amino-2-propanol to EAL eliminates the Pf state and lowers the quantum yield of Pc (0.03) relative to that of Ps (0.01) but does not significantly change the quantum yield or decay rate constant of Ps, relative to those of holo-EAL. The substrate analogue thus influences the quantum yield at 10−7 s by changing the cage escape rate from the geminate cob(II)alamin−5′-deoxyadenosyl radical pair state. However, the predicted substrate analogue binding-induced increase in the quantum yield is not observed. It is proposed that the substrate analogue does not induce the radical pair stabilizing changes in the protein that are characteristic of true substrates.
机译：在10−7−上研究了鼠伤寒沙门氏菌依赖AdoCbl的乙醇胺氨解酶（EAL）中的腺苷钴胺素（AdoCbl;辅酶B12）的脉冲激光光解形成的Cob（II）alamin的量子产率和动力学。通过使用瞬态紫外-可见吸收光谱在295 K下的10-1 s时间刻度。目的是探究Cob（II）alamin-5'-脱氧腺苷基对的形成和稳定机理，该对是EAL催化的关键中间体，以及底物结合对该过程的影响。底物结合是天然系统中钴碳键裂解所必需的。在EAL中AdoCbl的光解作用会导致10-7 s时cob（II）阿拉明的量子产率为0.08±0.01，这比水溶液中AdoCbl的量子产率（0.23±0.01）小三倍。因此，蛋白质结合位点抑制了光产物自由基对的形成。在全息EAL中，通过不同的Cob（II）alamin衰变动力学（下标分别表示快，慢和恒定）识别出三个光产物状态Pf，Ps和Pc。这些状态具有以下一阶衰减率常数和量子产率：Pf为2.2×103 s-1和0.02，Ps为4.2×102 s-1和0.01，且没有重组的恒定振幅和Pc为0.05，分别。底物类似物（S）-1-氨基-2-丙醇与EAL的结合可消除Pf状态并相对于Ps（0.01）降低Pc（0.03）的量子产率，但不会显着改变量子产率或衰减相对于全息EAL的Ps速率常数。因此，底物类似物通过改变笼状cob（II）alamin-5'-脱氧腺苷基对状态的笼逃逸率而影响10-7 s处的量子产率。然而，未观察到预期的底物类似物结合诱导的量子产率的增加。提出底物类似物不诱导蛋白质中作为真实底物特征的自由基对稳定化变化。

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