Solvent as a probe of active site motion and chemistry during the hydrogen tunnelling reaction in morphinone reductase
Solvent as a probe of active site motion and chemistry during the hydrogen tunnelling reaction in morphinone reductase
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DOI：
10.1002/cphc.200800303
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发表时间：
2008-09-15
期刊：
影响因子：
2.9
通讯作者：
Scrutton, Nigel S.
中科院分区：
文献类型：
--
作者：
Hay, Sam;Pudney, Christopher R.;Scrutton, Nigel S.
吗啡酮还原酶的还原半反应包括从酶结合的β -烟酰胺腺嘌呤二核苷酸（NADH）到黄素单核苷酸（FMN）的氢化物转移。我们之前已经证明了这一步是通过量子力学隧穿机制进行的。在此，我们探讨了溶剂对活性位点化学的影响。根据FMN中点电位的ph依赖性，还原后的FMN NI的pK(a)为7.4 +/- 0.7。我们排除了还原的FMN N1的质子化与之前的h转移相耦合，因为反应的速率和温度依赖性对高于或低于该pK的溶液pH变化不敏感(a)。此外，溶剂的动力学同位素效应近似于1.0,1度和2度的KIEs对溶液ph都不敏感。研究了溶剂介电常数的影响，发现介电常数在60和80之间变化不影响h转移速率。我们认为，虽然有晶体学证据表明活性部位存在一些水，但参与h隧穿反应的推定促进运动对这种变化不敏感。
The reductive half-reaction of morphinone reductase involves a hydride transfer from enzyme-bound beta-nicotinamide adenine dinucleotide (NADH) to a flavin mononucleotide (FMN). We have previously demonstrated that this step proceeds via a quantum mechanical tunnelling mechanism. Herein, we probe the effect of the solvent on the active site chemistry. The pK(a) of the reduced FMN NI is 7.4 +/- 0.7, based on the pH-dependence of the FMN midpoint potential. We rule out that protonation of the reduced FMN N1 is coupled to the preceding H-transfer as both the rate and temperature-dependence of the reaction are insensitive to changes in solution pH above and below this pK(a). Further, the solvent kinetic isotope effect is similar to 1.0 and both the 1 degrees and 2 degrees KIEs are insensitive to solution pH. The effect of the solvent's dielectric constant is investigated and the rate of H-transfer is found to be unaffected by changes in the dielectric constant between similar to 60 and 80. We suggest that, while there is crystallographic evidence for some water in the active site, the putative promoting motion involved in the H-tunnelling reaction is insensitive to such changes.