Bucci Anthony, Yu Tang-Qing, Vanden-Eijnden Eric, Abrams Cameron F
Department of Chemical and Biological Engineering, Drexel University , Philadelphia, Pennsylvania 19104, United States.
Courant Institute of Mathematical Sciences, New York University , New York, New York 10012, United States.
J Chem Theory Comput. 2016 Jun 14;12(6):2964-72. doi: 10.1021/acs.jctc.6b00071. Epub 2016 May 19.
The flavoenzyme monomeric sarcosine oxidase (MSOX) catalyzes a complex set of reactions currently lacking a consensus mechanism. A key question that arises in weighing competing mechanistic models of MSOX function is to what extent ingress of O2 from the solvent (and its egress after an unsuccessful oxidation attempt) limits the overall catalytic rate. To address this question, we have applied to the MSOX/O2 system the relatively new simulation method of Markovian milestoning molecular dynamics simulations, which, as we recently showed [ Yu et al. J. Am. Chem. Soc. 2015 , 137 , 3041 ], accurately predicted the entry and exit kinetics of CO in myoglobin. We show that the mechanism of O2 entry and exit, in terms of which possible solvent-to-active-site channels contribute to the flow of O2, is sensitive to the presence of the substrate-mimicking competitive inhibitor 2-furoate in the substrate site. The second-order O2 entry rate constants were computed to be 8.1 × 10(6) and 3.1 × 10(6) M(-1) s(-1) for bound and apo MSOX, respectively, both of which moderately exceed the experimentally determined second-order rate constant of (2.83 ± 0.07) × 10(5) M(-1) s(-1) for flavin oxidation by O2 in MSOX. This suggests that the rate of flavin oxidation by O2 is likely not strongly limited by diffusion from the solvent to the active site. The first-order exit rate constants were computed to be 10(7) s(-1) and 7.2 × 10(6) s(-1) for the apo and bound states, respectively. The predicted faster entry and slower exit of O2 for the bound state indicate a longer residence time within MSOX, increasing the likelihood of collisions with the flavin isoalloxazine ring, a step required for reduction of molecular O2 and subsequent reoxidation of the flavin. This is also indirectly supported by previous experimental evidence favoring the so-called modified ping-pong mechanism, the distinguishing feature of which is an intermediate complex involving O2, the flavin, and the oxidized substrate simultaneously in the cavity. These findings demonstrate the utility of the Markovian milestoning approach in contributing new understanding of complicated enyzmatic function.
黄素酶单体肌氨酸氧化酶（MSOX）催化一组复杂的反应，目前尚缺乏一致的机制。在权衡MSOX功能的相互竞争的机制模型时出现的一个关键问题是，溶剂中O2的进入（以及在氧化尝试失败后的逸出）在多大程度上限制了整体催化速率。为了解决这个问题，我们将马尔可夫里程碑分子动力学模拟这种相对较新的模拟方法应用于MSOX/O2系统，正如我们最近所展示的[Yu等人，《美国化学会志》2015年，137卷，3041页]，该方法准确预测了肌红蛋白中CO的进出动力学。我们表明，就哪些可能的溶剂到活性位点通道有助于O2的流动而言，O2进出的机制对底物位点中模拟底物的竞争性抑制剂2-糠酸的存在很敏感。计算得出结合态和脱辅基MSOX的二级O2进入速率常数分别为8.1×10(6)和3.1×10(6) M(-1) s(-1)，两者均适度超过实验测定的MSOX中O2氧化黄素的二级速率常数(2.83±0.07)×10(5) M(-1) s(-1)。这表明O2氧化黄素的速率可能不会受到从溶剂扩散到活性位点的强烈限制。计算得出脱辅基态和结合态的一级逸出速率常数分别为10(7) s(-1)和7.2×10(6) s(-1)。预测的结合态O2进入更快和逸出更慢表明其在MSOX中的停留时间更长，增加了与黄素异咯嗪环碰撞的可能性，这是还原分子O2和随后黄素再氧化所需的一步。先前支持所谓修正乒乓机制的实验证据也间接支持了这一点，其显著特征是在腔内同时存在涉及O2、黄素和氧化底物的中间复合物。这些发现证明了马尔可夫里程碑方法在促进对复杂酶功能的新理解方面的实用性。
