Kinetics of O2 Entry and Exit in Monomeric Sarcosine Oxidase via Markovian Milestoning Molecular Dynamics

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• 作者：Anthony Bucci ; Tang-Qing Yu ; Eric Vanden-Eijnden ; Cameron F. Abrams
• 刊名：Journal of Chemical Theory and Computation
• 出版年：2016
• 出版时间：June 14, 2016
• 年：2016
• 卷：12
• 期：6
• 页码：2964-2972
• 全文大小：500K
• 年卷期：0
• ISSN：1549-9626

文摘

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 × 10⁶ and 3.1 × 10⁶ 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) × 10⁵ 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 10⁷ s^–1 and 7.2 × 10⁶ 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.