Kinetics of O-2 Entry and Exit in Monomeric Sarcosine Oxidase via Markovian Milestoning Molecular Dynamics

摘要

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 O-2 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/O-2 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 O-2 entry and exit, in terms of which possible solvent-to-active-site channels contribute to the flow of O-2, is sensitive to the presence of the substrate-mimicking competitive inhibitor 2-furoate in the substrate site. The second-order O-2 entry rate constants were computed to be 8.1 x 10(6) and 3.1 x 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) x 10(5) M-1 s(-1) for flavin oxidation by O-2 in MSOX. This suggests that the rate of flavin oxidation by O-2 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 x 10(6) s(-1) for the apo and bound states, respectively. The predicted faster en-try and slower exit of O-2 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 O-2 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 O-2, 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.