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Spectral and kinetic characterization of the michaelis charge transfer complex in monomeric sarcosine oxidase.

ABSTRACT: Monomeric sarcosine oxidase is a flavoenzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine). Rapid reaction kinetic studies under anaerobic conditions at pH 8.0 show that the enzyme forms a charge transfer Michaelis complex with sarcosine (E-FAD(ox).sarcosine) that exhibits an intense long-wavelength absorption band (lambda(max) = 516 nm, epsilon(516) = 4800 M(-)(1) cm(-)(1)). Since charge transfer interaction with sarcosine as donor is possible only with the anionic form of the amino acid, the results indicate that the pK(a) of enzyme-bound sarcosine must be considerably lower than the free amino acid (pK(a) = 10.0). No redox intermediate is detectable during sarcosine oxidation, as judged by the isosbestic spectral course observed for conversion of E-FAD(ox).sarcosine to reduced enzyme at 25 or 5 degrees C. The limiting rate of the reductive half-reaction at 25 degrees C (140 +/- 3 s(-)(1)) is slightly faster than turnover (117 +/- 3 s(-)(1)). The kinetics of formation of the Michaelis charge transfer complex can be directly monitored at 5 degrees C where the reduction rate is 4.5-fold slower and complex stability is increased 2-fold. The observed rate of complex formation exhibits a hyperbolic dependence on sarcosine concentration with a finite Y-intercept, consistent with a mechanism involving formation of an initial complex followed by isomerization to yield a more stable complex. Similar results are obtained for charge transfer complex formation with methylthioacetate. The observed kinetics are consistent with structural studies which show that a conformational change occurs upon binding of methylthioacetate and other competitive inhibitors.

SUBMITTER: Zhao G

PROVIDER: S-EPMC2764459 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Spectral and kinetic characterization of the michaelis charge transfer complex in monomeric sarcosine oxidase.

Zhao Gouhua G Jorns Marilyn Schuman MS

Biochemistry 20060501 19

Monomeric sarcosine oxidase is a flavoenzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine). Rapid reaction kinetic studies under anaerobic conditions at pH 8.0 show that the enzyme forms a charge transfer Michaelis complex with sarcosine (E-FAD(ox).sarcosine) that exhibits an intense long-wavelength absorption band (lambda(max) = 516 nm, epsilon(516) = 4800 M(-)(1) cm(-)(1)). Since charge transfer interaction with sarcosine as donor is possible only with the ani ...[more]

PMID: 16681370

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Project description: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.

Dataset Information

Spectral and kinetic characterization of the michaelis charge transfer complex in monomeric sarcosine oxidase.

Publications

Spectral and kinetic characterization of the michaelis charge transfer complex in monomeric sarcosine oxidase.

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