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首页> 外文期刊>Biochemistry >Spectral and Kinetic Characterization of the Michaelis Charge Transfer Complex in Monomeric Sarcosine Oxidase
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Spectral and Kinetic Characterization of the Michaelis Charge Transfer Complex in Monomeric Sarcosine Oxidase

机译：单体肌肉氧化酶中迈克莱斯电荷复合物的光谱和动力学表征

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摘要

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)centre dot 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)centre dot sarcosine to reduced enzyme at 25 or 5 deg C.The limiting rate of the reductive half-reaction at 25 deg 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 deg 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.
机译：单体肌氨酸氧化酶是一种黄酮酶，其催化肌肉素（N-甲基甘氨酸）.RAPID反应动力学研究在pH8.0下的厌氧条件下的氧化剂表明，酶形成与肌肉（E-FAD_（牛（牛）形成电荷转移迈克莱斯络合物）中心点Sarcosine）表现出强烈的长波长吸收带（Lambda__（max）= 516nm，epsilon_（516）= 4800m〜（-1）cm〜（-1））。由于电荷转移与肌肉苷的相互作用仅通过氨基酸的阴离子形式可以实现供体，结果表明酶结合的肌氨酸的PK_A必须比游离氨基酸（PK_A = 10.0）。在触觉氧化期间不可检测NNO氧化还原中间体。判断观察到ESOSBestic光谱课程用于将E-FAD_（牛）中心点肌氨酸转化为25或5℃的降低酶。在25℃（140 + -3 s〜（ - 1））略微比周转更快（117 + -3 s〜（-1））。形式的动力学Michaelis电荷转移复合物可以在5℃下直接监测，其中减少率为4.5倍，复杂的稳定性增加2倍。观察到的复杂形成速率表现出对肌肉浓度的双曲线依赖性，有限-Intercept，与涉及形成初始复合物的机制，其次是异构化，得到更稳定的复合物。获得含甲基乙酸酯的电荷转移复合物的相似结果。观察到的动力学与结构研究一致，表明一致性变化发生了一致性变化结合甲基乙酸酯和其他竞争性抑制剂。

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  • 来源
    《Biochemistry》 |2006年第19期|共8页
  • 作者

    Gouhua Zhao; Marilyn Schuman Jorns;

  • 作者单位

    Department of Biochemistry and Molecular Biology Drexel University College of Medicine Philadelphia Pennsylvania 19102 Department of Biochemistry and Molecular Biology Drexel University College of Medicine Philadelphia Pennsylvania 19102;

    Department of Biochemistry and Molecular Biology Drexel University College of Medicine Philadelphia Pennsylvania 19102;

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  • 正文语种 eng
  • 中图分类 生物化学;
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