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Structural Characterization of Mutations at the Oxygen Activation Site in Monomeric Sarcosine Oxidase,

机译：单体肌氨酸氧化酶中氧激活位点突变的结构表征

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摘要

Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur atnseparate sites above the si- and re-faces, respectively, of the flavin ring.Mutagenesis studies implicate Lys265nas the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (Arg) residuenresults in an ∼104n- or 250-fold decrease, respectively, in the reaction rate. The overall structure ofMSOX andnresidue conformation in the sarcosine binding cavity are unaffected by replacement of Lys265 with Met ornArg. The side chain ofMet265 exhibits the same configuration in each molecule of Lys265Met crystals and isnnearly congruent with Lys265 in wild-typeMSOX. The side chain of Arg265 is, however, dramatically shiftedn(∼4-5A) compared with Lys265, points in the opposite direction, and exhibits significant conformationalnvariability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely toncontain a “flipped-out” Arg265 and exhibit negligible oxygen activation, similar to Lys265Met. The 400-foldnhigher oxygen reactivity observed with Lys265Arg is attributed to a minor (<1%) “flipped-in” Arg265nconformer whose oxygen reactivity is similar to that of wild-typeMSOX. A structural water (WAT1), foundnabove the si-face of the flavin ring in all previously determined MSOX structures, is part of an apparentnproton relay system that extends from FAD N(5) to bulk solvent. WAT1 is strikingly absent in Lys265Metnand Lys265Arg, a feature that may account for the apparent kinetic stabilization of a reductive half-reactionnintermediate that is detectable with the mutants but not wild-type MSOX.
机译：单体肌氨酸氧化酶（MSOX）中的氧还原和肌氨酸氧化分别发生在黄素环的正反面和反面上方。诱变研究表明Lys265nas的氧活化位点。用中性（Met，Gln或Ala）或碱性（Arg）残基取代Lys265可使反应速率分别降低约104n-或250倍。 Met ornArg取代Lys265不会影响肌氨酸结合腔中MSOX的整体结构和残基构象。在野生型MSOX中，Met265的侧链在Lys265Met晶体的每个分子中显示相同的构型，并且几乎与Lys265完全一致。然而，与Lys265相比，Arg265的侧链明显移位了n（〜4-5A），指向相反的方向，并且在同一晶体的分子之间显示出显着的构象可变性。与Lys265Met相似，Lys265Arg溶液中的主要物质很可能会包含“倒出的” Arg265，并且氧活化作用可忽略不计。用Lys265Arg观察到的400倍更高的氧反应性归因于次要（<1％）的“翻转” Arg265n构象异构体，其氧反应性与野生型MSOX相似。在所有先前确定的MSOX结构中的黄素环的正侧面都存在的结构水（WAT1）是从FAD N（5）扩展到本体溶剂的表观质子传递系统的一部分。在Lys265Metnand Lys265Arg中，WAT1显着缺失，这一特征可能解释了该突变体可检测到的还原半反应中间体的明显动力学稳定性，而野生型MSOX却检测不到。

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    《Biochemistry》 |2010年第17期|p.3631-3639|共9页
  • 作者

    Marilyn Schuman Jorns Zhi-wei Chen and F. Scott Mathews;

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    §Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, and) Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110;

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