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Kinetic Analysis of Product Inhibition in Human Manganese Superoxide Dismutase

机译：人锰超氧化物歧化酶产物抑制动力学分析

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摘要

Manganese superoxide dismutase (MnSOD) cycles between the Mn(II) and Mn(III) states during the catalyzed disproportionation of O_2~·, a catalysis that is limited at micromolar levels of superoxide by a peroxide-inhibited complex with the metal. We have investigated the role in catalysis and inhibition of the conserved residue Trp161 which forms a hydrophobic side o the active site cavity of MnSOD. Crystal structures of mutants of human MnSOD in which Trp161 was replaced with Ala or Phe showed significant conformational changes on adjacent residues near the active site, particularly Gln 143 and Tyr34 which in wild-type MnSOD participate in a hydrogen bond network believed to support proton transfer during catalysis. Using pulse radiolysis and observing the UV absorbance of superoxide, we have determined rate constants for the catalytic dimutation of superoxide. In addition, the rates of formation and dissociation of the product-inhibited complex of these mutants were determined by direct observation of the characteristic visible absorption of the oxidized and inhibited states. Catalysis by W161A and W161F MnSOD was associated with a decrease of at least 100-fold in the catalytic rate of reduction of superoxide, which then promotes a competing pathway leading to product inhibition. The structural changes caused by the mutations at position 161 led to small changes, at most a 6-fold decrease, in the rate constant for formation of the inhibited complex. Solvent hydrogen isotope effects support a mechanism in which formation of this complex, presumably the peroxide dianion bound to the manganese, involves no rate-contributing proton transfer; however, the dissociation of the complex requires proton transfer to generate HO_2~- or H_2O_2.
机译：在O_2〜·0℃的催化歧化期间，Mn（II）和Mn（III）态在催化歧化期间的锰超氧化物歧化酶（MNSOD）循环，其通过过氧化物抑制络合物与金属的过氧化物微摩尔水平限制的催化。我们研究了在催化的残留物TRP161的催化作用和抑制中的作用，它形成疏水侧的液体侧面的疏水侧。用ALA或PHE代替TRP161的人MNSOD突变体的晶体结构在活性位点附近的相邻残基，特别是GLN 143和TYR34附近的相邻残基进行了显着的构象变化，其中在野生型MNSOD中参与认为支持质子转移的氢键网络在催化期间。使用脉冲辐射分解并观察超氧化物的UV吸光度，我们已经确定了超氧化物催化桨级的速率常数。此外，通过直接观察氧化和抑制状态的特征可见的吸收来确定这些突变体的产物抑制复合物的形成和解离的速率。 W161A和W161F MNSOD的催化与超氧化物还原的催化速率下降至少100倍，然后促进导致产品抑制的竞争途径。由位置161的突变引起的结构变化导致较小的变化，最多为6倍，在形成抑制复合物的速率常数中。溶剂氢同位素作用支持这种复合物形成的机制，大概是过氧化锰的过氧化物脱叶，涉及贡献的质子转移;然而，复杂的解离需要质子转移以产生HO_2〜 - 或H_2O_2。

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  • 来源
    《Biochemistry》 |2001年第40期|共8页
  • 作者

    Amy S. Hearm; M. Elizabeth Stroupe; Diane E. Cabelli; James R. Lepock; John A. Tainer; Harry S. Nick; David N. Silverman;

  • 作者单位

    Department of Pharmacology and Neuroscience University of Florida Gainesville Florida 32610 Department of Molecular Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla California 92037 Department of Chemistry Brookhaven;

    Department of Pharmacology and Neuroscience University of Florida Gainesville Florida 32610 Department of Molecular Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla California 92037 Department of Chemistry Brookhaven;

    Department of Pharmacology and Neuroscience University of Florida Gainesville Florida 32610 Department of Molecular Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla California 92037 Department of Chemistry Brookhaven;

    Department of Pharmacology and Neuroscience University of Florida Gainesville Florida 32610 Department of Molecular Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla California 92037 Department of Chemistry Brookhaven;

    Department of Pharmacology and Neuroscience University of Florida Gainesville Florida 32610 Department of Molecular Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla California 92037 Department of Chemistry Brookhaven;

    Department of Pharmacology and Neuroscience University of Florida Gainesville Florida 32610 Department of Molecular Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla California 92037 Department of Chemistry Brookhaven;

    Department of Pharmacology and Neuroscience University of Florida Gainesville Florida 32610 Department of Molecular Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla California 92037 Department of Chemistry Brookhaven;

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  • 正文语种 eng
  • 中图分类 生物化学;
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