Hearn A S, Stroupe M E, Cabelli D E, Lepock J R, Tainer J A, Nick H S, Silverman D N
Department of Pharmacology, University of Florida, Gainesville, Florida 32610, USA.
Biochemistry. 2001 Oct 9;40(40):12051-8. doi: 10.1021/bi011047f.
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 of 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 Gln143 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 dismutation 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 H2O2.
锰超氧化物歧化酶（MnSOD）在催化超氧阴离子（O₂⁻）的歧化反应过程中，在Mn（II）和Mn（III）状态之间循环，这种催化作用在微摩尔水平的超氧化物存在时，会受到与金属形成的过氧化物抑制复合物的限制。我们研究了保守残基色氨酸161（Trp161）在催化和抑制过程中的作用，该残基构成了MnSOD活性位点腔的一个疏水侧。人MnSOD中色氨酸161被丙氨酸或苯丙氨酸取代的突变体的晶体结构显示，活性位点附近的相邻残基发生了显著的构象变化，特别是野生型MnSOD中的谷氨酰胺143（Gln143）和酪氨酸34（Tyr34），它们参与了一个氢键网络，据信在催化过程中支持质子转移。通过脉冲辐解并观察超氧化物的紫外吸收，我们测定了超氧化物催化歧化的速率常数。此外，通过直接观察氧化态和抑制态的特征可见吸收，确定了这些突变体的产物抑制复合物的形成和解离速率。W161A和W161F MnSOD的催化作用伴随着超氧化物还原催化速率至少降低100倍，这进而促进了一条导致产物抑制的竞争途径。161位突变引起的结构变化导致抑制复合物形成速率常数的变化很小，最多降低6倍。溶剂氢同位素效应支持一种机制，即这种复合物（可能是与锰结合的过氧化物二价阴离子）的形成不涉及对速率有贡献的质子转移；然而，复合物的解离需要质子转移以生成HO₂⁻或H₂O₂。
