Catalytic and structural effects of amino acid substitution at histidine 30 in human manganese superoxide dismutase:: Insertion of valine Cγ into the substrate access channel
Catalytic and structural effects of amino acid substitution at histidine 30 in human manganese superoxide dismutase:: Insertion of valine Cγ into the substrate access channel
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DOI：
10.1021/bi0266481
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发表时间：
2003-03-18
期刊：
影响因子：
2.9
通讯作者：
Silverman, DN
中科院分区：
文献类型：
--
作者：
Hearn, AS;Stroupe, ME;Silverman, DN
人锰超氧化物歧化酶（MnSOD）催化超氧化物歧化的特点是最初的催化爆发，随后是一个更慢的区域，在超氧化物中是零阶的，由于过氧化阴离子的产物抑制。我们制备了具有His30替换的位点特异性突变体，其侧链位于底物通路上，距离金属约5.8 A。通过脉冲辐射分解产生超氧化物，我们确定与野生型、H30N和H30Q MnSOD相比，H30V MnSOD的k(cat)/ k -m降低，产物抑制增加，幅度均为1-2个数量级。这些影响不是归因于氧化还原电位，所有这些变体的氧化还原电位都是相似的。H30V Mn(III)SOD与野生型、H30Q和H30N Mn(III)SOD的晶体结构比较表明，活性位点上有两个γ碳的Val30；在野生型中，Cgamma1与His30的Cgamma重叠，Cgamma2延伸到底物通路中，在野生型中占据了近似于水分子的位置。数据表明，Val侧链的Cgamma2明显地中断了这种重叠进入通道的催化作用，并可能与底物-产物结合位点重叠。H30V MnSOD的晶体结构与来自Thermus thermoophilus的叠氮化物与Mn(III)SOD结合的晶体结构的比较，以及可见吸收光谱显示H30V Mn(III)SOD中叠氮化物与金属的结合被消除，都支持了这一点。此外，与野生型相比，Val30的存在使产物抑制复合物的解离速率常数降低了100倍。
Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD) is characterized by an initial burst of catalysis followed by a much slower region that is zero order in superoxide and due to a product inhibition by peroxide anion. We have prepared site-specific mutants with replacements at His30, the side chain of which lies along the substrate access channel and is about 5.8 A from the metal. Using pulse radiolysis to generate superoxide, we have determined that k(cat)/K-m was decreased and product inhibition increased for H30V MnSOD, both by 1-2 orders of magnitude, compared with wild type, H30N, and H30Q MnSOD. These effects are not attributed to the redox potentials, which are similar for all of these variants. An investigation of the crystal structure of H30V Mn(III)SOD compared with wild type, H30Q, and H30N Mn(III)SOD showed the positions of two gamma carbons of Val30 in the active site; Cgamma1 overlaps Cgamma of His30 in wild type, and Cgamma2 extends into the substrate access channel and occupies the approximate position of a water molecule in the wild type. The data suggest that Cgamma2 of the Val side chain has significantly interrupted catalysis by this overlap into the access channel with possible overlap with the substrate-product binding site. This is supported by comparison of the crystal structure of H30V MnSOD with that of azide bound to Mn(III)SOD from Thermus thermophilus and by visible absorption spectra showing that azide binding to the metal in H30V Mn(III)SOD is abolished. Moreover, the presence of Val30 caused a 100-fold decrease in the rate constant for dissociation of the product-inhibited complex compared with wild type.