Role of tryptophan 161 in catalysis by human manganese superoxide dismutase
Role of tryptophan 161 in catalysis by human manganese superoxide dismutase
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DOI：
10.1021/bi9909142
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发表时间：
1999-09-07
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：
Cabelli, DE;Guan, Y;Silverman, DN
色氨酸161是一个高度保守的残基，它形成锰超氧化物歧化酶（MnSOD）活性位点空腔的疏水侧，其吲哚环邻近锰，距离锰约5埃。我们在人MnSOD中制备了一个含有Trp 161 → Phe保守取代的突变体（W161 F MnSOD），测定了它的晶体结构，并用脉冲辐解测定了所产生的突变体产生O-2（.在W161 F MnSOD的结构中，Phe 161的苯基侧链叠加在野生型中Trp 161的吲哚环上。然而，在突变体中，Tyr 34的羟基侧链距离锰3.9 iq，比野生型中更接近1.2埃。MnSOD中的色氨酸对于涉及锰还原的催化活性的半循环不是必需的：突变体W161 F MnSOD的k（cat）/K-m为2.5 x 10（8）M-1 s（-1），与野生型相比仅降低3倍。然而，该突变体表现出较强的产物抑制与超氧化物衰变的零级区域慢10倍，与野生型相比。在抑制状态下的W161 F MnSOD的可见吸收光谱是非常相似的，观察到的抑制野生型酶。抑制形式的出现需要2摩尔当量的O-2（.-）与W161 F Mn（III）SOD反应，一种形成金属的还原态，第二种形成抑制复合物，证实抑制复合物需要O-2（.-）反应。与酶的还原形式。这项工作表明，色氨酸161在活性位点的一个重要作用是促进产品过氧化物的解离，也许部分通过其对酪氨酸34的取向的影响。
Tryptophan 161 is a highly conserved residue that forms a hydrophobic side of the active site cavity of manganese superoxide dismutase (MnSOD), with its indole ring adjacent to and about 5 Angstrom from the manganese. We have made a mutant containing the conservative replacement Trp 161 --> Phe in human MnSOD (W161F MnSOD), determined its crystal structure, and measured the catalysis of the resulting mutant using pulse radiolysis to produce O-2(.-). In the structure of W161F MnSOD the phenyl side chain of Phe 161 superimposes on the indole ring of Trp 161 in the wild type. However, in the mutant, the hydroxyl side chain of Tyr 34 is 3.9 iq from the manganese, closer by 1.2 Angstrom than in the wild type. The tryptophan in MnSOD is not essential for the half-cycle of catalytic activity involving reduction of the manganese: the mutant W161F MnSOD had k(cat)/K-m at 2.5 x 10(8) M-1 s(-1), reduced only 3-fold compared with wild type. However, this mutant exhibited a strong product inhibition with a zero-order region of superoxide decay slower by 10-fold compared with wild type. The visible absorption spectrum of W161F MnSOD in the inhibited state was very similar to that observed for the inhibited wild-type enzyme. The appearance of the inhibited form required reaction of 2 molar equiv of O-2(.-) with W161F Mn(III)SOD, one to form the reduced state of the metal and the second to form the inhibited complex, confirming that the inhibited complex requires reaction of O-2(.-) With the reduced form of the enzyme. This work suggests that a significant role of Trp 161 in the active site is to promote the dissociation of product peroxide, perhaps in part through its effect on the orientation of Tyr 34.