Role of hydrogen bonding in the active site of human manganese superoxide dismutase
Role of hydrogen bonding in the active site of human manganese superoxide dismutase
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DOI：
10.1021/bi049888k
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发表时间：
2004-06-08
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：
Greenleaf, WB;Jefferson, J;Silverman, DN
Gln 143是锰超氧化物歧化酶（MnSOD）中的保守残基，其侧链与锰结合溶剂形成氢键，并且在维持催化活性方面至关重要。Tyr 34和Trp 123的侧链与Gln 143的甲酰胺形成氢键。我们已经取代Tyr 34和Trp 123与Phe在单和双突变体的人MnSOD和测定其催化活性的停流分光光度法和脉冲辐解。这些侧链的替换抑制了催化中多达50倍的步骤;此外，它们改变了催化和过氧化物复合物形成之间的门控，以产生更多的产物抑制酶。双突变体中Tyr 34和Trp 123的替换表明这两个残基在维持催化活性方面相互作用。Y34 F/W123 F人MnSOD在1.95埃分辨率下的晶体结构表明，这种效应与Gln 143侧链中的构象变化无关，Gln 143侧链不改变Y34 F/W123 F中的取向，而是由于Gln 143的羧酰胺侧链的氢键丧失而引起的更微妙的电子效应。含有Trp 123和Tyr 34的野生型MnSOD与在这些位置含有Phe的突变体相比具有大致相同的热稳定性，这表明由这些残基形成的氢键具有功能而不是结构作用。
The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F/W123F human MnSOD at 1.95 Angstrom resolution suggests that this effect is not related to a conformational change in the side chain of Gin 143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles.