A strategically positioned cation is crucial for efficient catalysis by chorismate mutase
A strategically positioned cation is crucial for efficient catalysis by chorismate mutase
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DOI：
10.1074/jbc.m006351200
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发表时间：
2000-11-24
影响因子：
4.8
通讯作者：
Hilvert, D
中科院分区：
文献类型：
--
作者：
Kast, P;Grisostomi, C;Hilvert, D
先前的组合诱变和体内选择实验提供了AroH类枯草芽孢杆菌分支酸盐的功能变体，其缺乏另外高度保守的活性位点残基Arg（90）。在这里，我们提出了一个详细的动力学和晶体学研究的几个这样的变体。去除精氨酸侧链（R90 G和R90 A）使催化效率降低超过5个数量级。通过赖氨酸取代将正电荷重新引入活性位点，使k（cat）恢复一千倍以上。值得注意的是，赖氨酸可以位于90位或更远的88位，只要配偶体位点存在空间上合适的残基。双突变体C88 S/R90 K和C88 K/R90 S的晶体结构显示赖氨酸采用扩展构象，其将其E-铵基团置于过渡态中结合分支酸酯的醚氧的氢键距离内。这些结果提供了支持的假设，即发展中的高度极化的过渡态的负电荷是稳定的静电由一个战略性地放置阳离子。这一发现的所有天然分支酸变异酶的机制和人工催化剂的设计的影响进行了讨论。
Combinatorial mutagenesis and in vivo selection experiments previously afforded functional variants of the AroH class Bacillus subtilis chorismate mutase lacking the otherwise highly conserved active site residue Arg(90). Here, we present a detailed kinetic and crystallographic study of several such variants. Removing the arginine side chain (R90G and R90A) reduced catalytic efficiency by more than 5 orders of magnitude. Reintroducing a positive charge to the active site through lysine substitutions restored more than a factor of a thousand in k(cat) Remarkably, the lysine could be placed at position 90 or at the more remote position 88 provided a sterically suitable residue was present at the partner site. Crystal structures of the double mutants C88S/R90K and C88K/R90S show that the lysine adopts an extended conformation that would place its E-ammonium group within hydrogen-bonding distance of the ether oxygen of bound chorismate in the transition state. These results provide support for the hypothesis that developing negative charge in the highly polarized transition state is stabilized electrostatically by a strategically placed cation. The implications of this finding for the mechanism of all natural chorismate mutases and for the design of artificial catalysts are discussed.