Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: The importance of electrostatic catalysis
Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: The importance of electrostatic catalysis
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DOI：
10.1073/pnas.93.10.5043
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发表时间：
1996-05-14
影响因子：
11.1
通讯作者：
Hilvert, D
中科院分区：
文献类型：
--
作者：
Kast, P;AsifUllah, M;Hilvert, D
氯酸变位酶(EC 5.4.99.5)催化分支酸分子内重排生成预苯酸。枯草芽孢杆菌酶活性部位的Arg-90与底物的乙醚氧非常接近，可能通过稳定C-O键断裂时可能产生的偶极过渡态而有助于有效的催化。为了验证这一想法，我们通过对部分芳香氨基酸生物合成途径的重组，开发了一种新的大肠杆菌中分支酸变位酶活性的互补系统。将Arg-90的密码子随机、单独和与Cys-88的密码子组合，选择活性克隆。结果表明，第88位(Lys)或第90位(Arg或Lys)上的正电荷残基是必需的，我们的数据支持了酶促分支酸重排过渡态稳定需要正电荷的假说。新的选择系统与组合诱变相结合，使天然酶(S)的作用机制得以进一步研究，并为改进具有分支酸变位酶活性的第一代催化抗体开辟了途径。
Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate. Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C-O bond. To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected. The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential, Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement. The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.