PROBING THE MECHANISM OF STAPHYLOCOCCAL NUCLEASE WITH UNNATURAL AMINO-ACIDS - KINETIC AND STRUCTURAL STUDIES
PROBING THE MECHANISM OF STAPHYLOCOCCAL NUCLEASE WITH UNNATURAL AMINO-ACIDS - KINETIC AND STRUCTURAL STUDIES
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DOI：
10.1126/science.8103944
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发表时间：
1993-09-17
期刊：
影响因子：
56.9
通讯作者：
SCHULTZ, PG
中科院分区：
文献类型：
--
作者：
JUDICE, JK;GAMBLE, TR;SCHULTZ, PG
葡萄球菌核酸酶是一种具有巨大催化能力的酶，可将磷酸二酯键水解加速至自发速率的 10(16) 倍。通过用非天然氨基酸类似物取代活性位点残基 Glu43、Arg35 和 Arg87，研究了这种速率加速的机制基础。两种 Glu43 突变体，一种含有谷氨酸的硝基类似物，另一种含有高谷氨酸，在 pH 9.9 下保留了高催化活性，但在较低 pH 值下的活性低于野生型酶。高谷氨酸突变体的X射线晶体结构表明，该残基的羧酸侧链占据的位置和方向与野生型酶中的Glu43相似。空间体积的增加是通过主链位移和扭转角的改变来调节的。硝基和高谷氨酸突变体表现出相似的 pH 与速率曲线，这与野生型酶不同。总而言之，这些研究表明，Glu43 可能不像之前认为的那样充当通用碱基，但可能在催化过程中发挥更复杂的结构作用。
Staphylococcal nuclease is an enzyme with enormous catalytic power, accelerating phosphodiester bond hydrolysis by a factor of 10(16) over the spontaneous rate. The mechanistic basis for this rate acceleration was investigated by substitution of the active site residues Glu43, Arg35, and Arg87 With unnatural amino acid analogs. Two Glu43 mutants, one containing the nitro analog of glutamate and the other containing homoglutamate, retained high catalytic activity at pH 9.9, but were less active than the wild-type enzyme at lower pH values. The x-ray crystal structure of the homoglutamate mutant revealed that the carboxylate side chain of this residue occupies a position and orientation similar to that of Glu43 in the wild-type enzyme. The increase in steric bulk is accommodated by a backbone shift and altered torsion angles. The nitro and the homoglutamate mutants display similar pH versus rate profiles, which differ from that of the wild-type enzyme. Taken together, these studies suggest that Glu43 may not act as a general base, as previously thought, but may play a more complex structural role during catalysis.