Nowlan Charity, Li Yingchun, Hermann Johannes C, Evans Timothy, Carpenter Joseph, Ghanem Eman, Shoichet Brian K, Raushel Frank M
Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012, USA.
J Am Chem Soc. 2006 Dec 13;128(49):15892-902. doi: 10.1021/ja0658618.
An array of 16 enantiomeric pairs of chiral phosphate, phosphonate, and phosphinate esters was used to establish the breadth of the stereoselective discrimination inherent within the bacterial phosphotriesterase and 15 mutant enzymes. For each substrate, the leaving group was 4-hydroxyacetophenone while the other two groups attached to the phosphorus core consisted of an asymmetric mixture of methyl, methoxy, ethyl, ethoxy, isopropoxy, phenyl, phenoxy, cyclohexyl, and cyclohexoxy substituents. For the wild-type enzyme, the relative rates of hydrolysis for the two enantiomers ranged from 3 to 5.4 x 10(5). Various combinations of site-specific mutations within the active site were used to create modified enzymes with alterations in their enantioselective properties. For the single-site mutant enzyme, G60A, the stereoselectivity is enhanced relative to that of the wild-type enzyme by 1-3 orders of magnitude. Additional mutants were obtained where the stereoselectivity is inverted relative to the wild-type enzyme for 13 of the 16 pairs of enantiomers tested for this investigation. The most dramatic example was obtained for the hydrolysis of 4-acetylphenyl methyl phenyl phosphate. The G60A mutant preferentially hydrolyzes the SP-enantiomer by a factor of 3.7 x 10(5). The I106G/F132G/H257Y mutant preferentially hydrolyzes the RP-enantiomer by a factor of 9.7 x 10(2). This represents an enantioselective discrimination of 3.6 x 10(8) between these two mutants, with a total of only four amino acid changes. The rate differential between the two enantiomers for any given mutant enzyme is postulated to be governed by the degree of nonproductive binding within the enzyme active site and stabilization of the transition state. This hypothesis is supported by computational docking of the high-energy, pentavalent form of the substrates to modeled structures of the mutant enzyme; the energies of the docked transition-state analogues qualitatively capture the enantiomeric preferences of the various mutants for the different substrates. These results demonstrate that the catalytic properties of the wild-type phosphotriesterase can be exploited for the kinetic resolution of a wide range of phosphate, phosphonate, and phosphinate esters and that the active site of this enzyme is remarkably amenable to structural perturbations via amino acid substitution.
使用16对磷酸酯、膦酸酯和次膦酸酯的对映体对来确定细菌磷酸三酯酶和15种突变酶固有的立体选择性区分的广度。对于每种底物，离去基团是4-羟基苯乙酮，而连接到磷核心的另外两个基团由甲基、甲氧基、乙基、乙氧基、异丙氧基、苯基、苯氧基、环己基和环己氧基取代基的不对称混合物组成。对于野生型酶，两种对映体的相对水解速率范围为3至5.4×10⁵。活性位点内位点特异性突变的各种组合用于创建对映选择性特性发生改变的修饰酶。对于单点突变酶G60A，其立体选择性相对于野生型酶提高了1至3个数量级。还获得了其他突变体，在所测试的16对对映体中的13对中，其立体选择性相对于野生型酶发生了反转。最显著的例子是4-乙酰基苯基甲基苯基磷酸酯的水解。G60A突变体优先水解SP-对映体，倍数为3.7×10⁵。I106G/F132G/H257Y突变体优先水解RP-对映体，倍数为9.7×10²。这代表了这两种突变体之间3.6×10⁸的对映选择性区分，总共只有四个氨基酸变化。假设任何给定突变酶的两种对映体之间的速率差异由酶活性位点内非生产性结合的程度和过渡态的稳定性决定。底物的高能五价形式与突变酶的模拟结构进行计算对接，支持了这一假设；对接的过渡态类似物的能量定性地捕捉了各种突变体对不同底物的对映体偏好。这些结果表明，野生型磷酸三酯酶的催化特性可用于多种磷酸酯、膦酸酯和次膦酸酯的动力学拆分，并且该酶的活性位点非常适合通过氨基酸取代进行结构扰动。
