期刊
BIOCHEMISTRY
卷 49, 期 37, 页码 7988-7997出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi101058z
关键词
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资金
- National Institutes of Health [GM 68550]
- National Science Foundation [CHE-0541587]
Wild-type phosphotriesterase (PTE) preferentially hydrolyzes the R-P enantiomers of the nerve agents sarin (GB) and cyclosarin (GF) and their chromophoric analogues. The active site of PTE can be subdivided into three binding pockets that have been denoted as the small, large, and leaving group pockets based on high-resolution crystal structures. The sizes and shapes of these pockets dictate the substrate specificity and stereoselectivity for catalysis. Mutants of PTE that exhibit substantial changes in substrate specificity and the ability to differentiate between chiral substrates have been prepared. For example, the G60A mutant is stereoselective for the hydrolysis of the R-P enantiomer of the chromophoric analogues of sarin and cyclosarin, whereas the H254G/H257W/L303T (GWT) mutant reverses the stereoselectivity for the enantiomers of these two compounds. Molecular dynamics simulations and high-resolution X-ray structures identified the correlations between structural changes in the active site and the experimentally determined kinetic parameters for substrate hydrolysis. New high-resolution structures were determined for the H257Y/L303T (YT), I106G/F132G/H257Y (GGY), and H254Q/H257F (QF) mutants of PTE. Molecular dynamics calculations were conducted using the S-P and R-P enantiomers of the analogues for sarin and cyclosarin for the wild-type PTE and the G60A, YT, GGY, QF, and GWT mutants. The experimental stereoselectivity correlated nicely with the difference in the computed angle of attack for the nucleophilic hydroxide relative to the phenolic leaving group of the substrate.
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