Abstract：
:The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S(P)-enantiomers over the corresponding R(P)-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k(cat)/K(a) for the R(P)-enantiomers of up to 2700-fold but had little effect on k(cat)/K(a) for the corresponding S(P)-enantiomers. The kinetic preferences for the S(P)-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k(cat)/K(a) for the S(P)-enantiomers, while the values of k(cat)/K(a) for the R(P)-enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R(P)-enantiomers were preferred as substrates over the corresponding S(P)-enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Chen-Goodspeed M,Sogorb MA,Wu F,Raushel FMdoi
10.1021/bi001549dsubject
Has Abstractpub_date
2001-02-06 00:00:00pages
1332-9issue
5eissn
0006-2960issn
1520-4995pii
bi001549djournal_volume
40pub_type
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