Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues

The factors that govern the substrate reactivity and stereoselectivity of p hosphotriesterase (PTE) toward organophosphotriesters containing various co mbinations of methyl, ethyl, isopropyl, and phenyl substituents at the phos phorus center were determined by systematic alterations in the dimensions o f the active site. The wild type PTE prefers the Sp-enantiomers over the co rresponding Rp-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine resi dues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improveme nts 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 pr eferences for the Sp-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the siz e of the large subsite with the mutant H257Y resulted in a reduction in kca t/K-a for the S-p-enantiomers, while the values of k(cat)/K-a for the R-p-e nantiomers were essentially unchanged. The initial stereoselectivity observ ed with the wild type enzyme toward the chiral substrate library was signif icantly reduced with the H257Y mutant. Simultaneous alternations in the siz es 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 ra tional reconstruction of the dimensions of the active site.