The nature of the transition state for enzyme-catalyzed phosphoryl transfer. Hydrolysis of O-aryl phosphorothioates by alkaline phosphatase.

摘要: There has been much speculation that enzymes change the nature of transition state for phosphoryl transfer from dissociative observed in solution reactions to an associative at enzyme's active site. This proposal can be tested by comparing linear free energy relationships (LFERs) nonenzymatic and enzymatic reactions, provided specificity binding site does not perturb dependence rate on intrinsic reactivity a series substrates. The shallow groove Escherichia coli alkaline phosphatase (AP) its wide suggest this enzyme may suited such approach. A second requirement approach is actual chemical step rate-limiting. Comparisons aryl phosphorothioates phosphates support previous conclusion nonchemical limits kc,$& phosphates, but cleavage rate-limiting phosphorothioates. We therefore determined AP-catalyzed large negative values group = -0.8 reaction (kcat/&) - 1.1 hydrolysis there considerable character both states. Despite AP, certain substrates deviate LFER, underscoring extreme care required applying LFERs reactions. value Pleaving pup suggests AP achieve substantial catalysis via with character,