| Abstract
| - It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 102-fold slower thanphosphate monoesters. This “thio effect” is similar to that observed for the nonenzymatic hydrolysis ofphosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphatemonoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions oftriesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes thehydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate.In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster thanp-nitrophenyl phosphorothioate. Despite this ∼23-fold change in the magnitude of the thio effects, themagnitudes of Brønsted βlg for the native AP (−0.77 ± 0.09) and the R166A mutant (−0.78 ± 0.06) arethe same. The identical values for the βlg indicate that the transition states are similar for the reactionscatalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thioeffect is not accompanied by a change in the βlg indicates that the thio effect is not a reliable reporter forthe transition state of the enzymatic phosphoryl transfer reaction. This result has important implicationsfor the interpretation of thio effects in enzymatic reactions.
|
