Mutation of Arg-166 of alkaline phosphatase alters the thio effect but notthe transition state for phosphoryl transfer. Implications for the interpretation of thio effects in reactions of phosphatases

It has been suggested that the mechanism of alkaline phosphatase (AP) is as sociative, or triester-like, because phosphorothioate monoesters are hydrol yzed by AP approximately 10(2)-fold slower than phosphate monoesters. This "thio effect" is similar to that observed for the nonenzymatic hydrolysis o f phosphate triesters, and is the inverse of that observed for the nonenzym atic hydrolysis of phosphate monoesters. The latter reactions proceed by lo ose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosph atase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mu tant alkaline phosphatase enzyme, in which the active site arginine at posi tion 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate on ly about 3 times faster than p-nitrophenyl phosphorothioate. Despite this s imilar to 23-fold change in the magnitude of the thio effects, the magnitud es of Bronsted beta(1g) for the native AP (-0.77 +/- 0.09) and the R166A mu tant (-0.78 +/- 0.06) are the same. The identical values for the beta(1g) i ndicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the beta(1g) in dicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has import ant implications for the interpretation of thio effects in enzymatic reacti ons.