Mutation of Arg-166 of AlkalinePhosphatase Alters the Thio Effect but Not the Transition State for Phosphoryl Transfer.Implications for the Interpretation of Thio Effects in Reactions of Phosphatases

Authors

K. M. Holtz
I. E. Catrina
Alvan C. Hengge, Utah State UniversityFollow
E. R. Kantrowitz

Document Type

Article

Journal/Book Title

Biochemistry

Publication Date

2000

Volume

39

First Page

9451

Last Page

9458

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 than phosphate monoesters. This “thio effect” is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis 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 position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this ∼23-fold change in the magnitude of the thio effects, the magnitudes of Brønsted β_lg for the native AP (−0.77 ± 0.09) and the R166A mutant (−0.78 ± 0.06) are the same. The identical values for the β_lg indicate 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 β_lg indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.

Recommended Citation

K. M. Holtz, I. E. Catrina, A. C. Hengge, and E. R. Kantrowitz. “Mutation of Arg-166 of Alkaline Phosphatase Alters the Thio Effect but Not the Transition State for Phosphoryl Transfer. Implications for the Interpretation of Thio Effects in Reactions of Phosphatases.” Biochemistry, 2000, 39, 9451-9458.