Mechanistic Study of Protein Phosphatase-1 (PP1), A Catalytically PromiscuousEnzyme

Authors

C. McWhirter
E. A. Lund
E. A. Tanifum
G. Feng
Q. L. Sheikh
Alvan C. Hengge, Utah State UniversityFollow
N. H. Williams

Document Type

Article

Journal/Book Title

Journal of the American Chemical Society

Publication Date

2008

Volume

130

Issue

41

First Page

13673

Last Page

13682

Abstract

The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for k_cat/K_M indicative of catalysis by both acidic and basic residues, with kinetic pK_a values of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Brønsted β_lg of −0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (−1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are ¹⁸(V/K)_bridge = 1.0170 and ¹⁵(V/K) = 1.0010, which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Brønsted β_lg of −0.30, smaller than the alkaline hydrolysis (−0.69) and similar to the β_lg measured for monoester substrates, indicative of similar transition states. The KIEs and the β_lg data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates.

Recommended Citation

C. McWhirter, E. A. Lund, E. A. Tanifum, G. Feng, Q. L. Sheikh, A. C. Hengge, and N. H. Williams “Mechanistic Study of Protein Phosphatase-1 (PP1), A Catalytically Promiscuous Enzyme,” J. Am. Chem. Soc. 2008; 130(41), 13673-13682.