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

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 s 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.

Stabilization of Different Types of Transition States in a Single Enzyme Active Site: QM/MM Analysis of Enzymes in the Alkaline Phosphatase Superfamily

“…In other systems, the enzyme may employ different catalytic groups for different substrates, 23 or impose transition states of similar nature for different substrates. 108 Therefore, there appear to be diverse mechanisms that underlie the phenomena of catalytic promiscuity in enzymes, 5,104–106 highlighting the need of employing an integrated experimental/computational approach for understanding and predicting promiscuous enzyme activities. Another intriguing research direction is to couple such mechanistic analysis with advances in ancestral gene recon-struction, 109,110 which allows one to explore how the interplay between catalytic promiscuity and specificity during evolution shapes the functional landscape of “modern” enzymes.…”

Efficient Catalytic Promiscuity for Chemically Distinct Reactions

“…AP, [27,28] many other phosphatases, [22,29] and a sulfatase [30] use similar mechanisms to those in solution, but an example of a promiscuous enzyme that hydrolyzes phosphate and phosphonate monoesters with transition states that are closer to one another than those of the non-enzymatic reaction was recently reported. [31] Efficient hydrolysis of phosphate diesters by PAS may result from active-site groups that could carry out similar roles during catalysis of both native and promiscuous reactions. Specifically, the proposed double displacement mechanism for sulfate hydrolysis [32] involves Lewis acid catalysis by a calcium ion, a reactive nucleophile, and efficient substrate binding and general acid catalysis by several cationic active site groups (Supporting Information, Figure S4).…”

Development and Validation of a Robust and Sensitive Assay for the Discovery of Selective Inhibitors for Serine/Threonine Protein Phosphatases PP1α (PPP1C) and PP5 (PPP5C)

“…These considerations dictate a compromise between maximizing the signal window and choosing a pH relevant to conditions in which newly identified inhibitors and their derivatives are expected to operate. In the case of several PPP family phosphatases-lambda phosphatase, 52 PP1, 53 calcineurin, 54 and PP5 (unpublished observations)-much research on the reaction mechanisms and kinetics of these enzymes has established the importance of two active-site ionizable groups for enzyme activity, producing inverted-U-shaped pH rate profiles with broad optima between *pH 6 and 7. The mammalian PPP family shows high sequence homology among its members, with the catalytically important residues being absolutely conserved throughout, suggesting similar pH optima for the entire family.…”