Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer Enzymes

: Enhanced phosphate binding by phosphatases upon removal of their anionic nucleophiles suggests that these enzymes use ground state destabilization by anionic active site nucleophiles as part of their catalytic arsenal. Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 1022-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound Pi was determined from pH dependencies of the binding of Pi and tungstate, a Pi analog lacking titratable protons over the pH range of 5–11, and from the 31P chemical shift of bound Pi. The results show that the Pi trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ≥108-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and Pi binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous Pi binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state.Author Summary: Enzymes use a variety of tools and strategies to enhance (catalyze) biological reactions; these include the use of general acids and bases, cofactors, and the employment of remote binding interactions to position substrates near reactive chemical groups. Phosphatases are some of Nature's best enzymes, affording exceptional rate enhancements to the biologically ubiquitous removal of a phosphate group from a substrate (dephosphorylation). The apparent challenge faced by nonspecific phosphatases is that their wide substrate specificity precludes the efficient use of remote binding interactions. Previous work suggested that phosphatases could use negatively charged chemical groups (anionic nucleophiles) at the active site to destabilize substrate binding without simultaneously destabilizing the transition state barrier—an elusive catalytic strategy known as preferential ground state destabilization. In this work, we test this ground state destabilization model of catalysis by removing the anionic active site nucleophile of alkaline phosphatase and observing the effects on the enzyme's affinity for a phosphate ligand. We find that alkaline phosphatase has an exceptionally strong affinity for phosphate, and provide clear evidence for ground state destabilization by the anionic active site nucleophile that, when present, forestalls substrate saturation and product inhibition, and enhances catalysis by at least a thousand fold.