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

Abstract

Author SummaryEnzymes 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.