The rate of hydrolysis of phosphomonoester dianions and the exceptional catalytic proficiencies of protein and inositol phosphatases

Abstract

To evaluate the proficiency of phosphatases as catalysts, the rate of the uncatalyzed hydrolysis of simple phosphate monoester dianions was estimated by extrapolating rates measured over a range of high temperatures. The rate of spontaneous hydrolysis of phenyl phosphate dianion indicates that a linear free energy relationship reported earlier is reliable for leaving groups whose conjugate acids have pKa values up to at least 10. Using Teflon reaction vessels, it proved possible to follow the hydrolysis of methyl phosphate and 3-(4-carboxy)-2,2-dimethylpropyl phosphate in strong alkali. Even in 1 M KOH, the reaction was found to be specific acid catalyzed. These results establish an upper limit for dianion reactivity, which had been overestimated earlier as a result of the leaching by alkali of silicic acid from quartz reaction vessels. The present findings indicate that the half-time for attack by water on alkyl phosphate dianions is 1.1 ؋ 10 12 years (k ‫؍‬ 2 ؋ 10 ؊20 s) at 25°C and that phosphatases involved in cell signaling and regulation produce the largest rate enhancements that have been identified thus far. Protein phosphatase-1 and inositol 1-phosphatase exceed all other known enzymes in their affinities for the altered substrates in the transition state.M ost enzyme reactions proceed with k cat ͞K m values in the neighborhood of 10 7 M Ϫ1 ⅐s Ϫ1 and appear to be similarly efficient according to that criterion. However, to assess the proficiency of an enzyme as a catalyst, and its corresponding affinity for the altered substrate in the transition state, it is necessary to compare k cat ͞K m with the rate constant of the corresponding reaction under the same conditions in the absence of a catalyst. In contrast to the relatively narrow range of values of k cat ͞K m observed for enzyme catalyzed reaction rates, the rates of these uncatalyzed reactions span a range of at least 19 orders of magnitude (1). Thus, differences in enzyme proficiency tend to reflect differences in the rates of the uncatalyzed reactions rather than the catalyzed reactions, and enzymes differ greatly from one another in their prowess as catalysts. Enzymes that catalyze the slowest reactions are of practical interest, in that they offer sensitive targets for inhibition by transition-state analogues. Their mechanisms of action are also particularly challenging to rationalize. Conspicuous among these slow reactions is the hydrolysis of monoesters of phosphoric acid.In biological systems, phosphate monoesters are cleaved by two groups of monoesterases that act by different mechanisms.One group of phosphomonoesterases, typified by bacterial alkaline phosphatase (2) and protein tyrosine phosphatases (3), uses an active-site nucleophile to displace the alcohol-leaving group from the substrate to form a phosphoryl-enzyme intermediate, which is subsequently hydrolyzed. Rate enhancement by enzymes that act through a double-displacement mechanism is not simple to analyze in terms of transition-state affinity or transition-state stabilization (...

Why nature chose phosphate to modify proteins

“…Any molecule with a phosphate ester, including phosphorylated proteins, has these same attributes. The enormous stability of phosphate esters in water at pH 7 (phosphate monoesters are estimated to have a half-life of 10 12 years at 258C [1]) allows the formation of very long polynucleotides that are remarkably stable despite the large number of phosphate ester bonds, an attribute that is essential for long-term storage of genetic information.…”

Theoretical Comparison ofp-Nitrophenyl Phosphate and Sulfate Hydrolysis in Aqueous Solution: Implications for Enzyme-Catalyzed Sulfuryl Transfer

“…Phosphate esters are generally rather unreactive under physiological conditions 8,9,[42][43][44][45] , making the enzymes that catalyze this class of reaction extremely proficient. As an example, the uncatalyzed hydrolysis of dimethyl phosphate is estimated to have an upper limit of ~1x10 -15 s -1 for spontaneous P-O cleavage at 25 C 46 , with enzymes such as Staphylococcal Nuclease accelerating the rate of reaction by at least 10 16 -fold 47 (in this particular case, for instance, computational studies find a barrier reduction of more than 35 kcal/mol compared to the corresponding uncatalyzed reaction in solution 48,49 , with the most detailed existing analysis of the molecular basis for catalysis by this enzyme being presented in Ref.…”

A Trojan horse transition state analogue generated by MgF 3 − formation in an enzyme active site

“…As phosphate esters are kinetically extremely stable, efficient catalysis is crucial for the control of these cellular processes. Although model studies have taught us much about the intrinsic chemical mechanisms (2), our understanding of the origins of the enormous enzymatic rate accelerations involved, up to a factor of 10 21 (3), is far from complete (4). A snapshot of an enzyme in a high-energy state would be immensely useful, as it would allow the very interactions that bring about catalysis to be observed (5).…”