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What Does the Bronsted Slope Measure in the Phosphoryl Transfer Transition State?

ABSTRACT: The structural and energetic features of phosphate and phosphonate hydrolysis in Protein Phosphatase-1 (PP1) and water are studied using quantum mechanical (QM) cluster models. The calculations are able to reproduce observed kinetic isotope effects and capture several key trends in the experimental Brønsted plots: the βlg values are rather different for phosphate and phosphonate ester hydrolysis in solution but are similar in PP1. Detailed analyses of structure, charge distribution and bond order of computed transition states support the general conclusion from experimental study that phosphoryl transfer transition states are different for the two classes of substrates in solution but similar in PP1. On the other hand, the microscopic models also highlight notable differences between the phosphate and phosphonate transition states, which are manifested in not only structure but also kinetic isotope effects. Overall, we find that while βlg/βEQ,lg generally correlates with the partial charge on leaving group oxygen and the fractional bond order of the breaking P- Olg bond, the precise mapping between βlg/βEQ,lg and P- Olg bond order in the transition state is difficult due largely to the cross talk between breaking and forming P-O bonds. Therefore, further supporting previous analyses of limitations of free energy relations, our results suggest that while free energy relation is a valuable tool for probing the nature of transition state, a quantitative mapping of βlg and βlg/βEQ,lg values to structure or charge in the transition state should be conducted with great care.

SUBMITTER: Lai R

PROVIDER: S-EPMC8457673 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 P(i) was determined from pH dependencies of the binding of Pi and tungstate, a P(i) analog lacking titratable protons over the pH range of 5-11, and from the ³¹P chemical shift of bound P(i). 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 ?10?-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 P(i) 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.

Project description:BACKGROUND: The kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes. RESULTS: A number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the ?-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH2 of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the ?-phosphate of the ATP and the substrate nucleophile. CONCLUSIONS: The "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the coordination of the adenyl moiety of ATP and the C8-H of the adenyl moiety.

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What Does the Bronsted Slope Measure in the Phosphoryl Transfer Transition State?

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