Phosphate Monoester Hydrolysis by Trinuclear Alkaline Phosphatase; DFT Study of Transition States and Reaction Mechanism

Chen, Shi-Lu; Liao, Rong‐Zhen

doi:10.1002/cphc.201402016

Cited by 31 publications

(31 citation statements)

References 82 publications

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“…This result was similar to findings from another study 72 of TS structure in AP. Here we find that this trend in TS structure translates into substantial differences in the magnitude of 1° KIEs for both monoesters and diesters (Table 5).…”

Section: Resultssupporting

confidence: 92%

Substrate and Transition State Binding in Alkaline Phosphatase Analyzed by Computation of Oxygen Isotope Effects

Roston

Cui

2016

J. Am. Chem. Soc.

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Enzymes are powerful catalysts and a thorough understanding of the sources of their catalytic power will facilitate many medical and industrial applications. Here we have studied the catalytic mechanism of alkaline phosphatase (AP), which is one of the most catalytically proficient enzymes known. We have used quantum mechanics calculations and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to model a variety of isotope effects relevant to the reaction of AP. We have calculated equilibrium isotope effects (EIEs), binding isotope effects (BIEs), and kinetic isotope effects (KIEs) for a range of phosphate mono- and diester substrates. The results agree well with experimental values, but the model for the reaction’s transition state (TS) differs from the original interpretation of those experiments. Our model indicates that isotope effects on binding make important contributions to measured KIEs on V/K, which complicated interpretation of the measured values. Our results provide a detailed interpretation of the measured isotope effects and make predictions that can test the proposed model. The model indicates that the substrate is deformed in the ground state (GS) of the reaction and partially resembles the TS. The highly preorganized active site preferentially binds conformations that resemble the TS and not the GS, which induces the substrate to adapt to the enzyme, rather than the other way around—as with classic “induced fit” models. The preferential stabilization of the TS over the GS is what lowers the barrier to the chemical step.

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Section: Resultssupporting

confidence: 92%

Substrate and Transition State Binding in Alkaline Phosphatase Analyzed by Computation of Oxygen Isotope Effects

Roston

Cui

2016

J. Am. Chem. Soc.

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“…54 An active site model of AP, on the other hand, did find a similar shift in TS structure to ours for the two substrates they examined. 55 Williams noted that in principle, all FERs should be curved, 5 but an analysis by Jencks indicated that the effects of changing substituent on TS structure will generally be difficult to observe. 4 Thus, a standard analysis of FERs calculates an effective charge at the TS based on the slope of the FER.…”

Section: Resultsmentioning

confidence: 99%

Leaving Group Ability Observably Affects Transition State Structure in a Single Enzyme Active Site

2016

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A reaction’s transition state (TS) structure plays a critical role in determining reactivity and has important implications for the design of catalysts, drugs, and other applications. Here we explore TS structure in the enzyme alkaline phosphatase using hybrid Quantum Mechanics/Molecular Mechanics simulations. We find that minor perturbations to the substrate have major effects on TS structure and the way the enzyme stabilizes the TS. Substrates with good leaving groups (LGs) have little cleavage of the phosphorus-LG bond at the TS while substrates with poor LGs have substantial cleavage of that bond. The results predict non-linear free energy relationships for a single rate-determining step, and substantial differences in kinetic isotope effects for different substrates; both trends were observed in previous experimental studies, although the original interpretations differed from the present model. Moreover, due to different degrees of phosphorous-LG bond cleavage at the TS for different substrates, the LG is stabilized by different interactions at the TS: while a poor LG is directly stabilized by an active site zinc ion, a good LG is mainly stabilized by active site water molecules. Our results demonstrate the considerable plasticity of TS structure and stabilization in enzymes. Furthermore, perturbations to reactivity that probe TS structure experimentally (i.e., substituent effects) may substantially perturb the TS they aim to probe, thus classical experimental approaches such as free energy relations should be interpreted with care.

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“…They used two substrates in the calculations, the methyl‐ and p ‐nitrophenyl phosphates. The calculations confirmed that the alkaline phosphatase reaction employed a mechanism involving two chemical displacement steps, the first being the displacement of the substrate leaving group by a Ser102 alkoxide and the second, the hydrolysis of the phosphoseryl intermediate by a Zn–OH moiety (Scheme ) …”

Section: The Water Molecule and The Study Of The Hydrolytic Metallomentioning

confidence: 65%

“…That a nucleophilic Zn–OH species is involved in the alkaline phosphatase reaction is now fully accepted, and many similar studies of the role of metal ions in alkaline phosphatase, and the proposed mechanism of action, have been reported , . In 2014 Chen et al investigated the mechanism of alkaline phosphatase‐catalyzed hydrolysis of phosphate monoesters using density functional theory (DFT) with a model based on a crystal structure of the enzyme. They used two substrates in the calculations, the methyl‐ and p ‐nitrophenyl phosphates.…”

Section: The Water Molecule and The Study Of The Hydrolytic Metallomentioning

confidence: 99%

Metal‐coordinated Hydroxide as a Nucleophile: a Brief History

Blackman

Gahan

2018

Zeitschrift anorg allge chemie

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It is well known that a metal‐bound hydroxido ligand is an efficient nucleophile around neutral pH. In a mechanistic sense, the presence and chemical reactivity of the coordinated hydroxido nucleophile are frequently invoked both in biomimetic chemistry and in numerous examples of hydrolytic metalloenzymes. In this review, we explore the background of this chemistry in order to attempt to trace the thinking which led to what is now a fully accepted mechanistic pathway.

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Phosphate Monoester Hydrolysis by Trinuclear Alkaline Phosphatase; DFT Study of Transition States and Reaction Mechanism

Cited by 31 publications

References 82 publications

Substrate and Transition State Binding in Alkaline Phosphatase Analyzed by Computation of Oxygen Isotope Effects

Substrate and Transition State Binding in Alkaline Phosphatase Analyzed by Computation of Oxygen Isotope Effects

Leaving Group Ability Observably Affects Transition State Structure in a Single Enzyme Active Site

Metal‐coordinated Hydroxide as a Nucleophile: a Brief History

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