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

Andrews, Logan D.; Fenn, Tim D.; Herschlag, Daniel

doi:10.1371/journal.pbio.1001599

Cited by 41 publications

(70 citation statements)

References 72 publications

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“…This difference may arise because longer W–O bonds optimize interactions between tungstate and the active site, or because differences in the properties, such as unit cell compactness and elemental composition, between small molecule and protein crystals bias the distributions of W–O bonds observed. The observation of a covalent bond between Ser102 and tungstate accounts for the favorable contribution of the serine nucleophile to tungstate affinity observed by Andrews et al [6]. …”

Section: Resultsmentioning

confidence: 92%

“…1c) [6]. The observation that the AP active site stabilizes tungstate in trigonal bipyramidal geometry is striking given how infrequently tungstate adopts this geometry in other enzyme active sites and small-molecule compounds.…”

Section: Resultsmentioning

confidence: 99%

“…Nevertheless, this energetic penalty may account for a surprising observation: ablation of the nucleophile (from AP Arg166Ser to Ser102Gly/Arg166Ser), which results in loss of a covalent bond, only causes a fivefold reduction in affinity (Fig. 1c) [6]. Thus, it is possible that the enthalpic contribution from forming the covalent bond with Ser102 is nearly fully offset by distorting tungstate to an energetically unfavorable geometry.…”

Section: Resultsmentioning

confidence: 99%

“…3 were observed (data not shown). We were unable to measure inhibition constants below pH 8.0 due to polymerization of vanadate and tungstate at neutral pH and above pH 9.0 due to an enzymatic p K a [6] that raises the inhibition constant above the threshold that we can measure for most mutants.…”

Section: Methodsmentioning

confidence: 99%

“…An intriguing observation from this study was that the binding affinity of the two ligands changes distinctly with mutation: specifically, while ablation of active site Arg166 reduces affinity for both anions at pH 8, additional removal of the Ser102 nucleophile increases phosphate affinity by 1200-fold, while it reduces tungstate affinity by 5-fold (Fig. 1c) [6]. Stronger tungstate binding in the presence of Ser102 could arise from the formation of a covalent adduct between the serine nucleophile and tungstate, a model that we test herein.…”

Section: Introductionmentioning

confidence: 99%

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Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase

Peck

Sunden

Andrews

et al. 2016

Journal of Molecular Biology

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The catalytic mechanisms underlying Escherichia coli alkaline phosphatase’s (AP) remarkable rate enhancement have been probed extensively. Past work indicated that whereas the serine nucleophile (Ser102) electrostatically repels the product phosphate, another oxyanion, tungstate, binds more strongly in the presence of Ser102. These results predict a covalent bond between the serine nucleophile and tungstate, a model that we test herein. The crystal structure of tungstate-bound alkaline phosphatase provides evidence for a covalent adduct model and further shows that the ligand adopts trigonal bipyramidal geometry, which is infrequently observed for tungstate in small molecules and other active sites but mirrors the geometry of the presumed phosphoryl transfer transition state. The AP active site is known to stabilize another oxyanion, vanadate, in trigonal bipyramidal geometry, but the extent to which binding of either ligand reproduces the energetics of the transition state cannot be deduced from structural inspection alone. To test for transition state analog behavior, we determined the relationship between catalytic activity and affinity for tungstate and vanadate for a series of 20 AP variants. Affinity and activity were highly correlated for tungstate (r2 = 0.89) but not vanadate (r2 = 0.23), indicating that the tungstate•AP complex may better mimic this enzyme’s transition state properties. The results herein suggest that tungstate will be a valuable tool for further dissecting AP catalysis and may prove helpful in mechanistic studies of other phosphoryl transfer enzymes.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase

Peck

Sunden

Andrews

et al. 2016

Journal of Molecular Biology

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Structural Dynamics Support Electrostatic Interactions in the Active Site of Adenylate Kinase

Lawal

Welborn

2022

ChemBioChem

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Electrostatic preorganization as well as structural and dynamic heterogeneity are often used to rationalize the remarkable catalytic efficiency of enzymes. However, they are often presented as incompatible because the generation of permanent electrostatic effects implies that the protein structure remains rigid. Here, we use a metric, electric fields, that can treat electrostatic contributions and dynamics effects on equal footing, for a unique perspective on enzymatic catalysis. We find that the residues that contribute the most to electrostatic interactions with the substrate in the active site of Adenylate Kinase (our working example) are also the most flexible residues. Further, entropy-tuning mutations raise flexibility at the picosecond timescale where more conformations can be visited on short time periods, thereby softening the sharp heterogeneity normally visible at the microsecond timescale.

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Efficient Synthetic Access to Stable Isotope Labelled Pseudouridine Phosphoramidites for RNA NMR Spectroscopy

Glänzer,

Pfeiffer,

Ribar

et al. 2024

Chemistry A European J

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Here we report the efficient synthetic access to 13C/15N‐labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75 nt E. coli tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo‐enzymatic synthesis. 13C/15N labelled uracils were produced via chemical synthesis and enzymatically converted to 5′‐monophosphate pseudouridine (ΨMP) by using YeiN, a Ψ‐5′‐monophosphate C‐glycosidase. Removal of the 5′‐phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ ‐building block carrying both a 13C and a 15N label was incorporated into a product RNA and the complex formation with a 63 nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75 nt E. coli tRNAGly CCmnm5U, identifying the TΨC‐loop 5‐methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI labelled Ψ building blocks will allow the solution and solid‐state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ‐modified nucleic acids.

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

Abstract: 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.

Cited by 41 publications

References 72 publications

Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase

Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase

Structural Dynamics Support Electrostatic Interactions in the Active Site of Adenylate Kinase

Efficient Synthetic Access to Stable Isotope Labelled Pseudouridine Phosphoramidites for RNA NMR Spectroscopy

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