Phosphoryl transfer to anionic oxygen nucleophiles. Nature of the transition state and electrostatic repulsion

Herschlag, Daniel; Jencks, William P.

doi:10.1021/ja00201a048

Cited by 102 publications

(118 citation statements)

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“…By using the sulfated forms of the fluorinated PNPs listed in Table 1, the ‡ The ␤nuc value for the neutral phenol was calculated at pH 6.2 for PNP, 2-fluoro-4-nitrophenol, and 3-fluoro-4-nitrophenol by using Eq. 1 (41,42). The data showed a linear relationship with a slope of Ϫ0.3.…”

Section: Tively ‡mentioning

confidence: 88%

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Mechanistic studies of β-arylsulfotransferase IV

Chapman

Bryan

Wong

2003

Proc. Natl. Acad. Sci. U.S.A.

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Sulfotransferases are an important class of enzymes that catalyze the transfer of a sulfuryl group to a hydroxyl or amine moiety on various molecules including small-molecule drugs, steroids, hormones, carbohydrates, and proteins. They have been implicated in a number of disease states but remain poorly understood, complicating the design of specific, small-molecule inhibitors. A linear free-energy analysis in both the forward and reverse directions indicates that the transfer of a sulfuryl group to an aryl hydroxyl group catalyzed by ␤-arylsulfotransferase IV likely proceeds by a dissociative (sulfotrioxide-like) mechanism. Values for the Brønsted coefficients (␤ nuc and ␤lg) are ؉0.33 and ؊0.45, giving Leffler ␣ values of 0.19 and 0.61 for the forward and reverse reactions, respectively.sulfotransferase ͉ linear free energy ͉ mechanism S ulfotransferases (STs) are an important class of enzymes that catalyze the transfer of a sulfuryl group from 3Ј-phospho-adenosyl-5Ј-phosphosulfate (PAPS) to a hydroxyl or, less frequently, an amine to give the sulfated product and the cofactor byproduct 3Ј-phosphoadenosyl-5Ј-phosphate (PAP) (Fig. 1). They can be divided into two groups: the cytosolic STs and the membrane-associated STs. Cytosolic STs catalyze the sulfonation of xenobiotics for detoxification and hormones for regulation. Membrane-associated STs catalyze the sulfonation of proteins and carbohydrates for processes such as cellular signaling and modulation of receptor binding (1-9). Recent studies have implicated the STs in a number of disease states including entry of the herpes virus (10), entry of HIV (11, 12), chronic inf lammation (13), and various forms of cancer (1-9). For this reason there is considerable interest in the design of small-molecule inhibitors that are specific for a particular ST. Although a number of inhibitor designs and syntheses have been published, to date the inhibitors are relatively weak and have always been discovered through semirational methods because of the lack of a complete mechanistic picture (14).A number of structural and mechanistic studies have been carried out on STs. Crystal structures have been solved for estrogen ST (15), catecholamine ST (16), dopamine ST (17), hydroxysteroid ST (18), and the ST domain of heparan sulfate N-deacetylase͞N- . Sequence alignment data from these structures as well as other STs show there to be a great deal of homology at the amino acid level for certain residues near the active site. Furthermore, mutagenesis studies carried out on these conserved residues show several of them to be critical to enzyme function. One such study of estrogen ST using a bound vanadate indicates an in-line attack of PAPS by the steroid hydroxyl group (20). Moreover, early studies of ␤-arylsulfotransferase IV (␤-AST-IV) show a random, rapidly equilibrating bi-bi mechanism (21). In the kinase field, the transition state of a protein tyrosine kinase has been illuminated through the use of linear free-energy studies (22). This information then was used to design spec...

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Section: Tively ‡mentioning

confidence: 88%

“…Under the reaction conditions, each of the phenols may not be in the fully deprotonated state, and thus the k cat ͞K m values shown for PNP and 2-fluoro-4-nitrophenol are adjusted to reflect the amount of unprotonated and neutral species by using Eq. 1 (41,42),…”

Section: Tively ‡mentioning

confidence: 99%

Mechanistic studies of β-arylsulfotransferase IV

Chapman

Bryan

Wong

2003

Proc. Natl. Acad. Sci. U.S.A.

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“…However, an oversimplified interpretation of this relationship suggests that a dissociative reaction would be expected to have a negative value of b i (due to a large b lg ), whereas, conversely, an associative reaction would have a positive value of b i , as b nuc is larger than b lg (Herschlag & Jencks, 1989b). From Eqs.…”

Section: Qualitative Brønsted-type Linear Free-energy Relationships (mentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

340

448

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Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, ‘Why Nature Chose Phosphate’ (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that naturereallychose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

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“…1). In the extremes, these are distinguished by dependence on versus independence from nucleophilicity of the phosphoryl acceptor, respectively, distances between the ␥-phosphorus atom and the attacking and leaving group oxygens, and other factors (1)(2)(3)(4)(5)(6)(7). The associative pathway would benefit from proton abstraction prior to formation of the transition state so that a more reactive oxygen anion is formed, whereas that proton is retained in the dissociative transition state.…”

mentioning

confidence: 99%

Probing the Catalytic Mechanism of the Insulin Receptor Kinase with a Tetrafluorotyrosine-containing Peptide Substrate

Ablooglu¹,

Till²,

Kim³

et al. 2000

Journal of Biological Chemistry

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The interaction of a synthetic tetrafluorotyrosyl peptide substrate with the activated tyrosine kinase domain of the insulin receptor was studied by steady-state kinetics and x-ray crystallography. The pH-rate profiles indicate that the neutral phenol, rather than the chemically more reactive phenoxide ion, is required for enzyme-catalyzed phosphorylation. The pK a of the tetrafluorotyrosyl hydroxyl is elevated 2 pH units on the enzyme compared with solution, whereas the phenoxide anion species behaves as a weak competitive inhibitor of the tyrosine kinase. A structure of the binary enzymesubstrate complex shows the tetrafluorotyrosyl OH group at hydrogen bonding distances from the side chains of Asp 1132 and Arg 1136 , consistent with elevation of the pK a . These findings strongly support a reaction mechanism favoring a dissociative transition state.

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Phosphoryl transfer to anionic oxygen nucleophiles. Nature of the transition state and electrostatic repulsion

Cited by 102 publications

References 1 publication

Mechanistic studies of β-arylsulfotransferase IV

Mechanistic studies of β-arylsulfotransferase IV

Why nature really chose phosphate

Probing the Catalytic Mechanism of the Insulin Receptor Kinase with a Tetrafluorotyrosine-containing Peptide Substrate

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