The reactivity of phosphate esters. Multiple structure–reactivity correlations for the reactions of triesters with nucleophiles

Khan, Safraz; Kirby, Anthony J.

doi:10.1039/j29700001172

Cited by 109 publications

(130 citation statements)

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“…In the case of the simple aryl esters, the reaction has been shown to proceed preferentially (although not exclusively) through a stepwise A N +D N mechanism involving a phosphorane intermediate (Khan & Kirby, 1970). However, studies on similar structures that vary both nucleophile and leaving groups have supported the proposition that both stepwise and concerted mechanisms are viable, and that the stereochemistry of the reaction (i.e.…”

Section: The Mechanism Of Phosphate Triester Hydrolysismentioning

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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Section: The Mechanism Of Phosphate Triester Hydrolysismentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

340

448

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“…1, red shading). The -103-fold slower nonenzymatic reactions of phosphate diesters than phosphate monoesters provide experimental evidence for such an "anticatalytic" effect, with the alkyl group of the diester providing a model for protonation (40). The destabilization of the transition state from protonation of a -y-phosphoryl oxygen atom described above would hold whether the proton transfer to the y-phosphoryl group were direct or involved an intermediary (20, 35-39, …”

Section: Previous Mechanistic Proposals For the Ras Gtpase Reactionmentioning

confidence: 99%

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

Maegley

Admiraal²,

Herschlag³

1996

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

205

263

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Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach Ras and other G proteins are active in signaling when GTP is bound. Hydrolysis of GTP to give bound GDP turns these signaling proteins off (1-3). For this reason, the mechanism of GTP hydrolysis by Ras and other G proteins has elicited much interest. Nevertheless, despite a wealth of structural, kinetic, and site-directed mutagenesis data, the catalytic mechanism of Ras remains controversial.Crucial to an understanding of enzymatic catalysis is knowledge of the nature of the transition state of the reaction and how that transition state differs from the ground state; transition state theory defines catalysis as stabilization of a reaction's transition state relative to its ground state. An underlying feature comrmon among the several distinct models presented for Ras catalysis is an explicit or implicit assumption that the transition state is associative in character. However, recent model studies have shown that the transition state for GTP hydrolysis in solution is dissociative rather than associative (4). This transition state information is used to evaluate previously proposed mechanisms from a chemical perspective.A new catalytic mechanism is then proposed, involving a hydrogen bond to the ,3-y bridge oxygen of GTP. This proposal is consistent with pre-existing structural, spectral, and energetic data.

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“…Triesters typically react much faster (10 3 -10 5 -fold) under nonacidic conditions with nucleophiles than diesters, probably because of the enhanced electrophilicity of the phosphorus in the former (27,28). Thus one might anticipate a large rate reduction to be associated with rendering the ␥-phosphate of ATP more diester-like in the enzyme active site.…”

Section: Catalytic Base and Csk 22107mentioning

confidence: 99%

The Role of the Catalytic Base in the Protein Tyrosine Kinase Csk

Cole

Grace

Phillips

et al. 1995

Journal of Biological Chemistry

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A potential distinguishing feature between protein tyrosine kinases and homologous serine/threonine kinases is the function of the catalytic base in these enzymes. In this study, we show that a peptide containing the unnatural amino acid trifluorotyrosine shows remarkably similar efficiency as a substrate of the tyrosine kinase Csk (C-terminal Src kinase) compared with the corresponding tyrosine-containing peptide despite a 4-unit change in the phenolic pK a . These results argue against the importance of early tyrosine deprotonation by a catalytic base in Csk. To further explore the role of the proposed catalytic base, the Csk mutant protein D314E was produced. This mutant displayed a significant reduction in k cat (approximately 10 4 ) but relatively little effect on substrate K m values compared with wildtype Csk. Examination of the thio effect (k cat -ATP/k catadenosine 5-O-(thiotriphosphate)) for D314E Csk led to the suggestion that a role of aspartate 314 may be to enhance the reactivity of the ␥-phosphate of ATP toward electrophilic attack. These results may have significant impact on protein tyrosine kinase inhibitor design.Protein tyrosine kinases play central roles in cell proliferation, cell differentiation, and signaling processes (1). While their enzymatic activity, transfer of a phosphoryl group from ATP to tyrosines in proteins, was first described over 15 years ago, details of tyrosine kinase catalytic mechanism have largely remained elusive. In contrast, their homologous family member protein serine/threonine kinases have been more intensively studied and are better characterized enzymologically (2-8). In order to rationally develop specific inhibitors for these enzymes (9) as well as to delineate the biochemical consequences of their in vivo regulation, a more complete understanding of tyrosine kinase catalysis is needed.Recently, we carried out a preliminary mechanistic evaluation of the protein tyrosine kinase Csk (C-terminal Src kinase) from humans (10), overproduced in and purified from Escherichia coli (11). It was shown that, like the better studied serine/threonine kinase protein kinase A, the Csk-catalyzed kinase reaction obeys a ternary complex mechanism, most likely with rapid and random binding of the substrates ATP and 4:1 poly(Glu,Tyr). This argued against a covalent phosphoenzyme intermediate. Moreover, using ATP␥S 1 and microviscosity effects, it was shown that in the Csk ATP reaction, product ADP diffusional release is partially rate-determining for the overall reaction (10). The lack of a large deuterium solvent kinetic isotope effect (actually a slightly inverse, k cat H/ k cat D ϭ 0.74, was seen) in the ATP␥S reaction (where the chemical step appeared fully rate-determining) suggested that tyrosine hydroxyl deprotonation is asymmetric in the reaction transition state. Because a tetrafluorotyrosine-containing peptide was previously reported to be a tight binding inhibitor (and not detectably a substrate) for insulin receptor tyrosine kinase (12), a phenoxide anion was reasoned...

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The reactivity of phosphate esters. Multiple structure–reactivity correlations for the reactions of triesters with nucleophiles

Cited by 109 publications

References 0 publications

Why nature really chose phosphate

Why nature really chose phosphate

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

The Role of the Catalytic Base in the Protein Tyrosine Kinase Csk

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