Pseudo-rotation in the hydrolysis of phosphate esters

Westheimer, F. H.

doi:10.1021/ar50003a002

Cited by 856 publications

(459 citation statements)

References 83 publications

(34 reference statements)

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“…This locking might be rationalized in terms of the ability of Vi to form a coordination complex with nucleophilic residues at the active site. The tetrahedral vanadate ion has the capacity either to exchange ligands or to accept a fifth ligand (as in crystalline metavanadates) to form a trigonal bipyramidal complex (13), which resembles the transition state for phosphoryl transfer (2,24,25). Although there is no evidence of a five-coordinate V1 species in free solution (13,(26)(27)(28), such a complex might be stabilized at the myosin active site.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of myosin ATPase by vanadate ion.

Goodno¹

1979

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

274

218

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Inhibition of the myosin ATPase by vanadate ion (V1) has been studied in 90 mM NaCl/5 mM MgCl2/20 mM Tris HCI, pH 8.5, at 250C. Although the onset of inhibition during the assay is slow and dependent upon Vi concentration (kap -t 0.3 M-1 s-'), the final level of inhibition approaches iOO4o, provided the V; concentration is in slight excess over the concentration of ATPase sites. Inhibition is not reversible by dialysis or the addition of reducing agents. The source of this irreversible inhibition consists of the formation of a stable, inactive complex with the composition MADP-V1 (where M represents a single myosin active site). The complex has been isolated, and its mechanism of formation from M, ADP, and Vi has been studied. Omission of ATP increases the rate of formation by about 35-fold (kapp 11 M-1 s-'), yet this rate is still low in comparison with the rates of simple protein-ligand association reactions. This slowness is interpreted in terms of a rate-limiting isomerization step that follows the association of M, ADP, and V1: M*ADP*V1 _ Mt.ADP.V1 (t indicates the inactive product of the isomerization). (9) and Weeds and Taylor (10), respectively. The HMM fraction that precipitated between 45 and 60% saturated ammonium sulfate was dialyzed free of ammonium sulfate, centrifuged 30 min at 40,000 X g, and used within 10 days. The concentration of HMM was expressed in terms of the ATPase-site concentration, which was determined spectrophotometrically by using a value of A2801% = 6.47 (11), assuming a molecular weight of 340,000 and two ATPase sites per molecule.ATPase assays were carried out in buffer A (0.09 M NaCl/5 mM MgCl2/20 mM Tris-HCI, pH 8.5) at 250C by the addition of MgATP (final concentration, 1 mM) to HMM at 1-7 AM sites.Assay times ranged from 0.1 to 5 hr. The reaction was stopped in aliquots (1 ml) of the assay solution with 1 ml of 10% trichloroacetic acid. The aliquots were then clarified by centrifugation and half of each was analyzed for Pi by the procedure of Taussky and Shorr (12). V1 concentrations below 10 mM caused less than 1% interference.Vanadium Analysis. Stock solutions of V1 were prepared from either Na3VO4 (adjusted to pH 10 with 6 M HCl) or V205 (adjusted to pH 10 with 10 M NaOH) and then boiled to destroy yellow polymeric species such as V100286-(13). Standard solutions were prepared by volumetric dilution. In order to minimize the pH-dependent polymerization of Vi, all studies were carried out under the alkaline conditions used for the ATPase assays (buffer A). UV-visible spectra of Vi standard solutions were obtained by using a Cary 14 spectrophotometer, and the extinction coefficient was determined: Xrnax = 265 nm, C265 = 2925 M-1 cm-1. The Vi concentration was determined spectrophotometrically wherever possible.Where this was unfeasible (e.g., in the presence of protein), vanadium was determined by a modification of the colorimetric procedure of Pribil (14), using the metallochromic dye PAR.To a 1-ml sample in buffer A was added 100 Al of 1 M imidazole (pH 6.0) and ...

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Section: Resultsmentioning

confidence: 99%

Inhibition of myosin ATPase by vanadate ion.

Goodno¹

1979

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

274

218

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“…Since the nucleophile attacks from an axial position, the leaving group has to depart from an axial position also, which is facilitated by the pseudo-rotation. Finally, while this pseudorotation mechanism is quite rare and specialized (Westheimer, 1968), semi-empirical studies of phosphate monoester hydrolysis at different levels of phosphate and nucleophile protonation have demonstrated that not only is the non-inline associative mechanism with pseudo-rotation a reasonable reaction pathway, but there are certain conditions under which it can even be the lowest energy pathway (Wilkie & Gani, 1996). The barrier for the pseudo-rotation step is so low, however, that it is difficult to observe it either computationally or experimentally.…”

Section: Non-inline Mechanisms With Pseudo-rotationmentioning

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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“…99 Trigonal bipyramidal species are stabilised to the greatest extent possible if the most electronegative atom(s) occupy the apical sites. This arrangement of the substituents 30 results in the HOM O being as low in energy as is possible.…”

Section: Oxaphosphetane Structure and Cycloreversion Mechanismmentioning

confidence: 99%

“…The OPA formed by the cycloaddition step is postulated to have the oxygen in the apical position, in accordance with Westheimer's rule on apical entry 15 and departure for a phosphorus-centred trigonal bipyramid. 99 This OPA, (which could be any one of a number of pseudorotamers with apical oxygen) is proposed to undergo rapid pseudorotation to a less stable pseudorotamer with ring carbon-3 in an apical position, and the ring oxygen in an equatorial site. This 20 pseudorotamer undergoes [2+2] cycloreversion to alkene and phosphine oxide.…”

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confidence: 99%