Metaphosphate Intermediate Research Articles

Ab initio dynamics of gas‐phase and aqueous‐phase hydrolysis of adenosine triphosphate

AbstractIn this study, we have modelled the non‐enzymatic hydrolysis of ATP in the gas‐phase and the aqueous‐phase by performing ab initio molecular dynamics simulations combined with an enhanced sampling technique. In the gas‐phase, we studied hydrolysis of fully protonated ATP molecule, and in the aqueous‐phase, we studied hydrolysis of ATP coordinated with: a) two H+ ions (H‐ATP), b) Mg2+ (Mg‐ATP), and c) Ca2+ (Ca‐ATP). We show that gas‐phase ATP hydrolysis follows a two‐step dissociative mechanism via a highly stable metaphosphate intermediate. The Adenine group of the ATP molecule plays a crucial role of a general base; temporarily accepting protons and thus helping in the elimination‐addition process. In the aqueous‐phase hydrolysis of ATP, we find that the cage of solvent molecules increases the stability of the terminal phospho‐anhydride bond through a well‐known cage‐effect. The nature of the ions has an important effect on the mechanism of the reaction. We find a two‐step dissociative‐type mechanism for H‐ATP, a single‐step dissociative‐type mechanism for Mg‐ATP, and an SN‐2 type concerted hydrolysis pathway for Ca‐ATP.

Mechanochemical Function of Myosin II: Investigation into the Recovery Stroke and ATP Hydrolysis.

Myosin regulates muscle function through a complex cycle of conformational rearrangements coupled with the hydrolysis of adenosine triphosphate (ATP). The recovery stroke reorganizes the myosin active site to hydrolyze ATP and cross bridge with the thin filament to produce muscle contraction. Engineered mutations K84M and R704E in Dictyostelium myosin have been designed to specifically inhibit the recovery stroke and have been shown to indirectly affect the ATPase activity of myosin. We investigated these mutagenic perturbations to the recovery stroke and generated thermodynamically correct and unbiased trajectories for native ATP hydrolysis with computationally enhanced sampling methods. Our methodology was able to resolve experimentally observed changes to kinetic and equilibrium dynamics for the recovery stroke with the correct prediction in the severity of these changes. For ATP hydrolysis, the sequential nature along with the stabilization of a metaphosphate intermediate was observed in agreement with previous studies. However, we observed glutamate 459 being utilized as a proton abstractor to prime the attacking water instead of a lytic water, a phenomenon not well categorized in myosin but has in other ATPases. Both rare event methodologies can be extended to human myosin to investigate isoformic differences from Dictyostelium and scan cardiomyopathic mutations to see differential perturbations to kinetics of other conformational changes in myosin such as the power stroke.

Detection of metaphosphate intermediates in reaction solutions of phosphate esters: Chromatographic and spectroscopic studies

GRAPHICAL ABSTRACT

Phosphoramidic acid monoesters as phosphorylating agents: steric effects and reluctance to form monomeric metaphosphate intermediates.

The formation of phosphate diesters (RO)(2)P(X)OH (R = Et, Pr(i) or Pr(i)(2)CH) by phosphorylation of ROH with ROP(X)(NPr(i)(2))OH is insensitive to steric effects when X = S but not when X = O; this is consistent with a unimolecular mechanism and a thiometaphosphate (ROPOS) intermediate when X = S but a bimolecular S(N)2(P) mechanism when X = O.

High-resolution structures of adenylate kinase from yeast ligated with inhibitor Ap5A, showing the pathway of phosphoryl transfer.

The structure of adenylate kinase from yeast ligated with the two-substrate-mimicking inhibitor Ap5A and Mg2+ has been refined to 1.96 A resolution. In addition, the refined structure of the same complex with a bound imidazole molecule replacing Mg2+ has been determined at 1.63 A. These structures indicate that replacing Mg2+ by imidazole disturbs the water structure and thus the complex. A comparison with the G-proteins shows that Mg2+ is exactly at the same position with respect to the phosphates. However, although the Mg2+ ligand sphere of the G-proteins is a regular octahedron containing peptide ligands, the reported adenylate kinase has no such ligands and an open octahedron leaving space for the Mg2+ to accompany the transferred phosphoryl group. A superposition of the known crystalline and therefore perturbed phosphoryl transfer geometries in the adenylate kinases demonstrates that all of them are close to the start of the forward reaction with bound ATP and AMP. Averaging all observed perturbed structures gives rise to a close approximation of the transition state, indicating in general how to establish an elusive transition state geometry. The average shows that the in-line phosphoryl transfer is associative, because there is no space for a dissociative metaphosphate intermediate. As a side result, the secondary dipole interaction in the alpha-helices of both protein structures has been quantified.

Metaphosphate intermediates from fluoroaromatic precursors

Flow pyrolysis of some 2-aryloxy-1,3,2-dioxaphospholanes with ortho-CF 3 or F substituents, either alone or in the presence of methanol, has provided evidence for the formation of metaphosphates as intermediates; these highly reactive species then undergo further reaction, depending on the nature of the substituent. Mass spectrometric studies of the starting materials have also suggested the elimination of ethylene and the formation of metaphosphates to a limited extent.

The mechanism of fragmentation of alkyl α-(oxyimino)benzylphosphonates; use of silica gel as a novel hydroxylic trapping reactant for an intermediate alkyl metaphosphate.

Silica gel was employed to react with the suspected metaphosphate intermediate of the acid-promoted cleavage of monoalkyl α-(oxyimino)benzylphosphonates. The silica gels displayed CP-MAS 31P NMR signals at about δ −8. A similar value was obtained for silica gel phosphorylated with ethyl metaphosphate from a different source (thermolysis of a P-ethoxy 2,3-oxaphosphabicyclo[2.2.2]octene derivative.

ChemInform Abstract: Free Monomeric Metaphosphate in Protic Solution: Complete Racemization at Phosphorus in the tert‐Butyl Alcoholysis of p‐Nitrophenyl Phosphate.

AbstractThe results of the solvolysis of bis(tetra‐n‐butylammonium) p‐nitrophenyl (16O,17O,18O)phosphate stereospecifically labeled in neat tBuOH at 30 °C demonstrate the intermediacy of a completely free, symmetrically solvated monomeric metaphosphate intermediate.

Pyrophosphate formation for acetyl phosphate and orthophosphate anions in concentrated aqueous salt solutions does not provide evidence for a metaphosphate intermediate

The formation of pyrophosphate (PPi) from the anions of acetyl phosphate (AcP) and orthophosphate (Pi) in concentrated aqueous sodium perchlorate is approximately first order in each reactant. The pH dependence of the reaction in the presence of 0.25 M Pi monoanion and 6.4 M sodium perchlorate at 54 OC shows different yields of PPi from AcP monoanion and dianion. This result is inconsistent with trapping of a free metaphosphate intermediate by Pi monoanion and water. The results are consistent with a concerted mechanism for PPI formation and yields of PPi from the different ionic species of 11.7% for AcP2- and Pi2-, 7.8% for AcPZ- and Pi-, and 4.2% for AcP- and Pi-. Measurement of rate constants and partitioning with AcP2-, 0.25 M Pi2-, and 6.4 M sodium perchlorate, in the presence and absence of pyridine, show that the phosphorylated pyridine monoanion yields 40% PPi, whereas AcP2- yields 10% PPi under the same conditions. The formation of PPi from AcP and Pi dianions is at least fourth order in sodium ion in concentrated sodium perchlorate solutions. The corresponding reaction of the monoanions is second order in sodium ion. The order of effectiveness of salts for facilitation of the formation of PPi from the dianions of AcP and Pi is NaCIO., > NaC1, KCI, CsC1, and NaCl > CsCI. However, increasing the concentration of sodium perchlorate from 0.1 to 6.6 M changes the rate constant for hydrolysis of AcP mono- and dianion by <30% at 39 and 54 OC. Values of AH* = 29 kcal mol-' and AS* = +I2 eu for PPi formation from the dianions of AcP and Pi and AH* = 19 kcal mol-' and AS* = -20 eu for PPi formation from the monoanions were obtained from the yields of PPi and the rates of hydrolysis at 39 and 54 OC. The high concentrations of sodium ion decrease electrostatic repulsion between the anions of AcP and Pi and may catalyze the formation of PPi by bridging the anions of AcP and Pi. These results provide no evidence for the existence of a metaphosphate intermediate in reactions in aqueous solution.

A Stereochemical and Positional Isotope‐Exchange Study of the Mechanism of Activation of Methionine by Methionyl‐tRNA Synthetase from Escherichia coli

Methionyl-tRNA synthetase from Escherichia coli catalyses the activation of [18O2]methionine by adenosine 5'-[(R)-alpha 17O]triphosphate with inversion of configuration at P alpha. Furthermore methionyl-tRNA synthetase does not catalyse positional isotope exchange in adenosine 5'-[beta-18O2]triphosphate in the absence of methionine or in the presence of the competitive inhibitor, methioninol, which eliminates the possibility of either adenylyl-enzyme or adenosine metaphosphate intermediates being involved. These observations require that methionyl-tRNA synthetase catalyses the activation of methionine by an associative 'in-line' nucleotidyl transfer mechanism. A kinetic study of positional isotope exchange in adenosine 5'-[beta-18O2]triphosphate in the presence of methionine, Mg2+ and methionyl-tRNA synthetase showed that torsional equilibration (18O exchange into the P alpha--O--P beta bridge) occurs faster than tumbling (18O exchange into P gamma by rotation about the C2 axis of Mg[18O2]PPi), demonstratings that the positional isotope exchange occurs at least in part in the E X Met-AMP X Mg[18O2]PPi complex.

Phosphoryl transfer from phenyl and 4-nitrophenyl phosphates in aprotic and protic solvents : Amine catalysis and formation of oxyphosphorane and metaphosphate intermediates

The behavior of 4-nitrophenyl dihydrogen phosphate, ArOPO 3H 2, and of its tetra-n-butylammonium and tetramethylammonium salts, ArOPO 3H −R 4N +, ArOPO 3 2−2(R 4N +), was studied in aprotic solvents, in the absence and in the presence of increasing amounts of alcohols or water. The reactions were investigated in the absence of amines, and in the presence of hindered and unhindered amines, diisopropylethylamine and quinuclidine. The course of the reactions was followed at 35° or at 70° by 31P and 1H NMR spectrometry. Values for the approximate half-times of the reactions were estimated (± 25 %) from the times at which reactant signal intensity becomes equal to product signal intensity. The mononitrophenyl ester transfers its phosphoryl group to alcohols and water from the diprotonated acid by the addition-elimination mechanism via oxyphosphorane intermediates, and from the monoanion and dianion by the elimination-addition mechanism via the monomeric metaphosphate intermediate, PO 3 −. Formation of PO 3 − is faster from dianion than from monoanion in acetonitrile and in alcohol solutions. Conversely, PO 3 − is generated at a faster rate from monoanion than from dianion in aqueous solution. This effect results from a decrease in the rate of formation of PO 3 − in the solvent series: acetonitrile > alcohols > water. The rate depression as a function of the medium is greater for the dianion than for the monoanion, and is attributed to greater solvation of the more polar phosphate ground state than of the less polar transition state in the more polar protic solvents. Unhindered amines add to 4-nitrophenyl phosphate monoanion, but not to the dianion. The oxyphosphorane intermediate thus formed collapses to aroxide ion and a protonated dipolar phosphoramide which is rapidly deprotonated by the relatively basic 4-nitrophenoxide: ArOPO 3H − + CH(CH 2CH 2) 3N(acetonitrile ⇌ CH(CH 2CH 2) 3N +P(O)(OH)O − + ArO −⇌ CH(CH 2 CH 2) 3N +PO 3 2−+ ArOH → CH(CH 2CH 2) 3N + PO 3 −. The postulated formation of PO 3 − by this route explains why the addition of quinuclidine to an acetonitrile solution containing the monoanion salt, ArOPO 3H −R 4N +, and t−BuOH produces t-butyl phosphate at a faster rate than the addition of diisopropylethylamine to the same solution. 2,4-Dinitrophenyl phosphate, which was previously studied by the same techniques, reacts via oxyphosphorane intermediates from the diprotonated and the monoanion forms, and via monomeric metaphosphate, from the dianion form.