MgF(3)(-) as a transition state analog of phosphoryl transfer

The formation of complexes between small G proteins and certain of their effectors can be facilitated by aluminum fluorides. Solution studies suggest that magnesium may be able to replace aluminum in such complexes. We have determined the crystal structure of RhoA.GDP bound to RhoGAP in the presence of Mg(2+) and F(-) but without Al(3+). The metallofluoride adopts a trigonal planar arrangement instead of the square planar structure of AlF(4)(-). We have confirmed that these crystals contain magnesium and not aluminum by proton-induced X-ray emission spectroscopy. The structure adopted by GDP.MgF(-) possesses the stereochemistry and approximate charge expected for the transition state. We suggest that MgF3(-) may be the reagent of choice for studying phosphoryl transfer reactions.     

Physical nature of intermolecular interactions within cAMP-dependent protein kinase active site: differential transition state stabilization in phosphoryl transfer reaction

The origin of enzyme catalytic activity may be effectively explored within the nonempirical theory of intermolecular interactions. The knowledge of electrostatic, exchange, delocalization, and correlation components of the transition state and substrates stabilization energy arising from each enzyme active site residue allows to examine the most essential physical effects involved in enzymatic catalysis. Consequently, one can build approximate models of the catalytic activity in a systematic and legitimate manner. Whenever the dominant role of electrostatic interactions is recognized or assumed, the properties of an optimal catalytic environment could be simply generalized and visualized by means of catalytic fields that, in turn, aids the design of new catalysts. Differential transition state stabilization (DTSS) methodology has been applied herein to the phosphoryl transfer reaction catalyzed by cAMP-dependent protein kinase (PKA). The MP2 results correlate well with the available experimental data and theoretical findings indicating that Lys72, Asp166, and the two magnesium ions contribute -22.7, -13.3, -32.4, and -15.2 kcal/mol to differential transition state stabilization, respectively. Although all interaction energy components except that of electron correlation contribution are meaningful, the first-order electrostatic term correlates perfectly with MP2 catalytic activity. Catalytic field technique was also employed to visualize crucial electrostatic features of an ideal catalyst and to compare the latter with the environment provided by PKA active site. The map of regional electronic chemical potential was used to analyze the unfavorable catalytic effect of Lys168. It was found that locally induced polarization of TS atoms thermodynamically destabilizes electrons, pulling them to regions displaying higher electronic chemical potential.     

Benchmark calculations on models of the phosphoryl transfer reaction catalyzed by protein kinase A

Protein phosphorylation has been proved to be of great importance in many stages of cell life. In the last few years, its reaction mechanism has been extensively studied. In this work we present the analysis of the performances of several computational methods with different computational costs (from multilevel to semiempirical) to point out the best method to be used at each level in the study of phosphoryl transfer. Finally, we center on the semiempirical methods, and mainly on the AM1/d Hamiltonian with different sets of parameters, which will permit hybrid quantum mechanics/molecular mechanics (QM/MM) free energy calculations on big models at an acceptable computational cost. We have used quite a large set of molecules and model reactions to test the computational methods, reproducing all the chemical steps involved in the mainly accepted reaction pathways for the protein phosphorylation. In the end, we also present the results for an enlarged model, cut out from an entire biological model: we compare the 2-D PES at the B3LYP and AM1/d levels with the purpose of obtaining a correction for the semiempirical method. The AM1/d-PhoT semiempirical parameterization corrected using single-point energy calculations at the B3LYP/MG3S level seems to be suitable to carry out reliable QM/MM calculations of the complete biological system.     

A QM/MM study of the phosphoryl transfer to the Kemptide substrate catalyzed by protein kinase A. The effect of the phosphorylation state of the protein on the mechanism

We present here a theoretical study of the phosphoryl transfer catalytic mechanism of protein kinase A, which is the best known member of the large protein kinase family. We have built different theoretical models of the complete PKA-Mg(2)-ATP-substrate system to explore the two most accepted reaction pathways, using for the first time in a reaction mechanism theoretical study, the heptapeptide substrate Kemptide, which is relevant for its high efficiency and small size. The effect of the protein configuration, as modeled by two different X-ray structures with different phosphorylation states and degrees of flexibility, has been analyzed. The results indicate that the environmental conditions can influence the availability of the pathways and thus the choice of the mechanism to be followed. In addition, the roles of the two active site conserved residues, Asp166 and Lys168, have been analyzed for each reaction mechanism.     

Computational study of the phosphoryl transfer catalyzed by a cyclin-dependent kinase

A cyclin-dependent kinase, Cdk2, catalyzes the transfer of the gamma-phosphate from ATP to a threonine or serine residue of its polypeptide substrates. Here, we investigate aspects of the reaction mechanism of Cdk2 by gas-phase density functional calculations, classical molecular dynamics, and Car-Parrinello QM/MM simulations. We focus on the role of the conserved Asp127 and on the nature of the phosphoryl transfer reaction mechanism catalyzed by Cdk2. Our findings suggest that Asp127 is active in its deprotonated form by assisting the formation of the near-attack orientation of the substrate serine or threonine. Therefore, the residue does not act as a general base during the catalysis. The mechanism for the phosphoryl transfer is a single SN2-like concerted step, which shows a phosphorane-like transition state geometry. Although the resulting reaction mechanism is in agreement with a previous density functional study of the same catalytic reaction mechanism (Cavalli et al., Chem. Comm. 2003, 1308-1309), the reaction barrier is considerably lower when QM/MM calculations are performed, as in this study ( approximately 42 kcal mol(-1) QM vs. approximately 24 kcal mol(-1) QM/MM); this indicates that important roles for the catalysis are played by the protein environment and solvent waters. Because of the high amino acid sequence conservation among the whole family of cyclin-dependent kinases (CDKs), these results could be general for the CDK family.     

Ab initio QM/MM studies of the phosphoryl transfer reaction catalyzed by PEP mutase suggest a dissociative metaphosphate transition state

The interconversion between phosphoenolpyruvate (PEP) and phosphonopyruvate (P-pyr) catalyzed by PEP mutase is investigated using an ab initio QM/MM method with the QM region treated at the B3LYP/6-31G* level of theory. Two-dimensional minimum energy path calculations were carried out for both the wild-type enzyme and the N122A mutant. The calculations suggest a dissociative transition state featuring metaphosphate and Mg(2+)-coordinating pyruvate enolate, stabilized by an extensive hydrogen bond network involving Asn122, Ser123, Arg159, His190, Ser46, and Leu48. It is also found that a substantial conformational change in the pyruvyl group is required for the interconversion.     

Anionic charge is prioritized over geometry in aluminum and magnesium fluoride transition state analogs of phosphoryl transfer enzymes

Phosphoryl transfer reactions are ubiquitous in biology and metal fluoride complexes have played a central role in structural approaches to understanding how they are catalyzed. In particular, numerous structures of AlFx-containing complexes have been reported to be transition state analogs (TSAs). A survey of nucleotide kinases has proposed a correlation between the pH of the crystallization solution and the number of coordinated fluorides in the resulting aluminum fluoride TSA complexes formed. Enzyme ligands crystallized above pH 7.0 were attributed to AlF3, whereas those crystallized at or below pH 7.0 were assigned as AlF4-. We use 19F NMR to show that for beta-phosphoglucomutase from Lactococcus lactis, the pH-switch in fluoride coordination does not derive from an AlF4- moiety converting into AlF3. Instead, AlF4- is progressively replaced by MgF3- as the pH increases. Hence, the enzyme prioritizes anionic charge at the expense of preferred native trigonal geometry over a very broad range of pH. We demonstrate similar behavior for two phosphate transfer enzymes that represent typical biological phosphate transfer catalysts: an amino acid phosphatase, phosphoserine phosphatase from Methanococcus jannaschii and a nucleotide kinase, phosphoglycerate kinase from Geobacillus stearothermophilus. Finally, we establish that at near-physiological ratios of aluminum to magnesium, aluminum can dominate over magnesium in the enzyme-metal fluoride inhibitory TSA complexes, and hence is the more likely origin of some of the physiological effects of fluoride.     

The role of the putative catalytic base in the phosphoryl transfer reaction in a protein kinase: first-principles calculations

Protein kinases are important enzymes controlling the majority of cellular signaling events via a transfer of the gamma-phosphate of ATP to a target protein. Even after many years of study, the mechanism of this reaction is still poorly understood. Among many factors that may be responsible for the 1011-fold rate enhancement due to this enzyme, the role of the conserved aspartate (Asp166) has been given special consideration. While the essential presence of Asp166 has been established by mutational studies, its function is still debated. The general base catalyst role assigned to Asp166 on the basis of its position in the active site has been brought into question by the pH dependence of the reaction rate, isotope measurements, and pre-steady-state kinetics. Recent semiempirical calculations have added to the controversy surrounding the role of Asp166 in the catalytic mechanism. No major role for Asp166 has been found in these calculations, which have predicted the reaction process consisting of an early transfer of a substrate proton onto the phosphate group. These conclusions were inconsistent with experimental observations. To address these differences between experimental results and theory with a more reliable computational approach and to provide a theoretical platform for understanding catalysis in this important enzyme family, we have carried out first-principles structural and dynamical calculations of the reaction process in cAPK kinase. To preserve the essential features of the reaction, representations of all of the key conserved residues (82 atoms) were included in the calculation. The structural calculations were performed using the local basis density functional (DFT) approach with both hybrid B3LYP and PBE96 generalized gradient approximations. This kind of calculation has been shown to yield highly accurate structural information for a large number of systems. The optimized reactant state structure is in good agreement with X-ray data. In contrast to semiempirical methods, the lowest energy product state places the substrate proton on Asp166. First-principles molecular dynamics simulations provide additional support for the stability of this product state. The latter also demonstrate that the proton transfer to Asp166 occurs at a point in the reaction where bond cleavage at the PO bridging position is already advanced. This mechanism is further supported by the calculated structure of the transition state in which the substrate hydroxyl group is largely intact. A metaphoshate-like structure is present in the transition state, which is consistent with the X-ray structures of transition state mimics. On the basis of the calculated structure of the transition state, it is estimated to be 85% dissociative. Our analysis also indicates an increase in the hydrogen bond strength between Asp166 and substrate hydroxyl and a small decrease in the bond strength of the latter in the transition state. In summary, our calculations demonstrate the importance of Asp166 in the enzymatic mechanism as a proton acceptor. However, the proton abstraction from the substrate occurs late in the reaction process. Thus, in the catalytic mechanism of cAPK protein kinase, Asp166 plays a role of a "proton trap" that locks the transferred phosphoryl group to the substrate. These results resolve prior inconsistencies between theory and experiment and bring new understanding of the role of Asp166 in the protein kinase catalytic mechanism.     

Extraction mechanism of Tc(IV) by Di-(2-ethylhexyl)phosphoric acid (HDEHP)

Journal of Radioanalytical and Nuclear Chemistry - Di-(2-ethylhexyl)phosphoric acid, HDEHP, is a well-known extractant used for metal ion extraction in an industrial scale and for research work....     

Measuring pK(a) values in protein folding transition state ensembles by NMR spectroscopy

Protein folding kinetic data have been obtained for the marginally stable N-terminal SH3 domain of the Drosophila protein drk as a function of pH in order to investigate the electrostatic properties of Asp8 in the folding transition state ensemble. The slow exchange between folded and unfolded forms of the protein gives rise to separate NMR resonances for both folded and unfolded states at equilibrium. As a result, kinetic data can be derived from magnetization transfer between these two states without the need for denaturants. Using the fact that ionization of Asp8 dominates the electrostatic behavior of the protein between pH 2 and 3, along with pKa values for titrating groups in both folded and unfolded states that have been determined in a previous study, values of 2.9 +/- 0.1 and 3.3 +/- 0.2 are obtained for the pKa of Asp8 in the transition state for the wild-type protein and for a His7Ala mutant, respectively. The data are consistent with the partial formation in the transition state ensemble of an Asp8 side chain carboxylate-a Lys21 backbone amide interaction that represents a highly conserved contact in folded SH3 domains.     

Nitrogen atom transfer from iron(IV) nitrido complexes: a dual-nature transition state for atom transfer

The mechanism of nitrogen atom transfer from four-coordinate tris(carbene)borate iron(IV) nitrido complexes to phosphines and phosphites has been investigated. In the absence of limiting steric effects, the rate of nitrogen atom transfer to phosphines increases with decreasing phosphine σ-basicity. This trend has been quantified by a Hammett study with para-substituted triarylphosphines, and is contrary to the expectations of an electrophilic nitrido ligand. On the basis of electronic structure calculations, a dual-nature transition state for nitrogen atom transfer is proposed, in which a key interaction involves the transfer of electron density from the nitrido highest occupied molecular orbital (HOMO) to the phosphine lowest unoccupied molecular orbital (LUMO). Compared to analogous atom transfer reactions from a 5d metal, these results show how the electronic plasticity of a 3d metal results in rapid atom transfer from pseudotetrahedral late metal complexes.     

Diabatic transition state theory and the concept of temperature

A modified transition state theory, appropriate for direct reactions, which incorporates dynamic constraints and can account for population inversion, is presented. It is shown that the distribution of vibrational states can be characterized by a lagrangian parameter that can be interpreted as temperature.     

Prioritization of charge over geometry in transition state analogues of a dual specificity protein kinase

The direct observation of a transition state analogue (TSA) complex for tyrosine phosphorylation by a signaling kinase has been achieved using (19)F NMR analysis of MEK6 in complex with tetrafluoroaluminate (AlF(4)(-)), ADP, and p38α MAP kinase (acceptor residue: Tyr182). Solvent-induced isotope shifts and chemical shifts for the AlF(4)(-) moiety indicate that two fluorine atoms are coordinated by the two catalytic magnesium ions of the kinase active site, while the two remaining fluorides are liganded by protein residues only. An equivalent, yet distinct, AlF(4)(-) complex involving the alternative acceptor residue in p38α (Thr180) is only observed when the Tyr182 is mutated to phenylalanine. The formation of octahedral AlF(4)(-) species for both acceptor residues, rather than the trigonal bipyramidal AlF(3)(0) previously identified in the only other metal fluoride complex with a protein kinase, shows the requirement of MEK6 for a TSA that is isoelectronic with the migrating phosphoryl group. This requirement has hitherto only been demonstrated for proteins having a single catalytic magnesium ion.     

Solvent extraction of Fe(III) by di-(2-ethylhexyl)phosphoric acid from phosphoric acid solutions

The extraction of iron(III) from aqueous phosphoric acid was studied using di-(2-ethylhexyl)phosphoric acid and trioctylphosphine oxide in nonaromatic hydrocarbon diluent. Distribution ratios have been investigated as a function of concentration of iron(III), phosphoric acid concentration, extractant concentration and extraction temperature. The apparent enthalpy change for the extraction reaction has been calculated from the temperature dependence data. It was found that the extractant dependency for iron(III) is first power indicating hydrolysis of iron(III) in the aqueous phase.     

Kinetic study of gas-phase reactions with CF2Cl2 and CFCl3. Analysis using the transition state theory (TST) of the chlorine atom transfer reactions by CF3 and CH3 radicals from halomethanes

The following gas-phase reactions: were studied by the competitive method with CF₃I as the source of radicals. The kinetic parameters obtained in the temperature range 533–613 K and 503–613 K respectively for chlorine atom transfer reactions are given by: where θ = 2.303 RT (cal mol^?1). The Arrhenius A values were calculated for seven chlorine atom transfer reactions (CF₂Cl₂, CFCl₃, CCl₄ with CF₃ radicals; CF₃Cl, CF₂Cl₂, CFCl₃ and CCl₄ with CH₃ radicals) by using the thermochemical kinetic version of the Transition State Theory (TST).     

Initial state versus transition state solvation effects on the kinetics of oxidation of nickel(II) dioxocyclam by peroxodisulfate

The kinetics of oxidation of 1,4,8,11-tetraazacyclotetradecane-5,7-diamidonickel(II) (nickel dioxocyclam) by peroxodisulfate have been measured in a range of binary aqueous solvent mixtures. Reaction rates are retarded by the presence of organic co-solvents, indicative of destabilization of the transition state (TS) relative to the initial state (IS) in the binary aqueous mixtures. Thus the TS is more hydrophilic than the IS. From solubility measurements of the nickel(II) macrocycle, the transfer chemical potential of the IS of the reaction has been estimated and compared with that of the TS. The transfer potential of the IS is dominated by the solvation of the peroxodisulfate ion.     

Kinetics and mechanism of oxidation of formic acid by bismuth(V) in aqueous phosphoric acid medium

The solution of bismuth(V) was prepared by digesting sodium bismuthate in aqueous phosphoric acid (3.0 mol dm⁻³), the resulting pink colour solution absorbs in the visible region at 530 nm (640 dm³ mol⁻¹ cm⁻¹). The stoichiometry of the oxidation of formic acid by bismuth(V) corresponds to the reaction as represented by the Eq. ( 1 ). (1) The observed kinetic rate law is given by the Eq. ( 2 ); (2) where Bi^V and [HCO₂H] are the gross analytical concentrations of bismuth(V) and formic acid respectively. A plausible reaction mechanism corresponding to the rate law (2) has been proposed. Also the pattern of reactivity of bismuth(V) in HCIOHF mixture and H₃PO₄ respectively has been compared. © 2000 John Wiley & Sons, Inc. Int J Chem Kinet 32: 491–497, 2000     

An uncharged amine in the transition state of the ribosomal peptidyl transfer reaction

The ribosome has an active site comprised of RNA that catalyzes peptide bond formation. To understand how RNA promotes this reaction requires a detailed understanding of the chemical transition state. Here, we report the Br?nsted coefficient of the alpha-amino nucleophile with a series of puromycin derivatives. Both 50S subunit- and 70S ribosome-catalyzed reactions displayed linear free-energy relationships with slopes close to zero under conditions where chemistry is rate limiting. These results indicate that, at the transition state, the nucleophile is neutral in the ribosome-catalyzed reaction, in contrast to the substantial positive charge reported for typical uncatalyzed aminolysis reactions. This suggests that the ribosomal transition state involves deprotonation to a degree commensurate with nitrogen-carbon bond formation. Such a transition state is significantly different from that of uncatalyzed aminolysis reactions in solution.