Slow Step Of Reaction Research Articles

NTP-driven Translocation by Human RNA Polymerase II

We report a "running start, two-bond" protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis delta antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure.

A Study of Charge-Transfer Reactions on (110) Single-Crystal Nickel Surfaces in Nickel Sulfamate Electrolyte Using Electrochemical Impedance Spectroscopy

Electrochemical impedance spectroscopy measurements under a small alternating current density were performed to investigate electrochemical reactions on (110) single-crystalline nickel surfaces in nickel sulfamate electrolyte. The reaction impedance shows a power law of the ac modulation frequency, and two exponents for the reaction resistance and reaction capacitance are found to approximately agree with the values predicted by a homogeneous charge-transfer reaction model. In addition, the Nyquist diagrams exhibit the absence of inductive loops for the frequency range from 50 mHz to 100 kHz. A condition for the existence of inductive loops derived in this study suggests that the electrocrystallization of Ni2+ ions may take place in the slow reaction step Ni2+ + e- → and in the fast reaction step + e- → Ni.

Surface-dependent Coagulation Enzymes: FLOW KINETICS OF FACTOR Xa GENERATION ON LIVE CELL MEMBRANES

The initial surface reactions of the extrinsic coagulation pathway on live cell membranes were examined under flow conditions. Generation of activated coagulation factor X (fXa) was measured on spherical monolayers of epithelial cells with a total surface area of 41-47 cm(2) expressing tissue factor (TF) at >25 fmol/cm(2). Concentrations of reactants and product were monitored as a function of time with radiolabeled proteins and a chromogenic substrate at resolutions of 2-8 s. At physiological concentrations of fVIIa and fX, the reaction rate was 3.05 +/- 0.75 fmol fXa/s/cm(2), independent of flux, and 10 times slower than that expected for collision-limited reactions. Rates were also independent of surface fVIIa concentrations within the range 0.6-25 fmol/cm(2). The transit time of fX activated on the reaction chamber was prolonged relative to transit times of nonreacting tracers or preformed fXa. Membrane reactions were modeled using a set of nonlinear kinetic equations and a lagged normal density curve to track the expected surface concentration of reactants for various hypothetical reaction mechanisms. The experimental results were theoretically predicted only when the models used a slow intermediate reaction step, consistent with surface diffusion. These results provide evidence that the transfer of substrate within the membrane is rate-limiting in the kinetic mechanisms leading to initiation of blood coagulation by the TF pathway.

Theoretical Study of the Proton Transfer between Water and [FeH(CO)4]- in Aqueous Solution and Relevance to the Water-Gas Shift Reaction Catalyzed by Iron Pentacarbonyl in the Condensed Phase

Proton transfer between H2O and FeH(CO)4-, the assumed slow step of the water-gas shift reaction catalyzed by Fe(CO)5, has been studied with Hartree−Fock and multiconfiguration CASSCF ab initio methods. The calculations have been performed in vacuo and in water solution at 298 K, simulating the solvent as a polarizable continuum medium. The process in solution has been found to be less endothermic than in the gas phase by about 50 kcal/mol. The valence bond analysis, made on the CASSCF wave function along the reaction path, explains the mechanism in terms of a single structure in which the transferred hydrogen is simultaneously bonded to iron and oxygen with a different polarization depending on the reaction coordinate.

Kinetics of wet oxidation of black liquor over a Pt–Pd–Ce/alumina catalyst

The catalytic oxidation of industrial wastewater from paper and pulp mills has been investigated in a slurry reactor at a temperature range 433–463 K and at pressures from 1.5 to 2.2 MPa. Adding Ce on alumina support promotes the catalytic activity for oxidation of black liquor. Pt–Pd–Ce/alumina catalyst shows a promising activity for wet catalytic oxidation of black liquor. The oxidation reaction over a Pt–Pd–Ce catalyst is characterized by an initial fast reaction step followed by a slow reaction step. The rate of total organic carbon (TOC) reduction was described by first-order kinetics with respect to TOC concentration in black liquor for both initial and later reaction steps. The activation energies were determined to be 54.53 and 50.13 kJ/mol for the initial and later oxidation steps, respectively. Comparison of the data with the generalized kinetic model was also presented.

Equations for progress curves of some kinetic models of enzyme-single substrate-single slow binding modifier system.

A procedure is described by means of which the equations for progress curves for the kinetic models that include fast and slow reaction steps can be derived. It is based on combined assumptions of equilibrium and steady-state and uses Laplace transformation for solving the systems of differential equations. The progress curve equations and the significance of the corresponding parameters are given for some most frequently occurring models describing the influence of a slow binding modifier on a single substrate enzyme reaction.

Thermal Desorption Spectroscopy of Alkanethiol Self-Assembled Monolayer on Au(111)

The adsorption and desorption of octadecanethiol ( CH3(CH2)17SH: monomer) self-assembled monolayers (SAMs) on Au(111) have been investigated by thermal desorption spectroscopy (TDS). Two desorption peaks of dioctadecyl disulfide ( (CH3(CH2)17S)2+: dimer+) exhibiting different sticking behaviors have been identified in the TDS spectra, while the desorption of octadecanethiol ( CH3(CH2)17SH+: monomer+) occurred at a lower temperature than that of the dimer+. The desorption activation energy and the pre-exponential factor for each peak were estimated assuming first- and second-order reactions. The analysis results suggest that both alkyl thiolate (RS–Au) with strong chemisorption and dialkyl disulfide (RS–SR: dimer) with relatively weak adsorption co-exist in the alkanethiol SAM on Au. The total amount of relatively weakly adsorbed dimers increased appreciably in a slow reaction step, while the monolayer thickness remained almost constant after SAM formation. This result suggests that dimer molecules are formed in the monolayer. This formation of weakly adsorbed dimers is strongly related to the self-exchange phenomena in SAM films reported by Schlenoff et al.. [J. Am. Chem. Soc. 117 (1995) 12528].

Stopped-flow fluorescence kinetics of bovine alpha 2-antiplasmin inhibition of bovine midiplasmin.

In the conversion of bovine plasminogen to bovine plasmin not only the expected urokinase-catalysed cleavage of Arg-557-Val-558, and the following autocatalytic cleavage separating the N-terminal peptide 1-77 from the heavy chain of plasmin, but also a cleavage at Arg-342-Met-343 between kringles 3 and 4 is seen. Here, kinetic studies of the interaction of bovine alpha 2-antiplasmin with bovine plasmin were performed on isolated bovine midiplasmin (lacking kringles 1-3) and on bovine plasmin containing all of the activation products from the bovine plasminogen. A series of experiments using stopped-flow fluorescence fast kinetics as well as conventional techniques suggests a reaction model in accordance with the one known for the human system. First, a tight complex (K1 in the nanomolar range) is formed in a fast reaction step; and second, a tightening of this complex occurs in a slow reaction step. The final complex is indeed so tight (Ki < or = pM), that the reaction for many practical purposes is legitimately considered irreversible. The stopped-flow method allows for the determination of reliable values of the second-order rate constant for the fast association step. At pH 7.4 and 25 degrees C, k+1 = 1.7 x 10(6) M-1 s-1 was obtained in the absence and k+1 = 0.9 x 10(6) M-1.s-1 in the presence of the kringles 1-3 domain of bovine plasmin. In contrast to this, substantial reductions of k+1 were seen in the presence of concentrations of 6-amino-hexanoic acid corresponding to lysine-binding-site interactions and far too low to be attributed to active-site interactions with the bovine plasmins (for each, Ki = 42 mM). All in all, the data indicated that the lysine-binding site(s) not of kringle 1, but of midiplasmin (those of kringles 4 and 5) are regulating the inhibition reaction.

Kinetics and thermodynamics of the mechanism of interaction of sodium phytate with alpha-globulin.

The precipitation mechanism of alpha-globulin in the presence of myo-inositol hexaphosphate (sodium phytate) was studied in detail. The maximum interaction was found at pH 2.3 where the protein was in a dissociated state having an 8.3S aggregate and a 1.5S monomer. This interaction was predominantly dependent upon the sodium phytate to protein ratio. Velocity sedimentation studies indicated polymer formation due to preferential progressive binding of ligand to polymer, whose size and concentration increased with an increase in sodium phytate concentration. The polymer formation was shown to be ligand mediated and exists independently in solution along with the monomer. The binding isotherm by equilibrium dialysis confirmed differential binding of sodium phytate to the polymer and the monomer as indicated by two sets of binding sites, one having 7 +/- 2 sites of a K value 1.3 x 10(-4) mol-1 and the other having 56 +/- 3 sites with a K value of 2.8 x 10(-3) mol-1. Binding resulted in perturbation of chromophores of protein due to charge effects. The kinetics of the polymer formation was shown to be a pseudo-first-order reaction having two steps. The initial fast reaction involving conformational changes has rate constants of k1 = 52.4 x 10(-3) s-1 and k' = 67.5 x 10(-3) s-1, followed by a slow reaction step of rate constants k2 = 4.3 x 10(-3) s-1 and k'2 = 2.9 x 10(-3) s-1 at sodium phytate concentrations of 1 x 10(-4) M and 5 x 10(-4) M, respectively.

The effect of the lattice spacing change on cross-bridge kinetics in chemically skinned rabbit psoas muscle fibers. II. Elementary steps affected by the spacing change

The actin-myosin lattice spacing of rabbit psoas fibers was osmotically compressed with a dextran T-500, and its effect on the elementary steps of the cross-bridge cycle was investigated. Experiments were performed at the saturating Ca (pCa 4.5-4.9), 200 mM ionic strength, pH 7.0, and at 20 degrees C, and the results were analyzed by the following cross-bridge scheme: [formula: see text] where A = actin, M = myosin head, S = MgATP, D = MgADP, and P = Pi = phosphate. From MgATP and MgADP studies on exponential process (C) and (D), the association constants of cross-bridges to MgADP (K0), MgATP (K1a), the rate constants of the isomerization of the AM S state (k1b and k-1b), and the rate constants of the cross-bridge detachment step (k2 and k-2) were deduced. From Pi study on process (B), the rate constants of the cross-bridge attachment (power stroke) step (k4- and k-4) and the association constant of Pi ions to cross-bridges (K5) were deduced. From ATP hydrolysis measurement, the rate constant of ADP-isomerization (rate-limiting) step (k6) was deduced. These kinetic constants were studied as functions of dextran concentrations. Our results show that nucleotide binding, the ATP-isomerization, and the cross-bridge detachment steps are minimally affected by the compression. The rate constant of the reverse power stroke step (k-4) decreases with mild compression (0-6.3% dextran), presumably because of the stabilization of the attached cross-bridges in the AM*DP state. The rate constant of the power stroke step (k4) does not change with mild compression, but it decreases with higher compression (> 6.3% dextran), presumably because of an increased difficulty in performing the power stroke. These results are consistent with the observation that isometric tension increases with a low level of compression and decreases with a high level of compression. Our results also show that the association constant K5 of Pi with cross-bridge state AM*D is not changed with compression. Our result further show that the ATP hydrolysis rate decreased with compression, and that the rate constants of the ADP-isomerization step (k6) becomes progressively less with compression. The effect of compression on the power stroke step and rate-limiting step implies that a large-scale molecular rearrangement in the myosin head takes place in these two slow reaction steps.

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl-tRNA synthetase from Escherichia coli.

A total rate equation was used to calculate the discrimination of valine by the isoleucyl-tRNA synthetase from Escherichia coli. The PPi present in the cell makes the backward reaction or the pyrophosphorolysis of the E.aa-AMP possible. If the E.Ile-AMP has been corrected for wrong aminoacyl adenylation by the pretransfer proofreading, the pyrophosphorolysis rapidly equilibrates the corrected E.Ile-AMP with E.Ile and thus spoils the effect of the proofreading. The loss of the corrected species is avoided if there is a barrier (perhaps conformational) formed by a slow reaction step between the noncorrected E.Ile-AMP and the corrected (*E)tRNA(Ile-AMP). If such a slow conformational change exists, the increase in accuracy from the pretransfer proofreading would be beneficial, and, in addition, the PPi increases the accuracy by optimizing the initial discrimination of the wrong amino acid.

Mechanistic studies of the oxidative coupling polymerization of phenols. VI. Comparison of reactivities of DMP, PPO‐dimer, and ‐trimer in the copper‐catalyzed oxidative coupling polymerization

AbstractIn the oxidative coupling polymerization, catalyzed by copper‐amine complexes, the oxidation rates of 2,6‐dimethylphenol (DMP) and its CO‐coupled dimer [4‐(2′,6′‐dimethylphenoxy)‐2,6‐dimethylphenol] and trimer [4‐(‐4′‐(2″,6″‐dimethylphenoxy)‐2′,6′‐dimethylphenoxy))‐2,6‐dimethylphenol] have been determined. The DMP concentration dependence shows a Michaelis–Menten‐type behavior. On the other hand, the dimer and trimer showed a first‐order rate‐dependence in the respective phenol concentrations. This indicates that the slow reaction step, following an equilibrium complex formation between DMP and copper complex, is relatively fast for both the dimer and the trimer. Therefore, coordination of dimer or trimer to the copper complex appears to be rate‐determining. Furthermore, the dimer and trimer gave overall reaction rates approximately eight times higher than found for DMP. Following the Flory principle of equal reactivity for functional groups of oligomers in polycondensations, all PPO oligomers can be assumed to have equally high oxidation rates as the dimer and trimer. The yield of undesired DPQ side product is strongly reduced when starting with the dimer (0.18%), or trimer (0.17%), compared to 3.3% for DMP. This is not unexpected, since DPQ can only be formed from two monomeric DMP residues. In fact, using a 1/10 molar mixture of dimer/DMP already results in a DPQ yield of only 1.7%. Furthermore, when starting from DMP, it has been observed that DPQ was predominantly formed during the first 30% conversion. Starting from dimer (or trimer) DPQ was formed at an almost constant very low rate during the whole course of the reaction. From these experiments it can be concluded that the most important polymerization reaction involves oxidation of copper‐coordinated DMP anion to its corresponding cations, followed by coupling with a copper coordinated PPO chain.

Membrane transport models with fast and slow reactions: General analytical solution for a single relaxation

Membrane transport models are usually expressed on the basis of chemical kinetics. The states of a transporter are related by rate constants, and the time-dependent changes of these states are given by linear differential equations of first order. To calculate the time-dependent transport equation, it is necessary to solve a system of differential equations which does not have a general analytical solution if there are more than five states. Since transport measurements in a complex system rarely provide all the time constants because some of them are too rapid, it is more appropriate to obtain approximate analytical solutions, assuming that there are fast and slow reaction steps. The states of the fast steps are related by equilibrium constants, thus permitting the elimination of their differential equations and leaving only those for the slow steps. With a system having only two slow steps, a single differential equation is obtained and the state equations have a single relaxation. Initial conditions for the slow reactions are determined after the perturbation which redistribute the states related by fast reactions. Current and zero-trans uptake equations are calculated. Curve fitting programs can be used to implement the general procedure and obtain the model parameters.

Activation and binding volumes of the sarcoplasmic reticulum transport enzyme activated by calcium or strontium

The effect of pressure on the hydrolysis of dinitrophenyl phosphate (DnpP) and p-nitrophenyl phosphate (NpP) by the sarcoplasmatic reticulum transport enzyme in permeabilized and native closed vesicles activated by calcium or strontium, respectively, in aqueous and Me2SO-containing media has been studied. At atmospheric pressure, the enzyme in permeabilized vesicles, saturated with respect to substrates and activating ions, hydrolyzes DnpP ten times faster than NpP; for both substrates, calcium activation exceeds that by strontium only a little (20%). In aqueous media the enzyme displays, under all activating conditions, an almost identical curvilinear relationship between the logarithm of enzyme activity and pressure. The data were analysed on the basis of a simplified reaction scheme, in which two unidirectionally proceeding substrate-driven pressure-dependent reactions (k2, k4) cyclically transfer high-affinity into low-affinity binding sites which are assumed to be in equilibrium with either calcium or strontium. The fitting procedure yielded two sets of positive activation volumes delta V2* = 90-110 ml/mol and delta V4* = 15-25 ml/mol. Substrate specificity, as well as the effect of temperature, are exclusively localized in the pressure-independent rate constants k'2 and k'4. Considerable different pressure/activity relations characterized by a single activation volume of 20 ml/mol were obtained for the strongly suppressed substrate hydrolysis of native closed vesicles. At atmospheric pressure DnpP hydrolysis of open vesicles is inhibited by Me2SO, while NpP hydrolysis is considerably activated, irrespective of its activation by calcium or strontium. In the presence of 22.5% Me2SO, the activation volumes are reduced by 50-70 ml/mol. The rate constants of DnpP and NpP hydrolysis are either augmented or reduced by rising Me2SO concentrations, depending on the corresponding supporting substrate. Me2SO has only a slight effect on the pressure dependence of substrate hydrolysis by native vesicles. The small activation volume observed for the activity of native vesicles could be assigned on account of the simplified reaction scheme of the slow reaction step k4, by which the enzyme is transferred from its low-affinity into its high-affinity binding state. Volume changes connected with the binding of calcium or strontium to the luminal binding site of the enzyme were deduced from the observed activation volume and the computed volume change of the slow reaction step (delta V4*).

The elimination of fast variables in complex chemical reactions. III. Mesoscopic level (irreducible case)

The master equation for a complex chemical reaction cannot always be reduced to a simpler master equation, even if there are fast and slow individual reaction steps. Nevertheless the elimination of intermediates can be carried out with the help of theΩ-expansion. This is illustrated with a well-known complex reaction: the dissociation of N2O5. It is shown that the intrinsic fluctuations in the N2O5 decay are larger than those implied by the master equation suggested by the macroscopic rate law.

Kinetics of spontaneous dissolution of copper-47.3 atom-% zinc brass with hydrogen depolarization: Slow reaction step

The spontaneous dissolution rate of Cu-47.3 atom-% Zn ordered β'-brass in deaerated 2.0 M sulphuric acid was measured at 65.75 and 85 °C. The simplified Erofe'ev equation was used for the discussion of the results obtained. A working hypothesis is put forward that formation of the metallic copper phase is the slow step of the dissolution process.

Regulation of cholesterol metabolism in rat adrenal mitochondria

Addition of cholesterol to rat adrenal mitochondria resulted in a stimulation of pregnenolone synthesis. The slow step of the mitochondrial cholesterol side-chain cleavage reaction could be the interaction of the sterol with cytochrome P-450. The rate of cholesterol binding to this enzyme as observed spectroscopically correlated with the equilibration period (20 min) of the mitochondria and exogenous cholesterol required for maximal rates of pregnenolone synthesis. It is suggested that translocation of cholesterol between different sterol pools occurs within the mitochondria. Potential intracellular effectors that could be of importance in the movement or regulation of mitochondrial cholesterol include bivalent metallic ions, prostaglandins, cyclic nucleotides, polyamines and polylysine. Of the effectors studied, only calcium ions and polylysine markedly stimulated pregnenolone synthesis. These effectors might stimulate steroidogenesis by lateral displacement of cholesterol in the mitochondrial membrane into a compartment easily accessible to the cholesterol side-chain cleavage enzyme complex.

Zur wirkung von zusatzstoffen bei der bildung von geordneten graphit-FeCl 3-einlagerungsverbindungen

Intercalation compounds with strong disturbances in the stacking sequences of the layers are normally formed in the reaction of ferric chloride vapour with graphite [6]. Fully periodic sequences can be prepared, however, by addition of either chlorine, oxygen or water vapour [5]. The effect of these added substances has been investigated. Oxygen and water vapour do not act directly but liberate chlorine from ferric chloride in a preliminary reaction. The produced chlorine was detected quantitatively by spectrophotometry. If no additional time is allowed for the preliminary reaction, the ordering effect of oxygen (Table 2) and of water vapour is less than that of chlorine. The stacking order has been determined by X-ray diffraction [6]. It is quantitatively represented by the “degree of order” (“Bildungsgrad”) of a stage, with a value between zero (completely random sequence) and one (strictly periodical sequence). If chlorine is added only in the initial phase of the intercalation, then the ordering effect will be lost with progressive reaction. Thus the effect of chlorine does not result in any permanent change of the graphite. The rate of intercalation is reduced a little by the addition of chlorine (Fig. 2), probably as the result of the diffusioncontrolled kinetics. Thus the chlorine does not act like a catalyst causing an acceleration of an otherwise slow reaction step. The ordered compounds cannot be interpreted as secondary products of the disordered ones. therefore, and also not as alternative products which are kinetically promoted by addition of chlorine. X-Ray diffractometer recordings (Fig. 1) show that the effect of chlorine is much stronger in the final phase than in the initial phase. Following up quantitatively the degree of order, it became evident that with additional chlorine in the final phase a supplementary increase in order is possible. This works as long as the graphite takes up ferric chloride. A subsequent annealing with additional chlorine does not affect the order any more. For the model conception of the effect of chlorine the following conclusions can be drawn: a close connection exists between the motion of ferric chloride in the interior of the crystal and the eventual formation of periodic arrangements. We assume that the chemisorbed chlorine at the surface of graphite weakens the bond of ferric chloride to graphite by charge transfer. Thereby the mobility of ferric chloride between the graphite layers increases. In each small region (domain) periodical arrangements then can be formed more easily.

The comparative study of CO 2 + H ads reaction on platinum electrode in H 2O and D 2O

The observed isotopic effect in CO 2 + H ads reaction showed that at 0.05 V the rate determining step is ▪ formation whereas at 0.2 V the reorientation of adsorbed intermediate: ▪ is probably the slowest step of reaction. The oxidation of adsorbed product is slower in D 2SO 4 than H 2SO 4 solution like the surface oxidation of platinum. The rate determining step of COOH ads oxidation is a reaction with OH ads.

Transients and Cooperativity: A SLOW TRANSITION MODEL FOR RELATING TRANSIENTS AND COOPERATIVE KINETICS OF ENZYMES

A model is proposed to account for the occurrence of both slow transient kinetics and cooperativity in a monomeric, single-site enzyme. This slow transition model incorporates one reaction of the enzyme, in addition to normal catalytic steps, which is slower than other steps in the mechanism and is influenced by substrate. The slow reaction step may be an isomerization (conformation change), a dissociation-association, or binding of another ligand (modifier). Analysis of steady state kinetics and simulation studies for the slow isomerization mechanism have demonstrated that both positive and negative cooperativity can occur with either bursts or lags of enzyme activity. Examination of various combinations of rate constants have illustrated the dependence of cooperativity of the system on changes in certain rate constants. For example, a necessary condition for cooperativity in this model requires that both the binding and the isomerization steps of the mechanism be in a steady state but not at equilibrium. Modifiers could potentially shift the extent or type of cooperativity. The combination of slow transitions with homotropic, sitesite interaction could generate several types of complex, cooperative kinetics. Whenever a transient is observed in a progress curve the contribution of the slow transition model to cooperativity of the kinetics should be assessed. The results of binding studies, initial (pre-transition) velocity measurements, and the effects of factors (e.g. pH, temperature, modifiers) on the half-time of the transient should be capable of determining the extent of the contribution of the slow transition to cooperativity. A table of 10 enzymes which show both transients and cooperativity suggests that these phenomena do occur together and may be related mechanistically through the slow transition mechanism. Thus, in those cases in which a slow transition occurs, the transition may be responsible for nonlinear, double reciprocal plots of the steady state kinetics without requiring interactions between more than one site.