The oscillating peroxidase-oxidase reaction in an open system. Analysis of the reaction mechanism.

1. The oscillations in the peroxidase-oxidase reaction in an open system with NADH as the hydrogen donor are caused by the reaction starting and stopping at critical concentrations of the substrates O2 and NADH. The existence of such critical concentrations is typical of branched chain reactions. 2. The critical concentrations of O2 and NADH that determine the initiation of the reaction are mutually dependent. 3. The branching reactions that determine these critical concentrations involve compounds I and II. 4. Superoxide may be involved in the branching reactions by reacting with NADH and ferriperoxidase. At pH 5.1 the rate constant for the latter reaction is determined as 1.5 . 10(5) M-1 . s-1, whereas for the former reaction only an upper limit for the rate constant of 3.5 . 10(4) M-1 . s-1 could be estimated. These relatively low rate constants suggest that alternative branching reactions may also be involved.     

Melatonin activates the peroxidase-oxidase reaction and promotes oscillations.

We have studied the peroxidase-oxidase reaction with NADH and O2 as substrates and melatonin as a cofactor in a semibatch reactor. We show for the first time that melatonin is an activator of the reaction catalyzed by enzymes from both plant and animal sources. Furthermore, melatonin promotes oscillatory dynamics in the pH range from 5 to 6. The frequency of the oscillations depends on the pH such that an increase in pH was accompanied by a decrease in frequency. Conversely, an increase in the flow rate of NADH or an increase in the average concentration of NADH resulted in an increase in oscillation frequency. Complex dynamics were not observed with melatonin as a cofactor. These results are discussed in relation to observations of oscillatory dynamics and the function of melatonin and peroxidase in activated neutrophils.     

Hydrogen/deuterium isotope effect in the oscillating peroxidase-oxidase reaction.

The oscillatory peroxidase-oxidase reaction has been investigated by using NADH deuterated in the nicotinamide 4-A position. A considerable kinetic hydrogen/deuterium isotope effect on the oscillatory behavior was revealed, which may provide an additional valuable tool for mechanistic studies and for discriminating between various mechanistic models of the peroxidase-oxidase reaction. Particularly, this effect manifests in different oscillation frequencies. A sequence of simple and aperiodic oscillations was found between two stable steady states.     

Oscillatory reaction of alcohol dehydrogenase in an oil/water system

With the use of an oil/water system, oscillatory reactions of an enzyme have been demonstrated. This reaction system has been conceived as an example of the metabolic oscillations of living cells. When a substrate (ethanol) in the oil phase of toluene or chloroform slowly migrated into the aqueous phase containing alcohol dehydrogenase and NAD+, oscillations were observed in the concentration of NADH produced. The gradual entry of substrate into the aqueous phase was essential for the oscillatory reactions to occur. A possible mechanism to account for the appearance of oscillatory reactions of enzymes is proposed, which differs from that presented previously.     

Mechanism of melatonin-induced oscillations in the peroxidase-oxidase reaction

Melatonin induces oscillations in the peroxidase-oxidase (PO) reaction catalyzed by horseradish peroxidase. We present here studies of the effect of pH, enzyme concentration, and concentration of melatonin on the oscillation frequency. We also present a mechanistic model to explain the experimentally observed changes in oscillation frequency. Using the data obtained here we are able to predict that oscillations will also occur in the PO reaction catalyzed by myeloperoxidase. Myeloperoxidase is an important protein in activated neutrophils and we provide evidence that the oscillations of NAD(P)H, superoxide and hydrogen peroxide in these cells may involve this enzyme. Thus, our experimental system can be considered a model system for the nonrespiratory oxygen metabolism in activated neutrophils and other similar cells participating in the defence against invading pathogens.     

Computer simulation of sustained oscillations in peroxidase-oxidase reaction

A system of differential equations of second order exhibiting transitional behaviour and sustained oscillations has been obtained for a complete scheme of the peroxidase-oxidase reaction. The concentrations of hydrogen peroxide and of hydrogen donor radicals are slow variables of the system. The most essential reactions responsible for oscillations have been selected. Analysis of the system in phase plane and in parameter space has been carried out. The dependence of oscillation period and amplitude on the parameter values has been investigated.     

Three steady state situation in an open chemical reaction system. I

In an isothermal continuously stirred tank reactor (open chemical reaction system) fed by sulphuric acid solutions of bromate, bromide and cerium)(III) bistability (three steady state situation) is experimentally observed. This remarkable behavior, based on the instability of one steady state, has important consequences for the understanding of excitability and biochemical control mechanisms. The mass-balance equations for the reactor and the chemical mechanism of the reaction are combined into a simple mathematical model. The behavior of the resulting nonlinear differential equations is examined analytically and by a graphical integration procedure (method of isoclines). Using realistic kinetic data, the model shows the same behavior as observed in the experiment.     

Stochastic models for an enzyme reaction in an open linear system

A stochastic model is developed for an enzyme reaction in an open linear system. The proposed model assumes that the open system maintains the concentration of substrate and inhibitor at constant levels and that the product molecules are removed from the system by a first order reaction. Stochastic models for several enzyme reactions occurring in this open system are shown to correspond to special cases of theGI/M/∞ queue. Takács’ (1958) results for this queueing system are used to obtain the stochastic properties of the enzyme systems. A specific model we studied assumed completely competitive inhibition in an open system. The stationary distribution for the number of product molecules in the system is obtained. The enzyme reaction which incorporated the “intermediate chain hypothesis” can also be investigated by the queueing theory approach. It is shown that for this open system, if the model which incorporated the intermediate chain hypothesis has the same deterministic properties as the Michaelis-Menten model, then the latter has greater stochastic variation than the former. Research supported by the NIH Training Grant No. GM 1237-05 awarded to the Department of Statistics, The Johns Hopkins University. This paper in whole or in part may be reproduced for any purpose of the United States Government.     

A generalized theoretical treatment of the kinetics of an enzyme-catalysed reaction in the presence of an unstable irreversible modifier

A generalized theoretical treatment of the kinetics of an enzyme-catalysed reaction in the presence of an unstable irreversible inhibitor (or activator) is presented. Analytical expressions describing the time-dependence of product formation have been derived in coefficient form amenable to non-linear regression analysis for two operationally distinct types of reaction mechanism dependent on whether the reaction of the unstable modifier (X) with either or both the free enzyme (E) and enzyme-substrate complex (ES) occurs as a simple bimolecular process, or proceeds through the intermediacy of either or both adsorptive enzyme-modifier (EX) and enzyme-modifier-substrate (EXS) complexes in what may be considered as an extension of the Botts-Morales general modifier mechanism for (stable) reversible enzyme inhibitors and activators. Special cases of both models are classified in an analogous way to the traditional naming of reversible enzyme modifications, and guidelines concerning tests of mechanism and determination of kinetic parameters are given. In particular, it has been shown that kinetic constants describing enzyme inactivation by an unstable site-specific inhibitor forming a reversible EX complex prior to covalent modification step may be determined from a single progress curve. Kinetic analysis of the extended Botts-Morales mechanism describing irreversible enzyme inactivation has demonstrated that analytical expressions describing the time-course of product formation may be derived for a stable modifier by retaining the usual steady-state assumptions regarding the fluxes around ES and EXS provided quasi-equilibrium modifier binding to E and ES is assumed, but for unstable modifiers all of the binding steps must be assumed to be at quasi-equilibrium in the steady-state, except under restrictive circumstances.     

Experimental and theoretical studies of the conformational perturbations induced by an abasic site

The three-dimensional structure of the natural undecamer duplex d(CGCACACACGC). d(GCGTGTGTGCG) has been determined by the combined use of NMR spectroscopy and restrained molecular dynamics (rMD) and also by molecular mechanics calculations using the JUMNA program without experimental distance constraints. Both procedures have also been used to model the abasic structure d(CGCACOCACGC).d(GCGTGTGTGCG), where 'O' indicates a modified abasic site: 3-hydroxy-2-(hydroxymethyl) tetrahydrofuran. For the natural duplex, 134 interproton distances have been obtained by complete relaxation matrix analysis of the NOESY cross-peaks intensities, using MARDIGRAS software. These distances along with 100 torsion angles for sugar ring and additional data derived from canonical A and B-DNA, have been used for structures refinement by restrained molecular dynamics. Comparison of the natural oligomer with the abasic structure obtained earlier by NMR/rMD (Y. Coppel, N. Berthet, C. Coulombeau, Ce. Coulombeau, J. Garcia and J. Lhomme, Biochemistry 36, 4817-4830, 1997) confirms that the creation of an abasic site, in this sequence context, leads to marked helix kinking. It is also shown that the JUMNA procedure is capable of reproducing the overall structural features of the natural and damaged DNA conformations without the use of experimental constraints.     

Experimental and theoretical analysis of the invasive signal amplification reaction

The invasive signal amplification reaction is a sensitive method for single nucleotide polymorphism detection and quantitative determination of viral load and gene expression. The method requires the adjacent binding of upstream and downstream oligonucleotides to a target nucleic acid (either DNA or RNA) to form a specific substrate for the structure-specific 5' nucleases that cleave the downstream oligonucleotide to generate signal. By running the reaction at an elevated temperature, the downstream oligonucleotide cycles on and off the target leading to multiple cleavage events per target molecule without temperature cycling. We have examined the performance of the FEN1 enzymes from Archaeoglobus fulgidus and Methanococcus jannaschii and the DNA polymerase I homologues from Thermus aquaticus and Thermus thermophilus in the invasive signal amplification reaction. We find that the reaction has a distinct temperature optimum which increases with increasing length of the downstream oligonucleotide. Raising the concentration of either the downstream oligonucleotide or the enzyme increases the reaction rate. When the reaction is configured to cycle the upstream instead of the downstream oligonucleotide, only the FEN1 enzymes can support a high level of cleavage. To investigate the origin of the background signal generated during the invasive reaction, the cleavage rates for several nonspecific substrates that arise during the course of a reaction were measured and compared with the rate of the specific reaction. We find that the different 5' nuclease enzymes display a much greater variability in cleavage rates on the nonspecific substrates than on the specific substrate. The experimental data are compared with a theoretical model of the invasive signal amplification reaction.     

Experimental and theoretical microdialysis studies of in situ metabolism

Microdialysis sampling was performed to monitor localized metabolism in vivo and in vitro. A mathematical model that accounts for analyte mass transport during microdialysis sampling was used to predict metabolite concentrations in the microdialysis probe during localized metabolism experiments. The model predicts that metabolite concentrations obtained in the microdialysis probe are a function of different experimental parameters including membrane length, perfusion fluid flow rate, and sample diffusive and kinetic properties. Different microdialysis experimental parameters including membrane length and perfusion fluid flow rate were varied to affect substrate extraction efficiency (E(d)), or loss to the sample matrix, in vivo and in vitro. Local hepatic metabolism was studied in vivo in male Sprague-Dawley rats by infusing acetaminophen through the microdialysis probe. Acetaminophen sulfate concentrations increased linearly with respect to acetaminophen E(d) in contrast to modeling predictions. Xanthine oxidase was used as an in vitro model of localized metabolism. In vitro experimental results partially matched modeling predictions for 10-mm probes. These results suggest that monitoring local metabolism using microdialysis sampling is feasible. It is important to consider system parameters such as dialysis flow rate, membrane length, and sample properties because these factors will affect analyte concentrations obtained during local metabolism experiments.     

A simple assembly for anaerobic spectrophotometric reaction kinetics studies.

Details are given for the construction and use of a simple spectrophotometer cuvetteclosure that makes possible the study of reaction rates and enzyme assays under anaerobic conditions. The preparation of solutions and assembly of the cuvette unit is carried out in a N2 atmosphere in a glove box, but the spectrophotometric studies are conducted in a spectrophotometer with no modification except for the cell compartment cover.     

Experimental and theoretical studies of the structure of tellurate-borate glasses network

The structural properties of the xTeO₂·(1-x)B₂O₃ glasses (x = 0.6; 0.7) were investigated by FT-IR spectroscopy. From the analysis of the FTIR spectra, it is reasonable to assume that by the increasing of boron ions content, the tetrahedral [BO₄] units are gradually replaced by the trigonal [BO₃] units. The increase in the number of non-bridging oxygen atoms would decrease the connectivity of the glass network and will yield the depolymerization of the borate chains. The molecular structure and vibrational frequencies of the proposed structural models have been studied by exploring the density functional theory (DFT) calculations. The FTIR spectra of the xTeO₂·(1-x)B₂O₃ vitreous systems were compared with the calculated spectrum. This procedure allowed us to assign most of the observed IR bands.     

Bistability in the isocitrate dehydrogenase reaction: an experimentally based theoretical study.

The enzyme isocitrate dehydrogenase (IDH, EC 1.1.1.42) can exhibit activation by one of its products, NADPH. This activation is competitively inhibited by the substrate NADP+, whereas NADPH competes with NADP+ for the catalytic site. Experimental observations briefly presented here have shown that if IDH is coupled to another enzyme, diaphorase (EC 1.8.1.4), which transforms NADPH into NADP+, the system can attain either one of two stable states, corresponding to a low and a high NADPH concentration. The evolution toward either one of these stable states depends on the time of addition of diaphorase to the medium containing IDH and its substrate NADP+. We present a theoretical and numerical analysis of a model for the IDH-diaphorase bienzymatic system, based on the regulatory properties of IDH. The results confirm the occurrence of bistability for parameter values derived from the experiments. Depending on the total concentration of NADP+ plus NADPH and the concentration of IDH, the system can either admit a single steady state or display bistability. We obtain an expression for the critical time t*, before which diaphorase addition leads to the lower steady state and after which addition of the enzyme leads to the upper steady state of NADPH. The analysis is extended to the case where the second substrate of IDH, isocitrate, is consumed in the course of the reaction without being regenerated. Bistability occurs only as a transient phenomenon in these conditions.     

Experimental and theoretical studies of rate constant evaluation for the solute-matrix interaction in affinity chromatography.

In an investigation of the problem of determining kinetic parameters for the interaction of a solute with immobilized ligand sites on an affinity matrix, a combination of experimental studies and numerical simulations of frontal chromatography of methyl orange on Sephadex G-25 has yielded a simpler method than existing procedures for characterizing solute-matrix kinetics. A significant change in approach has entailed the direct evaluation of the kinetic contribution to boundary spreading from the flow-rate dependence of boundary variance under conditions of concentration-independent chromatographic migration (linear kinetics). This kinetic contribution is then interpreted in terms of an experimentally more appropriate form of a quantitative relationship for diffusion-free chromatographic migration (H. W. Hethcote and C. DeLisi, 1982, J. Chromatogr. 240, 269-281). Finally, the results of numerical simulations of concentration-dependent chromatographic migration (Langmuir kinetics) have indicated that rate constants should also be determinable under these conditions by extrapolation of their apparent values obtained by the above procedure to infinite dilution.     

Experimental and theoretical studies on the excess capacity of Photosystem II

It has been recently suggested that compensatory changes in Photosystem II (PS II) electron turnover rates can protect photosynthesis from photoinhibition [Behrenfeld et al. (1998) Photosynth Res 58: 259–268]. We have further explored this feature of PS II using a rate electrode for simultaneous measurements of the steady-state rate of oxygen evolution and the oxygen flash yield depending on the background irradiance in both control and photoinhibited algal cells of Chlorella Böhm. Theoretical simulations based on the two-electron gate model agree qualitatively with experimental data if we assume an increase of the electron turnover rate in the remaining functional PS II centers of the photoinhibited sample. Our results confirm the hypothesis that the compensatory effect enables cells to maintain the maximal rates of photosynthesis even in the presence of moderate photoinhibition (decrease of up to 50% in the number of functional centers) and that the effect originates from the inner capacity of electron transport through PS II. The origin of the compensatory effect is briefly discussed.     

Rapid biocatalytic polytransesterification: reaction kinetics in an exothermic reaction

Biocatalytic polytransesterification at high concentrations of monomers proceeds rapidly and is accompanied by an increase in the temperature of the reaction mixture due to liberation of heat of reaction during the initial phase. We have used principles of reaction calorimetry to monitor the kinetics of polymerization during this initial phase, thus relating the temperature to the extent of polymerization. Rate of polymerization increases with the concentration of monomers. This is also reflected by the increase in the temperature of the reaction mixture. Using time-temperature-conversion contours, a differential method of kinetic analysis was used to calculate the energy of activation ( approximately 15.1 Kcal/mol). Copyright 1998 John Wiley & Sons, Inc.