Partial characterization of peroxidase isoenzymes from rust-affected wheat leaves

Four anodic peroxidase isoenzymes from wheat leaves were purified by column chromatography and their kinetic behavior with common substrates were examined. One isoenzyme is more active in wheat resistant to stem rust fungi and differed from the others in carbohydrate content and also by a specific activity 2–4-fold higher with non-physiological electron donors. As a substrate, eugenol exhibited kinetic behavior different from p-phenylenediamine, guaiacol or o-dianisidine with all isoenzymes. All four isoenzymes showed similar pH and temperature optima and kinetic behavior and apparent K_m values for both H₂O₂ and non-physiological electron donors.     

Carbon capture and sequestration by a treatment wetland

An understanding of detailed kinetic of CO₂ removal by plants can lead to an effective design of the phytoremediation process for anthropogenic CO₂ reduction. This study examines the CO₂ removal rates of five wetland plants (Cyperus alternifolius, Dracaena fragrans, Iris ensata, Iris setosa and Thalia dealbata) by using saturation reaction and first-order reaction kinetic equations. It was determined that the elevation of CO₂ levels stimulated the plant-CO₂ uptake rate. The maximum CO₂ removal rates (k) of plants were found to range between 0.76 and 1.21 g m^?2 h^?1. The magnitude of first-order kinetic coefficient of plants (k′) had a close relationship with CO₂ level at half-velocity (K). For consistency, the same kinetics were applied to the continuous flow experiment. A saturation kinetic approach was well suited to estimate the removal rate of CO₂ in continuous flow system, while a first-order kinetic approach was limited to inflow CO₂ levels below 500 ppm.     

Investigating protein unfolding kinetics by pulse proteolysis

Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed “pulse proteolysis” to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins. Electrophoretic separation after proteolysis allows monitoring protein unfolding without protein purification. We employed pulse proteolysis to determine unfolding kinetics of E. coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H). The unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for unfolding of MBP•maltose complex, we have determined the dissociation equilibrium constant (K_d) of the complex from unfolding kinetic constants, which is also in good agreement with known K_d values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics.     

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. XI. Comparison of kinetic properties of the purified with the impure membrane-bound enzyme from Squalus acanthias

A variety of kinetic parameters have been compared in the membrane-bound and purified forms of the (sodium + potassium)-activated adenosinetriphosphatase (NaK ATPase) from the rectal gland of the spiny dogfish, Squalus acanthias. The kinetic parameters which have been studied have been temperature optima, pH optima, Mg-activation curves, optimum ATP/Mg ratios, K_m for ATP, ouabain-inhibition curves, and Na and K-activation curves. All kinetic parameters were remarkably similar for both forms of the enzyme. This encourages us to believe that information obtained from the pure enzyme can be extrapolated to the enzyme in its native membrane environment and should throw light on the molecular mechanism of Na and K transport.     

Kinetics of hydrogen peroxide decomposition by catalase: hydroxylic solvent effects

The effect of water–alcohol (methanol, ethanol, propan-1-ol, propan-2-ol, ethane-1,2-diol and propane-1,2,3-triol) binary mixtures on the kinetics of hydrogen peroxide decomposition in the presence of bovine liver catalase is investigated. In all solvents, the activity of catalase is smaller than in water. The results are discussed on the basis of a simple kinetic model. The kinetic constants for product formation through enzyme–substrate complex decomposition and for inactivation of catalase are estimated. The organic solvents are characterized by several physical properties: dielectric constant (D), hydrophobicity (log P), concentration of hydroxyl groups ([OH]), polarizability (α), Kamlet-Taft parameter (β) and Kosower parameter (Z). The relationships between the initial rate, kinetic constants and medium properties are analyzed by linear and multiple linear regression.     

Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors

The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1–3 follows a sequential ordered Bi–Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in k_cat for PKN1 and PKN2, and a decrease in peptide K_M for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (K_i<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.     

Equimolar interaction between calmodulin and the Ca2+ ATPase from human erythrocyte membranes

The interaction between calmodulin and the pure, solubilized Ca²⁺ ATPase from human erythrocyte membranes was examined by kinetic titration. The data indicated that the two proteins interacted in a molar ratio of 1:1 with a K_d of 4.2 nm. The dependence of enzyme activity on calmodulin concentration agreed quantitatively with that predicted by kinetic theory.     

Determination of the Catalytic Mechanism for Mitochondrial Malate Dehydrogenase

The kinetics of malate dehydrogenase (MDH) catalyzed oxidation/reduction of L-malate/oxaloacetate is pH-dependent due to the proton generated/taken up during the reaction. Previous kinetic studies on the mitochondrial MDH did not yield a consensus kinetic model that explains both substrate and pH dependency of the initial velocity. In this study, we propose, to our knowledge, a new kinetic mechanism to explain kinetic data acquired over a range of pH and substrate concentrations. Progress curves in the forward and reverse reaction directions were obtained under a variety of reactant concentrations to identify associated kinetic parameters. Experiments were conducted at physiologically relevant ionic strength of 0.17 M, pH ranging between 6.5 and 9.0, and at 25°C. The developed model was built on the prior observation of proton uptake upon binding of NADH to MDH, and that the MDH-catalyzed oxidation of NADH may follow an ordered bi-bi mechanism with NADH/NAD binding to the enzyme first, followed by the binding of oxaloacetate/L-malate. This basic mechanism was expanded to account for additional ionic states to explain the pH dependency of the kinetic behavior, resulting in what we believe to be the first kinetic model explaining both substrate and pH dependency of the reaction velocity.     

An error analysis for two-state protein-folding kinetic parameters and phi-values: progress toward precision by exploring pH dependencies on Leffler plots

The interpretation of φ-values has led to an understanding of the folding transition state ensemble of a variety of proteins. Although the main guidelines and equations for calculating φ are well established, there remains some controversy about the quality of the numerical values obtained. By analyzing a complete set of results from kinetic experiments with the SH3 domain of α-spectrin (Spc-SH3) and applying classical error methods and error-propagation formulas, we evaluated the uncertainties involved in two-state-folding kinetic experimental parameters and the corresponding calculated φ-values. We show that kinetic constants in water and m values can be properly estimated from a judicious weighting of fitting errors and describe some procedures to calculate the errors in Gibbs energies and φ-values from a traditional two-point Leffler analysis. Furthermore, on the basis of general assumptions made with the protein engineering method, we show how to generate multipoint Leffler plots via the analysis of pH dependencies of kinetic parameters. We calculated the definitive φ-values for a collection of single mutations previously designed to characterize the folding transition state of the α-spectrin SH3 domain. The effectiveness of the pH-scanning procedure is also discussed in the context of error analysis. Judging from the magnitudes of the error bars obtained from two-point and multipoint Leffler plots, we conclude that the precision obtained for φ-values should be ∼25%, a reasonable limit that takes into account the propagation of experimental errors.     

Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation

Industrial biocatalytic reduction processes require the efficient regeneration of reduced cofactors for the asymmetric reduction of prochiral compounds to chiral intermediates which are needed for the production of fine chemicals and drugs. Here, we present a new engineering strategy for improved NADH regeneration based on the Pichia pastoris methanol oxidation pathway. Studying the kinetic properties of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) and using the derived kinetic data for subsequent kinetic simulations of NADH formation rates led to the identification of FLD activity to constitute the main bottleneck for efficient NADH recycling via the methanol dissimilation pathway. The simulation results were confirmed constructing a recombinant P. pastoris strain overexpressing P. pastoris FLD and the highly active NADH-dependent butanediol dehydrogenase from S. cerevisiae. Employing the engineered strain, significantly improved butanediol production rates were achieved in whole-cell biotransformations.     

Biosorption of uranium by magnetically modified Rhodotorula glutinis

Adsorption of uranium from aqueous solution onto the magnetically modified yeast cell, Rhodotorula glutinis, was investigated in a batch system. Factors influencing sorption such as initial solution pH, biomass dosage, contact time, temperature, initial uranium concentration and other common cations were analyzed. Sorption isotherm, kinetic and thermodynamic studies of uranium on magnetically modified R. glutinis were also carried out. The temperature dependent equilibrium data agreed well with the Langmuir model. Kinetic data obtained at different temperatures were simulated using pseudo-first-order and pseudo-second-order kinetic models, the pseudo-second-order kinetic model was found to describe the data better with correlation coefficients near 1.0. The thermodynamic parameters, ΔH°, ΔS° and ΔG° were calculated from the sorption data gained at different temperatures. These thermodynamic parameters showed that the sorption process was endothermic and spontaneous. All results indicated that magnetically modified R. glutinis can be a potential sorbent for uranium wastewater treatment.     

Kinetic modelling of isomerization and anaerobic degradation of n- and i-butyrate

Very clear experimental evidence of isomerization between n- and i-butyrate during their anaerobic degradation was presented. A first experiment in the presence of bromoethane sulfonic acid (BESA), an inhibitor of methanogenesis, allowed the equilibrium distribution of n-, i-butyrate and acetate to be determined. To elucidate the mechanism of the isomerization process, a kinetic analysis was employed. The results suggested that i-butyrate was isomerized into n-butyrate prior to being oxidized to acetate. A mechanism for butyrate degradation, based on the values of the kinetic parameters obtained, was proposed.     

Comparative effect of different aerobic pretreatments on the kinetics and macroenergetic parameters of anaerobic digestion of olive mill wastewater in continuous mode

A comparative kinetic study was carried out on the anaerobic digestion of olive mill wastewater (OMW) and OMW that was previously fermented with Geotrichum candidum, Azotobacter chroococcum and Aspergillus terreus. The reactors used were continuously fed and contained sepiolite as support for the mediating bacteria. A kinetic model for multicomponent substrate removal by anaerobic digestion has been used. The model is based on the linear removal concept which is a special case of the broader Monod equation. The second-order kinetic constant, k _{2( s )}, was found to be influenced by the pretreatment carried out, and was 4.2, 4.0 and 2.5 times higher for Aspergillus, Azotobacter and Geotrichum-pretreated OMWs than that obtained in the anaerobic digestion of untreated OMW. This was significant at 95% confidence level. This behaviour is believed to be due to the lower levels of phenolic compounds and biotoxicity present in the pretreated OMWs. In fact, the kinetic constant increased when the phenolic compound content and biotoxicity of the pretreated OMWs decreased. In addition, the macroenergetic parameters of the anaerobic digestion of OMW, i.e. the specific rate of substrate uptake for cell maintenance, m, and the yield coefficient for the biomass, Y, decreased by a factor of 2.4, 3.6 and 5.1 and increased by a factor of 1.9, 2.2 and 2.4 respectively, for the OMWs previously treated with Geotrichum candidum, Azotobacter chroococcum and Aspergillus terreus in relation to the observed values for the untreated OMW.     

An improved kinetic model for lactic acid fermentation

A kinetic model for the production of lactic acid by Lactobacillus delbruckii from glucose has been developed using the batch kinetic data of Luedeking. This model incorporates the inhibitory effects of undissociated lactic acid and of hydrogen ion concentration upon cellular growth and production processes.     

Steady-state kinetics of indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis

Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for MtIGPS-catalized chemical reaction is proposed.     

Peroxidase-mediated oxidation of l-dopa: A kinetic approach

A kinetic model able to adequately describe the accumulation of p-topaquinone in peroxidase-mediated oxidation of l-dopa was developed, and the rate constants for both enzymatic and non-enzymatic branch were estimated either experimentally or using a computing program for detailed kinetic simulation. It is demonstrated that the accumulation of p-topaquinone in significant amounts occurred in excess of hydrogen peroxide or during auto-oxidation of l-dopa, but changes in the ratio of initial concentrations of the reactants can reduce considerably the production of this intermediate.     

Chemo-enzymatic asymmetric synthesis of S-citalopram by lipase-catalyzed cyclic resolution and stereoinversion of quaternary stereogenic center

A chemo-enzymatic synthesis method of S-citalopram was developed to overcome the disadvantage of relatively low selectivity of enzyme towards tertiary alcohols. The combination of kinetic resolution, cyclic resolution and stereoinversion synthesis was successfully applied in the asymmetric synthesis of the S-citalopram. Using the kinetic model to predict the cyclic resolution, R-diol with high ee value was obtained by controlling the conversion rate. Subsequently, the unwanted R-diol was inverted to S-citalopram by stereoinversion of chiral quaternary center with 98.0 % yield and ee value of 91.0 %. Based on dynamic simulation and experiments, the kinetic resolution was scaled up from 10 mL to 1 L and 14 L, gradually. There was no significant scale-up effect and the dynamic simulation result fitted the experimental data well, with an error of 12.5 and 14.0 %, respectively. This chemo-enzymatic synthesis route is a promising model system for the production of pharmaceuticals with the chiral tertiary alcohols intermediate.     

A kinetic characterization of the rabbit plasmin isozymes.

Steady-state and presteady-state kinetic parameters for plasmins derived from the two rabbit plasminogen isozymes have been determined. Steady-state kinetic experiments with N-α-tosyl-l-arginine methyl ester indicate that each isozyme has a similar V. Plasmin isozyme 2 has a higher K_m value for this substrate as well as a higher K_i, for the competitive inhibitor, benzamidine-HCl. Presteady-state kinetic experiments, using the p-nitrophenyl esters of p-(methylethylsulfoniummethyl)benzoate, p-(pyridiniummethyl) benzoate, p-(thiouroniummethyl)benzoate and p-(guanidinium)benzoate, indicate that each plasmin has similar rate constants of acylation (k₂). However, values of the dissociation constant (K_S) indicate that plasmin isozyme 1 has a greater binding affinity for these substrates than does isozyme 2. The magnitude of this difference varies with the substrate and is the greatest for those containing analogs of the guanidino moiety of l-arginine.     

Kinetics of Mixed Microbial Assemblages Enhance Removal of Highly Dilute Organic Substrates

Our experiments with selected organic substrates reveal that the rate-limiting process governing microbial degradation rates changes with substrate concentration, S, in such a manner that substrate removal is enhanced at lower values of S. This enhancement is the result of the dominance of very efficient systems for substrate removal at low substrate concentrations. The variability of dominant kinetic parameters over a range of S causes the kinetics of complex assemblages to be profoundly dissimilar to those of systems possessing a single set of kinetic parameters; these findings necessitate taking a new approach to predicting substrate removal rates over wide ranges of S.