Kinetic model for enzymatic hydrolysis in reverse micelles

A bound water model is developed for the interpretation of kinetic data of b-galactosidase in reverse micelles. Assessing the kinetic parameters of p-nitrophenyl-b-D-galactopyranoside hydrolysis in aqueous and reverse micellar system reveals that the major effect on hydrolytic rate is owing to the amount of free water in reverse micelles, not the enzyme molecules' structural change.     

Kinetic and enzymatic adsorption model in a recirculation hollow-fibre bioreactor

A general procedure has been developed to model the behaviour of enzymatic reactions in a membrane bioreactor. This procedure unifies the kinetics of the reaction and the adsorption of the enzyme or enzymatic complexes on the membrane, enabling the selection of the most appropriate kinetic model. The general procedure proposed has been particularized and applied to experimental results obtained with two enzymatic reactions carried out in a hollow-fibre reactor, enzymatic hydrolysis of lactose by β-galactosidase and glucose–fructose isomerization by glucose isomerase. The application of the general model has allowed us to determine the mechanism of the reaction for both kinetic reactions, assuming the adsorption of the enzymatic complex EGa for lactose hydrolysis and the adsorption of the free enzyme onto the membrane for glucose–fructose isomerization.     

A model for enzymatic isomerization of D-glucose to D-fructose in a batch reactor.

Using whole cells containing glucose isomerase, mathematical models for the enzymatic conversion of D-glucose to D-fructose and for the inactivation of the enzyme catalyst have been postulated and verified experimentally. The heat of reaction, the equilibrium constant, and the individual rate constants and their activation energies have been estimated. The model can be used to predict the time course for the enzymatic production of fructose in a batch reactor within the tested experimental range of 40-80 degrees C.     

Kinetic model of enzymatic hydrolysis of steam-exploded wheat straw

The hydrolysis kinetics of steam-exploded wheat straw treated with cellulase NS 50013 enzyme complex in combination with β-glucosidase NS 50010 is studied. The time dependence of the reducing sugars amount is followed at varying the temperature value and the amount of the enzyme introduced. The activation energy determined on the ground of the rate temperature dependence stays unchanged in the course of the process. The preexponential factor decreases with the increase of the degree of hydrolysis and is responsible for the process rate decrease. A new expression for the dependence of degree of hydrolysis of one of carbohydrate polymers (cellulose) in wheat straw on the time, the enzyme concentration and the temperature is obtained. It is of practical importance as well because it provides estimation of the degree of hydrolysis required at predetermined values of the temperature, the enzyme concentration and the time used. The expression can be used for control of the enzyme hydrolysis of cellulose in the wheat straw.     

Kinetic model of laccase-catalyzed oxidation of aqueous phenol

Laccase from Trametes versicolor (EC 1.10.3.2) catalyzes the oxidation of aqueous phenol by oxygen and has demonstrated good potential for applications in various industrial and environmental processes. A kinetic model of this system has been developed to facilitate a better understanding of the mechanisms and rate-limiting steps of enzyme-catalyzed transformation and to eventually assist in the choice and design of suitable reactor systems. A kinetic model was derived based on the differential and mass balance equations that describe the interactions of various forms of the enzyme with the aromatic substrate and oxygen. This model also incorporated an expression accounting for enzyme inactivation over time due to the reaction environment. The model was validated by comparing model predictions with experimental observations of phenol transformation and oxygen consumption over time at a variety of enzyme concentrations. Excellent agreement was found between experimental data and predictions of the kinetic model. Sensitivity analyses demonstrated that the reaction between oxidized-laccase and phenol was the rate-limiting step.     

Kinetic model of disaccharide oxidation by Agrobacterium tumefaciens

Disaccharides were microbaially transformed to their corresponding 3-keto-derivatives by resting cells of Agrobacterium tumefaciens NCPPB 396. The kinetics and yield of this highly specific oxidation depend on several factors. The oxygen concentration especially has a major influence on the production of 3-keto-derivatives and was investigated kinetically with respect to low stationary oxygen concentrations in solution. Experiments showed unconventional results that conflicted with normal Michaelis-Menten kinetics. A kinetic model was developed and the kinetic constants were calculated. The model and experimental data for sucrose, maltose, iso-maltulose (palatinose), and leucrose are in good agreement with each other. Initial reaction rates with different sugars using constant oxygen concentrations resulted in a Michaelis-Mentent type function. The complete kinetics, including the effect of disaccharide and oxygen concentrations, are presented. (c) 1995 John Wiley & Sons, Inc.     

Anomeric specificity of D-glucose phosphorylation and oxidation in human erythrocytes

1. In human erythrocytes, alpha-D-[U-14C]glucose is more efficiently oxidized than beta-D-[U-14C]glucose at a low concentration of the hexose (0.1 mM), but not so at higher glucose concentrations. 2. This unexpected situation may be attributable in part to the lower Km of hexokinase for alpha- than beta-D-glucose, this difference in affinity compensating for the higher maximal velocity found with the beta- rather than alpha-anomer. 3. A contributive role for aldose reductase in the anomeric control of D-glucose 6-phosphate circulation in the pentose phosphate pathway should not be ruled out, since aldose reductase inhibitors decrease the production of 14CO2 by erythrocytes exposed to D-[U-14C]glucose. 4. Nevertheless, the essential role of hexokinase in such an anomeric control is supported by the finding that, in the presence of menadione, which augments considerably D-[U-14C]glucose oxidation but fails to affect D-[5-3H]glucose utilization, the anomeric alpha/beta ratio in 14CO2 production from D-[U-14C]glucose follows, at increasing concentrations of the hexose, the same pattern as that found for its phosphorylation.     

Structural model of the mAb 806-EGFR complex using computational docking followed by computational and experimental mutagenesis

In this work, we combined computational protein-protein docking with computational and experimental mutagenesis to predict the structure of the complex formed by monoclonal antibody 806 (mAb 806) and the epidermal growth factor receptor (EGFR). We docked mAb 806, an antitumor antibody, to its epitope of EGFR residues 287-302. Potential mAb 806-EGFR orientations were generated, and computational mutagenesis was used to filter them according to their agreement with experimental mutagenesis data. Further computational mutagenesis suggested additional mutations, which were tested to arrive at a final structure that was most consistent with experimental mutagenesis data. We propose that this is the EGFR-mAb 806 structure, in which mAb 806 binds to an untethered form of the receptor, consistent with published experimental results. The steric hindrance created by the antibody near the EGFR dimer interface interferes with receptor dimerization, and we postulate this as the structural origin for the antitumor effect of mAb 806.     

Cytochrome c sorption-desorption effects on the external NADH oxidation by mitochondria: experimental and computational study

The rupture of the outer mitochondrial membrane is known to be critical for cell death, but the mechanism, specifically its redox-signaling aspects, still needs to be studied in more detail. In this work, the external NADH oxidation by rat liver mitochondria was studied under the outer membrane rupture induced by the mitochondria hypotonic treatment or the inner membrane permeability transition. The saturation of the oxidation rate was observed as a function of mitochondrial protein concentration. This effect was shown to result from cytochrome c binding to the mitochondrial membranes. At a relatively high concentration of mitochondria, the oxidation rate was strongly activated by 4 mm Mg(2+) due to cytochrome c desorption from the membranes. A minimal kinetic model was developed to explain the main phenomena of the external NADH oxidation modulated by cytochrome c and Mg(2+) in mitochondria with the ruptured outer membrane. The computational behavior of the model closely agreed with the experimental data. We suggest that the redox state of the released cytochrome c, considered by other authors to be important for apoptosis, may strongly depend on its oxidation by the fraction of mitochondria with the ruptured outer membrane and on the cytoplasmic cytochrome c reductase activity.     

Kinetic studies of the enzymatic isomerization of xylose

d-Xylose isomerase catalyses the conversion of the common pentose, d-xylose, to its keto-isomer, d-xylose. This reaction is of interest because many microorganisms that are unable to metabolize d-xylose can utilize d-xylulose. The kinetics of a commonly used immobilized whole-cell isomerase, Sweetzyme Q, have been determined from initial rate studies on the forward and reverse reactions. The effect of pH, temperature, and substrate and product concentration on enzyme activity have all been examined. Reaction rates were modelled with the Michaelis-Menten equation. Using constants determined from Lineweaver-Burk plots, the rate equation accurately simulated experimental conversion data.     

Robustness analysis and behavior discrimination in enzymatic reaction networks

Characterizing the behavior and robustness of enzymatic networks with numerous variables and unknown parameter values is a major challenge in biology, especially when some enzymes have counter-intuitive properties or switch-like behavior between activation and inhibition. In this paper, we propose new methodological and tool-supported contributions, based on the intuitive formalism of temporal logic, to express in a rigorous manner arbitrarily complex dynamical properties. Our multi-step analysis allows efficient sampling of the parameter space in order to define feasible regions in which the model exhibits imposed or experimentally observed behaviors. In a first step, an algorithmic methodology involving sensitivity analysis is conducted to determine bifurcation thresholds for a limited number of model parameters or initial conditions. In a second step, this boundary detection is supplemented by a global robustness analysis, based on quasi-Monte Carlo approach that takes into account all model parameters. We apply this method to a well-documented enzymatic reaction network describing collagen proteolysis by matrix metalloproteinase MMP2 and membrane type 1 metalloproteinase (MT1-MMP) in the presence of tissue inhibitor of metalloproteinase TIMP2. For this model, our method provides an extended analysis and quantification of network robustness toward paradoxical TIMP2 switching activity between activation or inhibition of MMP2 production. Further implication of our approach is illustrated by demonstrating and analyzing the possible existence of oscillatory behaviors when considering an extended open configuration of the enzymatic network. Notably, we construct bifurcation diagrams that specify key parameters values controlling the co-existence of stable steady and non-steady oscillatory proteolytic dynamics.     

A kinetic study of D-glucose oxidation by bromine in aqueous solutions

The kinetics of the oxidation of D-glucose to D-gluconic acid by bromine in aqueous solution were studied using potentiometric techniques and theoretical considerations of complex bromine-bromide-pH equilibria. The pH has a strong influence on reaction rate. At pH < 8 the reaction is very slow, while in the pH range pH 8-9.5 the reaction is sufficiently fast and seems optimal for the reaction. The proposed active species at that pH region is hypobromous acid. At pH > 9.5, the reaction is further accelerated due to the formation of hypobromite. The proposed kinetics expression for gluconic acid formation, based on the determined kinetic parameters at pH 9.24, is of the form dc(GA)/dt = 160c(2)(G)c(o)(HOBr)c(o)(H(+)c(o)(Br)     

Substrate diffusion and oxidation in GMC oxidoreductases: an experimental and computational study on fungal aryl-alcohol oxidase

AAO (aryl-alcohol oxidase) provides H?O? in fungal degradation of lignin, a process of high biotechnological interest. The crystal structure of AAO does not show open access to the active site, where different aromatic alcohols are oxidized. In the present study we investigated substrate diffusion and oxidation in AAO compared with the structurally related CHO (choline oxidase). Cavity finder and ligand diffusion simulations indicate the substrate-entrance channel, requiring side-chain displacements and involving a stacking interaction with Tyr?2. Mixed QM (quantum mechanics)/MM (molecular mechanics) studies combined with site-directed mutagenesis showed two active-site catalytic histidine residues, whose substitution strongly decreased both catalytic and transient-state reduction constants for p-anisyl alcohol in the H502A (over 1800-fold) and H546A (over 35-fold) variants. Combination of QM/MM energy profiles, protonation predictors, molecular dynamics, mutagenesis and pH profiles provide a robust answer regarding the nature of the catalytic base. The histidine residue in front of the FAD ring, AAO His??2 (and CHO His???), acts as a base. For the two substrates assayed, it was shown that proton transfer preceded hydride transfer, although both processes are highly coupled. No stable intermediate was observed in the energy profiles, in contrast with that observed for CHO. QM/MM, together with solvent KIE (kinetic isotope effect) results, suggest a non-synchronous concerted mechanism for alcohol oxidation by AAO.     

Kinetic model studies on the chemical ligation of oligonucleotides via hydrazone formation

We report on the suitability of hydrazone formation for activator-free ligation of oligonucleotides. 5'-Acyl hydrazides were synthesized using a previously described phosphoramidite modifier, whereas 3'-hydrazides resulted from a hydrazinolysis of an ester group serving as a linker to the solid support. Aromatic aldehydes could be directly introduced on the 5'-terminus via the respective phosphoramidates. Aliphatic aldehydes were generated by periodate cleavage of the corresponding 3'- and 5'-modified diol precursors. Ligation of a 3'-hydrazide-modified oligonucleotide with oligonucleotides bearing an aromatic aldehyde in 5'-position showed a fast reaction kinetics (k(1) about 10(-1) M(-1)s(-1)) [corrected] and irreversible hydrazone formation. The ligation of a 5'-hydrazide-modified oligonucleotide and a 3'-ribobisaldehyde appeared to proceed reversibly at the beginning, but became irreversible with increasing reaction time. Hydrazide-modified oligonucleotides were found to be somewhat unstable in aqueous solutions.     

Kinetic model of elemental sulfur oxidation by Thiobacillus thiooxidans in batch slurry reactors

The kinetics of sulfur oxidation by T. thiooxidans has been studied in a batch well-mixed reactor and in shaker flasks. A mathematical model is proposed, which considers the attachment of the cells onto the sulfur particles' surface following Freundlich isotherm, growth of the attached bacteria, and growth inhibition by sulfates accumulation. Best-fit values of the model parameters have been calculated from the experimental data. Results show that the addition of dimethyl-dichloro-silane in the aerated reactor to prevent the formation of foam reduces the maximum specific growth rate of attached bacteria, probably because of the resulting changes in surface properties of the sulfur particles. The other model parameters are not significantly affected. The formation of clusters of sulfur particles has been observed at an initial sulfur concentration of 5% . This phenomenon reduces the rate of sulfur conversion due to the reduction of the total surface area of the particles, and the model therefore over-estimates the formation of sulfates. At lower initial sulfur concentration, the phenomenon has not been observed and the model simulations are then satisfactory.     

Kinetic modeling of the enzymatic hydrolysis of pretreated cellulose

The production of sugars by the enzymatic hydrolysis of cellulose is a two-step process that includes conversion of the intermediate cellobiose to glucose by beta-glucosidase. The hydrolysis was followed by analyzing the two sugar products (cellobiose and glucose). The enzyme showed maximum activity at pH 4.8. Thermal deactivation was significant at temperatures above 45 degrees C. At 50 degrees C (optimum temperature) thermal deactivation was found to follow first-order kinetics. Several models were tested by modeling the kinetics of the reaction. Their parameter values were determined by numerical optimization, including temperature dependence. The best fitting model was a competitive product inhibition for the two reactions in the operational range.     

Kinetic determinants of high-fidelity tRNA discrimination on the ribosome

The ribosome selects aminoacyl-tRNA (aa-tRNA) matching to the mRNA codon from the bulk of non-matching aa-tRNAs in two consecutive selection steps, initial selection and proofreading. Here we report the kinetic analysis of selection taking place under conditions where the overall selectivity was close to values observed in vivo and initial selection and proofreading contributed about equally. Comparison of the rate constants shows that the 350-fold difference in stabilities of cognate and near-cognate codon-anticodon complexes is not used for tRNA selection due to high rate of GTP hydrolysis in the cognate complex. tRNA selection at the initial selection step is entirely kinetically controlled and is due to much faster (650-fold) GTP hydrolysis of cognate compared to near-cognate substrate.