A semi-integrated method for the determination of enzyme kinetic parameters and graphical representation of the Michaelis-Menten equation

A semi-integrated method for the determination of the enzyme kinetics parameters (Km and V) and graphical representation of the Michaelis-Menten equation is proposed as a variation of determination of initial reaction rate (v) as a function of initial substrate concentration ([S]0). The method is based on the determination of the time required to exhaust half of the initial substrate concentration as a function of the initial substrate concentration. The advantages and limitations of this method are discussed.     

A method for determining kinetic parameters at high enzyme concentrations.

A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.     

A microcomputer method for designing optimal experiments for estimating enzyme kinetic parameters

A computer program, written in BASIC, for designing optimal experiments with the aim of evaluating estimates of the parameters for any enzyme kinetic model is given. This computer program can be run on any microcomputer with less than 32 Kbytes of random access memory. The program uses the termed D-optimization design criterion, which minimizes the determinant of the variance-covariance matrix. The user only supplies the rate equation, the maximum and minimum concentrations of substrates and inhibitors, the weighting pattern, and the best possible values of the parameters. The computer supplies the optimal substrate and inhibitor concentrations (one for each parameters), for estimating the parameter values, and the determinant of the variance-covariance matrix. Likewise, the microcomputer supplies the eigenvalues and eigenvectors of information and redundancy matrices, the sensitivity and the global redundancy.     

A flow method for determination of the kinetic parameters for immobilized enzymes.

A flow method is described for determination of the kinetics parameters (V-m and K-m) for enzymes that are bound to particles, to membranes, and to the interior surfaces of tubes. Substrate solution is pumped through Tygon tubing to a microvolume flow cell and back into the reaction mixture, the flow rate being adjusted to be faster than the rate of formation of product. To illustrate the technique, it is applied to the determination of the parameters for electric-eel acetylcholinesterase attached to particles, to membranes, and to the inner surface of nylon tubing.     

A new, sensitive method for enzyme kinetic studies of scarce glucosides

The maize β-glucosidase Zm-p60.1 is important for the regulation of plant development through its role in the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. Enzyme kinetics studies using these scarce substrates close to physiological concentrations are difficult due to two reasons: (a) Available methods are mainly suited for end-point kinetics. (b) These methods are not sufficiently sensitive when using scarce glucoside substrates.We developed a glucose assay using a system comprising three enzymes β-glucosidase, glucose oxidase and horseradish peroxidase, with the new substrate N-acetyl-3,7-dihydroxyphenoxazine-Amplex Ultra Red reagent (Molecular Probes). A calibration curve was constructed for resorufin and validation was carried out by comparing our method with the standard spectrophotometric method using p-nitrophenyl-β-d-glucopyranoside. In comparison with the other methods, this method is more sensitive, precise and accurate. The assay is rapid and hence suited for continuous kinetics, it is readily adapted to suit automated procedures, and potential applications include its use in studying the physiological role(s) of enzymes that cleave scarce glucoside substrates.     

The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters

A new plot is described for analysing the results of kinetic experiments in which the Michaelis-Menten equation is obeyed. Observations are plotted as lines in parameter space, instead of points in observation space. With appropriate modifications the plot is applicable to most problems of interest to the enzyme kineticist. It has the following advantages over traditional methods of plotting kinetic results: it is very simple to construct, because it is composed entirely of straight lines and requires no calculation or mathematical tables; the kinetic constants are read off the plot directly, again without calculation; it may be used during the course of an experiment to judge the success of the experiment, and to modify the experimental design; it provides clear and accurate information about the quality of the observations, and identifies aberrant observations; it provides a clear indication of the precision of the kinetic constants; constructed with care, it provides unbiased estimates of the kinetic constants, the same as those provided by a computer program; it may be used to simulate results for illustrative purposes very rapidly and simply.     

A kinetic method for determination of arylsulfatase activity

A new assay for arylsulfatase activity is described, which consists of direct kinetic measurements of pseudo-first-order rate constants by means of a spectrophotometric procedure. The assay is applicable for reactions occurring at different pH conditions and it can be used for a wide range of activities.     

Influence of the experimental setup on the determination of enzyme kinetic parameters

For the design of bioconversion processes parallel experimentation in microtiter plates is commonly applied to reduce the experimental load, although data accuracy and reproducibility are often reduced. In an effort to quantify the impact of different microscale experimental systems on the estimation of enzyme kinetic parameters from progress curves, we comprehensively evaluated the enzymatic reduction of acetophenone in both open and closed polystyrene and quartz microtiter plates as well as quartz cuvettes. Differences in conversion of up to 50% over time were observed increasing from polystyrene MTPs to quartz MTPs to quartz cuvettes. Initial reaction velocities increased systematically from polystyrene to quartz MTPs and cuvettes. The experimental errors decreased in the same order showing highest experimental error of about 20% in polystyrene. We further evaluated reasons causing the deviations within one system as well as between the systems. The choice of reaction vessel material, temperature effects and substrate cross contaminations in MTPs were shown to be of importance in the experimental results. Although the experimental data differed between the reaction vessels, no distinct trends in estimated kinetic parameters were found. While the microkinetic parameters vary up to an order of magnitude between different systems, the corresponding macrokinetic parameters lie in the same range for all systems varying by 29–118%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:87–95, 2017     

Principle and method of kinetic microphotometric enzyme activity determination in situ

Summary An advanced apparative set-up is described for multipositional microphotometric recording of histochemical enzyme reactions in cryostat sections. It consists of a computer controlled microscope photometer with scanning stage. Measurements on the same tissue section may be performed at 12 preselected positions. These are repeatedly brought into the measuring beam in several measuring cycles. The complete measuring process, storage of measuring position coordinates, movements of the stage and statistical evaluation of the data is under computer control. By use of the gel film technique, extinction changes in tetrazolium coupled enzyme reactions can be measured continuously at initial rate conditions. Measurements are performed at identical conditions and can thus be analysed as relative enzyme activities.     

Principle and method of kinetic microphotometric enzyme activity determination in situ

An advanced apparative set-up is described for multipositional microphotometric recording of histochemical enzyme reactions in cryostat sections. It consists of a computer controlled microscope photometer with scanning stage. Measurements on the same tissue section may be performed at 12 preselected positions. These are repeatedly brought into the measuring beam in several measuring cycles. The complete measuring process, storage of measuring position coordinates, movements of the stage and statistical evaluation of the data is under computer control. By use of the gel film technique, extinction changes in tetrazolium coupled enzyme reactions can be measured continuously at initial rate conditions. Measurements are performed at identical conditions and can thus be analysed as relative enzyme activities.     

Evaluation of distribution-free confidence limits for enzyme kinetic parameters

Monte Carlo experiments have been used to test the robustness of distribution-free confidence limits for the parameters of the Michaelis-Menten equation (Porter & Trager, 1977). When used in conjunction with the modified form of the direct linear plot (Cornish-Bowden & Eisenthal, 1978), they prove to be more robust than least-squares confidence limits. In circumstances where the least-squares assumptions are correct, the distribution-free confidence limits define the parameters somewhat less precisely than the corresponding least-squares confidence limits, but this effect is negligible unless there are eight or fewer observations.     

A linearization method for low catalytic activity enzyme kinetic analysis

A kinetic analysis was made and a linear plot based on the general rate equation derived by Laidler [Can. J. Chem. 33, 1614-1624] is proposed. This linearization method allows determining the kinetic parameters (K(m), k(cat)) and [E](0) for enzymes with low catalytic activity. The method was applied to chloroperoxidase from Caldariomyces fumago [EC 1.11.1.10], whose kinetic parameters K(m)(app), k(cat)(app), and [E](0) with monochlorodimedone as substrate, were obtained by using the linearization plot and the V(max) value (calculated by Eadie-Hofstee plot). This plot could also be useful to the study of abenzyme kinetics provided the concentration of the latter is either higher or equal than K(m) value.     

A new kinetic assay method for quinone-reducing enzymes

A new kinetic method is described for the assay of quinone-reducing enzymes in various biological materials. It is based on polarographic determination of oxygen uptake in spontaneous oxidation of the diphenol formed as a result of 4-anilino-5-methoxybenzoquinone-1,2 (AMOBQ) enzymic reduction. The stoichiometry of the reducing equivalent transfer in the reaction sequence from NAD(P)H to oxygen has been analyzed. Data are presented on quinone-reducing activity distributions in different tissues.     

A new amperometric enzyme electrode for alcohol determination

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.     

Ill-conditioning associated with the "end-point" method for the determination of kinetic parameters describing irreversible enzyme inactivation by an unstable inhibitor

Study of the complete time-course of irreversible enzyme inhibition by an unstable inhibitor yields more information than can be obtained by recording data only at the end point of reaction. Time-course analysis of co-operative irreversible enzyme inhibition by an unstable inhibitor has been shown to be considerably less susceptible to ill-conditioning than the "end-point" method for the determination of kinetic parameters describing inactivation. As a result, mechanisms that cannot be distinguished by the "end-point" method are readily differentiated by time-course analysis without the need to isolate intermediate species.     

A new plot for the kinetic analysis of dead-end enzyme inhibitors

A new procedure to characterize reversible dead-end inhibitors is presented. Preliminary identification of the inhibitor type is made by plotting vo/vi against the inhibitor concentration at different substrate concentrations. The inhibition constants for competitive, uncompetitive and mixed dead-end inhibitors are determined by secondary plots of l/(slope) vs [S], l/(slope) vs l/[S] and (slope)(Ks + [S] vs [S] respectively. These secondary plots render straight lines only for their corresponding type of inhibitor. For noncompetitive inhibitors all the secondary plots used yield straight lines. Therefore, the application of this plotting procedure leads to unambiguous diagnosis of the inhibitor type. An important feature of the procedure presented here is that the variable used (vo/vi) is independent on Vmax values. Therefore, experimental values obtained from enzyme preparations showing significant differences in their specific activities -i.e. enzyme coming from different purification steps- can be used.     

Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method

An isoconversional method is proposed in order to calculate the kinetic parameters of enzyme inactivation. The method provides an efficient and low-cost procedure to describe both operational and thermal inactivation. Unlike the ordinary kinetic assays performed at constant enzyme concentration and at various substrate concentrations, the isoconversional method requires several extended kinetic curves for constant initial substrate concentration and different enzyme concentrations. The procedure was tested and validated using simulated data obtained for several kinetic models frequently discussed in the literature. After the validation, the isoconversional method was used for the investigation of the thermoinactivation of urease during urea hydrolysis in self buffered medium and the operational inactivation (destructive oxidation by excess peroxide) of catalase at high concentration of hydrogen peroxide. The results showed that the isoconversional method gives good results of global inactivation constant for both simple and more complex models.     

Evaluation of true kinetic parameters for reversible immobilized enzyme reactions

For a reversible one-substrate reaction system that follows the Haldane reaction mechanism, a new and effective method has been proposed to extract true or intrinsic kinetic parameters of immobilized enzymes from diffusion limited rate data. The method utilizes the effectiveness factors correlated in terms of the general modulus defined by Aris and Bischoff, and a new modulus defined in the present study. It requires a trial-and-error calculation, but only a few data points. Furthermore, it provides a saving of materials such as substrates and enzymes, and takes less time for experiments compared to the initial rate methods. The usefulness of the method is demonstrated by determining the kinetic parameters for membrane bound fumarase which catalyzes the reaction of the conversion of fumarate to L-malate, for which the equilibrium constant is ca. 4.