Enzyme kinetics characterization

Zhang Z-Y, Poorman RA, Maggiora LL et al (1991) Dissociative inhibition of dimeric enzymes. Kinetic characterization of the inhibition of HI V-1 protease by its COOH-terminal tetrapeptide. J Biol Chem 266 15591-15594... 

TABLE 13.3 Data transformations for enzyme kinetic characterization. 

The Michaehs-Menten equation and other similar nonhnear expressions characterize immobihzed enzyme kinetics. Therefore, for a spherical porous carrier particle with enzyme molecules immobilized on its external as well as internal surfaces, material balance of the substrate will result in the following ... 

Fleischmann, R, K. Studer et al. (2002). Partial purification and kinetic characterization of a carotenoid cleavage enzyme from quince fruit (Cydonia oblonga). J. Agric. Food Chem. 50(6) 1677-1680. 

Volume 354. Enzyme Kinetics and Mechanisms (Part F Detection and Characterization of Enzyme Reaction Intermediates)... 

The next section describes the utilization of //PLC for different applications of interest in the pharmaceutical industry. The part discusses the instrumentation employed for these applications, followed by the results of detailed characterization studies. The next part focuses on particular applications, highlighting results from the high-throughput characterization of ADMET and physicochemical properties (e.g., solubility, purity, log P, drug release, etc.), separation-based assays (assay development and optimization, real-time enzyme kinetics, evaluation of substrate specificity, etc.), and sample preparation (e.g., high-throughput clean-up of complex samples prior to MS (FIA) analysis). 

This section mainly builds upon classic biochemistry to define the essential building blocks of metabolic networks and to describe their interactions in terms of enzyme-kinetic rate equations. Following the rationale described in the previous section, the construction of a model is the organization of the individual rate equations into a coherent whole the dynamic system that describes the time-dependent behavior of each metabolite. We proceed according to the scheme suggested by Wiechert and Takors [97], namely, (i) to define the elementary units of the system (Section III. A) (ii) to characterize the connectivity and interactions between the units, as given by the stoichiometry and regulatory interactions (Sections in.B and II1.C) and (iii) to express each interaction quantitatively by... 

In conclusion, this approach was more or less a characterization method adopted from a biochemistry (enzyme kinetics) textbook. 

Thereafter, a reference text such as Enzyme Kinetics (Segel, 1993) should be consulted to determine whether or not the proposed mechanism has been described and characterized previously. For the example given, it would be found that the proposed mechanism corresponds to a system referred to as partial competitive inhibition, and an equation is provided which can be applied to the experimental data. If the data can be fitted successfully by applying the equation through nonlinear regression, the proposed mechanism would be supported further secondary graphing approaches to confirm the mechanism are also provided in texts such as Enzyme Kinetics, and values could be obtained for the various associated constants. If the data cannot be fitted successfully, the proposed reaction scheme should be revisited and altered appropriately, and the whole process repeated. 

The GRK family consists of seven well-characterized enzymes. These enzymes are distinguished by (1) the structural homology within the family (2) the specific amino acid sequences that a given GRK can phosphorylate (3) enzyme kinetics (169,181) and (4) GPCR disease phenotypes that are often manifested by dysregula-tion of GRK activity. Gain-of-function GPCR mutations are frequently found to be constitutively phosphorylated. Conversely, inadequate receptor desensitization and sequestration often result. 

Our spectral and kinetic characterization of the oxidized intermediates of MnP compound I (MnPI), and MnP compound II (MnPII) indicate (Fig. 1) that the oxidation states and catalytic cycle of MnP are similar to LiP and HRP (6,15,16,19). MnPl contains two oxidizing equivalents over the native feme enzyme. The first equivalent resides in the Fe =0... 

Yang and Schulz also formulated a treatment of coupled enzyme reaction kinetics that does not assume an irreversible first reaction. The validity of their theory is confirmed by a model system consisting of enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) with 2,4-decadienoyl coenzyme A as a substrate. Unlike the conventional theory, their approach was found to be indispensible for coupled enzyme systems characterized by a first reaction with a small equilibrium constant and/or wherein the coupling enzyme concentration is higher than that of the intermediate. Equations based on their theory can allow one to calculate steady-state velocities of coupled enzyme reactions and to predict the time course of coupled enzyme reactions during the pre-steady state. 

In standard kinetic studies, the initial velocity (v) should be directly proportional to the total enzyme concentration. Indeed, this plot should be one of the first items analyzed in the kinetic characterization of an enzyme. The enzyme concentration range in this plot should span from below to above the concentration to be used in the inital rate studies (and, ideally, the line should go through the origin). When curvature is observed in such plots, there are several potential explanations . ... 

Isozymes are also a common presence in enzyme preparations and they can often be detected via polyacrylamide gel electrophoresis. The detected presence of isozymes may result in the need for further purification steps and the kinetic characterization of each isozyme. It may be necessary to use nondenaturing electrophoretic procedures to separate the different isozymes. See Isozymes Enzyme Concentration... 

The initial rate assumption is one of the most powerful and widely used assumptions in the kinetic characterization of enzyme action. The proper choice of reaction conditions that satisfy the initial rate assumption is itself a challenge, but once conditions are established for initial rate measurements, the kinetic treatment of an enzyme s rate behavior becomes much more tractablek In reporting initial rate data, investigators would be well advised to provide the following information ... 

Rose and co-workers first demonstrated that a proteo-lyzed form of hexokinase forms a sticky (or sluggishly dissociable) complex with glucose. The generalized application of this approach to the kinetic characterization of multisubstrate enzymes has been treated in detail. See also Partition Coefficient Radiospecific Activity Stickiness... 

Cleland introduced the net rate constant method to simplify the treatment of enzyme kinetic mechanisms that do not involve branched pathways. This method can be applied to obtain rate laws for isotope exchange, isotope partitioning, and positional isotope exchange. Since the net-rate constant method allows one to obtain VraaJKra and in terms of the individual rate constants, this method has greatest value for the characterization of isotope effects on and Kj. Because only... 

AGIRE computer program for, 249, 79-81, 225-226 comparison to analysis based on rates, 249, 61-63 complex reactions, 249, 75-78 experimental design, 249, 84-85 inhibitor effects, 249, 71-75 potato acid phosphatase product inhibition, 249, 73-74 preliminary fitting, 249, 82-84 prephenate dehydratase product inhibition, 249, 72-73 product inhibition effects, 249, 72-73 prostate acid phosphatase phenyl phosphate hydrolysis, 249, 70 reactions with two substrates, 249, 75-77 reversible reactions, 249, 77-78 with simple Michaelian enzyme, 249, 63-71 [fitting equations, 249, 63] with slow-binding inhibitors, 249, 88 with unstable enzymes, for kinetic characterization, 249, 85-89. 

If enzymes are described under tbe aspect of reaction mechanisms, the maximal rate of turnover Vmax. the Michaelis and Menten constant Km, the half maximal inhibitory concentration ICso, and tbe specific enzyme activity are keys of characterization of the biocatalyst. Even though enzymes are not catalysts in a strong chemical sense, because they often undergo an alteration of structure or chemical composition during a reaction cycle, theory of enzyme kinetics follows the theory of chemical catalysis. 

Schneider, C. Stull, G.A. Apple, F.S. Kinetic characterization of human heart and skeletal muscle CK isoenzymes. Enzyme, 39, 220-226 (1988)... 

Saier, M.H. Schmidt, M.R. Lin, P. Phosphoryl exchange reaction catalyzed by enzyme I of the bacterial phosphoenolpyruvate sugar phosphotransferase system. Kinetic characterization. J. Biol. Chem., 255, 8579-8584 (1980)... 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Lipases are unusual hydrolytic enzymes because they act on substrates providing an interface (with few exceptions). This feature has been historically used to distinguish lipases from esterases, the latter of which act on substrates in true solution (Jensen, 1983). The distinction of lipases as interfacial catalysts can make kinetic characterization a challenge, because relevant substrate concentrations are expressed in terms of area and not concentration. 

Kinetic analysis was used to characterize enzyme-catalyzed reactions even before enzymes had been isolated in pure form. As a rule, kinetic measurements are made on purified enzymes in vitro. But the properties so determined must be referred back to the situation in vivo to ensure they are physiologically relevant. This is important because the rate of an enzymatic reaction can depend strongly on the concentrations of the substrates and products, and also on temperature, pH, and the concentrations of other molecules that activate or inhibit the enzyme. Kinetic analysis of such effects is indispensable to a comprehensive picture of an enzyme. 

Enzyme kinetics deals with the rate of enzyme reaction and how it is affected by various chemical and physical conditions. Kinetic studies of enzymatic reactions provide information about the basic mechanism of the enzyme reaction and other parameters that characterize the properties of the enzyme. The rate equations developed from the kinetic studies can be applied in calculating reaction time, yields, and optimum economic condition, which are important in the design of an effective bioreactor. 

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