Enzyme kinetic parameters

Table 9.2 Summary of enzyme kinetic parameters and inhibitor potencies for 11 human CYP activities in pooled human liver microsomes [117]. 

This simplifies greatly the concepts of first-pass hepatic metabolism and systemic clearance referred to previously. Most importantly Cli is directly evolved from the enzyme kinetic parameters, V ax and K ... 

Selected entries from Methods in Enzymology [vol, page(s)] Computer programs, 240, 312 infrared S-H stretch bands for hemoglobin A, 232, 159-160 determination of enzyme kinetic parameter, 240, 314-319 kinetics program, in finite element analysis of hemoglobin-CO reaction, 232, 523-524, 538-558 nonlinear least-squares method, 240, 3-5, 10 to oxygen equilibrium curve, 232, 559, 563 parameter estimation with Jacobians, 240, 187-191. 

Data analysis flow chart, 240, 314-315 data point number requirements, 240, 314 determination of enzyme kinetic parameters multisubstrate, 240, 316-319 single substrate, 240, 314-316 enzyme mechanism testing, 240, 322 evaluation of binding processes, 240, 319321 file transfer protocol site, 240, 312 instructions for use, 240, 312-313. 

A detailed account of the relationship between redox potentials and enzyme kinetic parameters is given by Ikeda and Kano [44]. For example, for a mediated substrate oxidation reaction, the rate Vg of the enzyme-catalyzed reaction can be measured and the kinetic parameters determined from ... 

The contributions of hydrogen bond donors to catalysis can be estimated by site-directed mutagenesis studies in cases where the hydrogen bond donor is located in the amino acid side chain. Deletion of the main chain NH is only possible by substituting the amino acid with a proUne. In all cases, the effects of the substitution to key enzyme kinetic parameters, and K, should be checked. Typically, the oxyanion hole residues contribute only Uttle to the binding of substrate [19-21]. This is reflected in the values, which typically remain very similar... 

The direction of a reaction can be assessed straightforwardly by comparing the equilibrium constant (Keq) and the ratio of the product solubility to the substrate solubility (Zsat) [39]. In the case of the zwitterionic product amoxicillin, the ratio of the equilibrium constant and the saturated mass action ratio for the formation of the antibiotic was evaluated [40]. It was found that, at every pH, Zsat (the ratio of solubilities, called Rs in that paper) was about one order of magnitude greater in value than the experimental equilibrium constant (Zsat > Keq), and hence product precipitation was not expected and also not observed experimentally in a reaction with suspended substrates. The pH profile of all the compounds involved in the reaction (the activated acyl substrate, the free acid by-product, the antibiotic nucleus, and the product) could be predicted with reasonable accuracy, based only on charge and mass balance equations in combination with enzyme kinetic parameters [40]. 

Enzyme kinetics were evaluated in a PDMS-glass chip using a continuous-flow system. A biotinylated enzyme (HRP or (5-galactosidase) was coupled to streptavidin-coated beads via the amide coupling of an aminocaproyl spacer. These beads (15.5 pm) were retained by a weir in the chip. The channel wall was passivated by 1 mg/mL BSA. The apparent enzyme kinetic parameters were evaluated using the Lilly-Homby model, as developed for the packed-bed enzymatic reactor systems. It was found that the apparent Michaelis constant (Km) approached the tme Km value of the free enzyme at zero-flow rate of a homogeneous reaction [845]. 

These studies of protein structure, like those of urea and methylamine effects on enzyme kinetic parameters, reveal that physiological mixtures of counteracting solutes achieve essentially the same end result as use of compatible solutes regulating cell volume through adjusting concentrations of compatible solutes or counteracting solutes (at the appropriate ratio of concentrations) conserves critical aspects of protein structure and function as well as cell volume per se. 

The simplest Michaelis-Menten-type scheme for showing analytically the effect of enzyme ionization on enzyme kinetic parameters is... 

Cornish-Bowden, A., Eisenthal, R. (1974) Statistical Considerations in the Estimation of Enzyme Kinetic Parameters by the Direct Linear Plot and other Methods, Biochem.J. 139, 721-730. 

Figure 30.6 shows a prediction of the plasma concentration of ARA-C and total radioactivity (ARA-C plus ARA-U) following administration of two separate bolus intravenous injections of 1.2 mg/kg to a 70-kg woman. All compartment sizes and blood flow rates were estimated a -priori, and all enzyme kinetic parameters were determined from published in vitro studies. None of the parameters was selected specifically for this patient only the dose per body weight was used in the simulation. The prediction has the correct general shape and magnitude. It can be made quantitative by relatively minor changes in model parameters with no requirement to adjust the parameters describing metabolism. 

The sensitivity of this method is directly related to the apparent molar enthalpy of reaction, so that very endo- or exothermic reactions will be most readily followed. Examples of the application of this method to the determination of enzyme kinetic parameters include dihydrofolate reductase, creatine phosphokinase, hexo-kinase, urease, trypsin, HIV-1 protease, heparinase, and pyruvate carboxylase. 

Metabolic networks can be quantitatively and qualitatively studied without enzyme kinetic parameters by using a constraints-based approach. Metabolic networks must obey the fundamental physicochemical laws, such as mass, energy, redox balances, diffusion, and thermodynamics. Therefore, when kinetic constants are unavailable, cellular function can still be mathematically constrained based on the mass and energy balance. Flux balance analysis (FBA) is a mathematical modeling framework that can be used to study the steady-state metabolic capabilities of cell-based physicochemical constraints. ... 

The flux balance approach is an elegant technique for analyzing biological pathways in that the approach allows metabolic networks to be quantitatively and qualitatively studied without knowledge of specific enzyme kinetic parameters. A combination of FBA and bioinformatics provides a rapid, economical, and predictive in silico screening method for potential strategies to produce a desired compound. 

Data for the matrix of values of Rg as a function of values of both 9 and c is conventionally visualised in a Zimm plot (Figure 4.30). As with families of double-reciprocal plots in steady-state enzyme kinetics, parameters are now extracted computationally, but presented graphically. The functions, however, are not simple and need not be linear. The ordinate is KcjRg, but the abscissa is sin (0/2) + kc, where c is the concentration of the polymer and k is an arbitrary constant chosen so that the y intercept of families of plots at constant c or constant 9 is constant. 

Enzyme Biochemical properties molecular mass, prosthetic groups, functional groups on protein-surface, purity (inactivating/protective function of impurities) Enzyme kinetic parameters specific activity, pH-, temperature profiles, kinetic parameters for activity and inhibition, enzyme stability against pH, temperature, solvents, contaminants, impurities... 

One of the critical factors in the design of experiments to determine the values of steady-state enzyme kinetic parameters is the optimal choice of a range of substrate concentrations. On the basis of the data in Fig. 5-28, suggest a possible optimal range of substrate concentrations. 

When substrates are used at low, radiotracer concentrations, the Michaelis-Menten equation defining reaction rate in terms of substrate concentration and enzyme kinetic parameters reduces to ... 

Finally, investigators also have used the Eadie-Hofstee plot to estimate enzyme kinetic parameters (Fig. 4.4). In this last case, v is plotted along the y-axis and v/[S] along the x-axis. The slope of the best-fit line is equal to —K, the y-intercept = Umax and the x-intercept = Vmax/ m- As opposed to the double-reciprocal plot, the Eadie-Hofstee plot can make good data look worse (Dowd and Riggs, 1965). Interestingly, another use of the Eadie-Hofstee plot is to diagnose atypical kinetic profiles based on the shape of the data-fit obtained. This will be discussed in detail later in the chapter. 

As with estimation of enzyme kinetic parameters, the most accurate method for determining inhibition kinetic parameters is through nonlinear regression fitting of the data set. However, it may be beneficial to plot the data graphically... 

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