Michaelis constant determining

Acetylcholineesterase, urease, glucose oxidase and butyryl chloinesterase. Immobilized enzyme on to the sensor chip by corsslinking with glutaraldehyde and BSA. Conductivity changes, produced by the enzyme-catalyzed hydrolysis of ACh were measured for the analysis. Detection limits for ACh was 0.07 mM with corresponding sensitivity of 5.6 0.2 pS/mM. The device could be also used for apparent Michaelis constant determination. [85]... 

Here, Km = K,. K is the Michaelis constant determined in the presence of an excess of inhibitor, k /k may be found from the maximum velocity in the presence of an excess of substrate and inhibitor. 

Discuss in detail how the current measured with these biosensors is related to the glucose reaction rate. I low was the Michaelis constant determined ... 

Substitutions at the active site specifically R392A, M153A and Q384N (Figure 5.26B-D) give rise to voltammetry that is distinctly different from the wild-type enzyme (Figure 5.26A) both in waveform and in nitrate concentration dependence. The Michaelis constants determined from solution assays vary considerably (from 400 pM for the WT enzyme to 66 mM for the R392A variant) and this is reflected in the nitrate concentration dependence of the voltammetry. Another outcome of this study was the connection made between the potential dependence of the catalytic current and the observation that the rate of nitrate reduction in solution assays has been found to increase as the concentration of reductant decreases. 

Michaelis constant An experimentally determined parameter inversely indicative of the affinity of an enzyme for its substrate. For a constant enzyme concentration, the Michaelis constant is that substrate concentration at which the rate of reaction is half its maximum rate. In general, the Michaelis constant is equivalent to the dissociation constant of the enzyme-substrate complex. 

Km for an enzymatic reaction are of significant interest in the study of cellular chemistry. From equation 13.19 we see that Vmax provides a means for determining the rate constant 2- For enzymes that follow the mechanism shown in reaction 13.15, 2 is equivalent to the enzyme s turnover number, kcat- The turnover number is the maximum number of substrate molecules converted to product by a single active site on the enzyme, per unit time. Thus, the turnover number provides a direct indication of the catalytic efficiency of an enzyme s active site. The Michaelis constant, Km, is significant because it provides an estimate of the substrate s intracellular concentration. 

Saturation kinetics are also called zero-order kinetics or Michaelis-Menten kinetics. The Michaelis-Menten equation is mainly used to characterize the interactions of enzymes and substrates, but it is also widely applied to characterize the elimination of chemical compounds from the body. The substrate concentration that produces half-maximal velocity of an enzymatic reaction, termed value or Michaelis constant, can be determined experimentally by graphing r/, as a function of substrate concentration, [S]. 

Michaelis constant) which determines the deviation from proportionality a small D (compared to M) corresponds to a weak dependence of S on M and a large D corresponds to a near proportional dependence. 

Sato et al. (1991) expanded their earlier PBPK model to account for differences in body weight, body fat content, and sex and applied it to predicting the effect of these factors on trichloroethylene metabolism and excretion. Their model consisted of seven compartments (lung, vessel rich tissue, vessel poor tissue, muscle, fat tissue, gastrointestinal system, and hepatic system) and made various assumptions about the metabolic pathways considered. First-order Michaelis-Menten kinetics were assumed for simplicity, and the first metabolic product was assumed to be chloral hydrate, which was then converted to TCA and trichloroethanol. Further assumptions were that metabolism was limited to the hepatic compartment and that tissue and organ volumes were related to body weight. The metabolic parameters, (the scaling constant for the maximum rate of metabolism) and (the Michaelis constant), were those determined for trichloroethylene in a study by Koizumi (1989) and are presented in Table 2-3. 

The Michaelis constant (Km) and the maximum rate (Fmax) of PGX for PGA, reduced and non-reduced oligogalacturonates with increasing degree of polymerization (DP=2-7) were determined. Data are presented in Table 2. 

Maximal speed (Vmax) and supposed Michaelis constant (K ) of pectin hydrolysis reaction (catalyzed by the studied pectinesterase) were determined in Zinewedwer — Berk coordinated, They were determined in the range of substrate concentration values that was below optimum one V = 14.7 10 M min K = 5.56 10 M. The value of dissociated constant (KJ of the triple enzyme—substrate complex was determined from the experimental data at high substrate concentration. It was the following Kj= 0.22 M. Bunting and Murphy method was used for determination. 

E I is a kinetic chimera Kj and kt are the constants characterizing the inactivation process kt is the first-order rate constant for inactivation at infinite inhibitor concentration and K, is the counterpart of the Michaelis constant. The k,/K, ratio is an index of the inhibitory potency. The parameters K, and k, are determined by analyzing the data obtained by using the incubation method or the progress curve method. In the incubation method, the pseudo-first-order constants /cobs are determined from the slopes of the semilogarithmic plots of remaining enzyme activity... 

The kinetic data below were reported for an enzyme catalyzed reaction of the type E + S ES E + P. Since the data pertain to initial reaction rates, the reverse reaction may be neglected. Use a graphical method to determine the Michaelis constant and Fmax for this system at the enzyme concentration employed. 

The turnover number of an enzyme is defined as the maximum number of moles of substrate reacted per mole of enzyme (or molecules per molecule) per minute under optimum conditions (i.e., saturating substrate concentration, optimum pH, etc). If 2 mg/cm3 of a pure enzyme (50,000 molecular weight, Michaelis constant Km = 0.03 mole/m3) catalyzes a reaction at a rate of 2.5 jumoles/nUksec when the substrate concentration is 5 x 10 3 moles/m3, determine the turnover number corresponding to this definition and the actual number of moles of substrate reacting per minute per mole of enzyme. 

The ratio k 1/k1 is effectively the dissociation equilibrium constant3 for ES in step (1), and is usually designated by Km (Michaelis constant, with units of concentration). The rate of formation of the product, P, is determined from step (2) 4... 

Determine the values of Vmax and the Michaelis constant Km in the Michaelis-Menten... 

In the absence of S2, the true Michaelis constant, Kml, is obtained. Consequently, the values of f and Vmax determined in a kinetics study depend upon the composition (csl and cS2) and total substrate concentration within the system. 

Determine the maximum reaction velocity (Vmax) and the Michaelis constant (Km) from... 

If a reaction that must be investigated follows a reaction sequence as in Scheme 10.1, and if the reaction order for the substrate equals unity, it means that (with reference to Eq. (4 b)), the observed rate constant (k0bs) is a complex term. Without further information, a conclusion about the single constants k2 and fCM is not possible. Conversely, from the limiting case of a zero-order reaction, the Michaelis constant cannot be determined for the substrate. For particular questions such as the reliable comparison of activity of various catalytic systems, however, both parameters are necessary. If they are not known, the comparison of catalyst activities for given experimental conditions can produce totally false results. This problem is described in more detail for an example of asymmetric hydrogenation (see below). 

Figure 3. Schematic view of the substrate uptake rate versus concentration relationship as described by the whole-cell Michaelis-Menten kinetics. Q is the substrate uptake rate, <2max the biologically determined maximum uptake rate per biomass, c the substrate concentration, and Kj the whole-cell Michaelis constant, i.e. the concentration resulting in 2max/2 (mass of substrate per volume). At c

It is perhaps wise to begin by questioning the conceptual simplicity of the uptake process as described by equation (35) and the assumptions given in Section 6.1.2. As discussed above, the Michaelis constant, Km, is determined by steady-state methods and represents a complex function of many rate constants [114,186,281]. For example, in the presence of a diffusion boundary layer, the apparent Michaelis-Menten constant will be too large, due to the depletion of metal near the reactive surface [9,282,283], In this case, a modified flux equation, taking into account a diffusion boundary layer and a first-order carrier-mediated uptake can be taken into account by the Best equation [9] (see Chapter 4 for a discussion of the limitations) or by other similar derivations [282] ... 

The value for the maximum velocity is related to the amount of enzyme used but the Michaelis constant is peculiar to the enzyme and is a measure of the activity of the enzyme. Enzymes with large values for Km show a reluctance to dissociate from the substrate and hence are often less active than enzymes with low Km values. The substrate concentration required for a particular enzyme assay is related to and when developing an assay, the value for Km should be determined. 

Procedure K.l Determination ofthc Michaelis constant (A ni) for the enzyme -amino acid oxidase (KC 1.4.3.3) using the I.ineweaver-Burk method... 

Equation 11.57 signifies that when the competitive method is used (i.e., both iso-topomers are present simultaneously in the reaction mixture) the experimentally determined kinetic isotope effect corresponds to the isotope effect on V/K regardless of the actual concentration of the substrate. In other words, one cannot measure the isotope effect on Vmax using this method even when concentration is much larger than the Michaelis constant Km-... 

Determination of the Michaelis constant for the cofactor NAD (A m.NAo) was carried out by measuring the initial rate of the reduction of NAD as a function of its concentration, at a constant concentration of glucose. All solutions were prepared in 0.1 M phosphate buffer pH 7.55. 

Determination of the reaction mechanism and the Michaelis constants for G6P (A m oep) and the cofactor NADP (A m.NADp) was carried out by measuring... 

For determination of the Michaelis constant, the activity of purified lignin peroxidase was measured by using the standard activity assay method except that the concentration of veratryl alcohol was varied between 7 fiM and 2.67 mM. 

There are many examples of first-order reactions dissociation from a complex, decompositions, isomerizations, etc. The decomposition of gaseous nitrogen pentoxide (2N2O5 4NO2 + O2) was determined to be first order ( d[N205]/dt = k[N205j) as is the release of product from an enzyme-product complex (EP E -t P). In a single-substrate, enzyme-catalyzed reaction in which the substrate concentration is much less than the Michaelis constant (i.e., [S] K ) the reaction is said to be first-order since the Michaelis-Menten equation reduces to... 

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