Enzyme kinetics Lineweaver-Burk plots

Michaelis-Menten Kinetics Lineweaver-Burk Plots Enzyme Inhibition... 

The three reversible mechanisms for enzyme inhibition are distinguished by observing how changing the inhibitor s concentration affects the relationship between the rate of reaction and the concentration of substrate. As shown in figure 13.13, when kinetic data are displayed as a Lineweaver-Burk plot, it is possible to determine which mechanism is in effect. 

Lineweaver-Burk plot a graphical means for evaluating enzyme kinetics, (p. 638)... 

Lineweaver-Burk plot Method of analyzing kinetic data (growth rates of enzyme catalyzed reactions) in linear form using a double reciprocal plot of rate versus substrate concentration. 

The Michaelis-Menten equation is, like Eq. (3-146), a rectangular hyperbola, and it can be cast into three linear plotting forms. The double-reciprocal form, Eq. (3-152), is called the Lineweaver-Burk plot in enzyme kinetics. ... 

First draw both Lineweaver-Burk plots and Hanes-Woolf plots for the following a Monod-Wyman-Changeux allosteric K enzyme system, showing separate curves for the kinetic response in (1) the absence of any effectors (2) the presence of allosteric activator A and (3) the presence of allosteric inhibitor I. Then draw a similar set of curves for a Monod-Wyman-Changeux allosteric Uenzyme system. 

The enzymatic activities of intercalated GOx-AM P layered nanocomposites at various pH values and temperatures were compared with the native enzyme in aqueous solution. In both cases, characteristic linear plots consistent with Michalis-Menton kinetics were obtained. The Lineweaver-Burk plots indicated that the reaction rates (Vmax) for free and intercalated GOx (3.3 and 4.0 pM min 1 respectively), were comparable, suggesting that the turnover rate at substrate saturation was only marginally influenced by entrapment between the re-assembled organoclay sheets. However, the dissociation constant (Km) associated with the activity of the enzyme was higher for intercalated GOx (6.63 mM) compared to native GOx (2.94 mM), suggesting... 

First, the activity of the enzyme was measured and kinetic parameters were determined by Lineweaver-Burk plots, using phenyhnalonic acid as the substrate. The results are summarized in Tabled. Among four mutants, C188S showed a drastic decrease in the activity (k -jt/Kn,). The low activity was due to a decrease in the catalytic tirrnover number (k(.jt) rather than in affinity for the substrate (Km). 

The main plots used in enzyme kinetics and receptor binding studies are the Scatchard plot, the Lineweaver-Burk plot, and the linearization for estimation of the Hill coefficient. This chapter gives a short survey of these transformations of enzyme kinetics or receptor binding data. 

Figure 8.4 The Lineweaver-Burk plot (A) and the Hanes plot (B) of typical enzyme kinetics in presence of a competitve (a) noncompetive (b), mixed type (c) and uncompetitive (d) inhibitor.

Bimolecular reactions of two molecules, A and B, to give two products, P and Q, are catalyzed by many enzymes. For some enzymes the substrates A and B bind into the active site in an ordered sequence while for others, bindingmay be iii a random order. The scheme shown here is described as random Bi Bi in a classification introduced by Cleland. Eighteen rate constants, some second order and some first order, describe the reversible system. Determination of these kinetic parameters is often accomplished using a series of double reciprocal plots (Lineweaver-Burk plots), such as those at the right. 

A Lineweaver-Burk plot of enzyme kinetics in the presence and absence of a noncompetitive inhibitor is shown in Figure E5.5. Umax in the presence of a noncompetitive inhibitor is decreased, but KM is unaffected. The effect of a competitive inhibitor on the direct linear plot is shown in Figure E5.6. 

Second, an enzyme assay may be used to measure the kinetic properties of an enzyme such as Ku, Vmax, and inhibition characteristics. In this situation, different experimental conditions must be used. If Ku for a substrate is desired, the assay conditions must be such that the measured initial rate is first order in substrate. To determine Ku of a substrate, constant amounts of enzyme are incubated with varying amounts of substrate. A Lineweaver-Burk plot (1/v vs. 1/[S]) or direct linear plot may be used to determine Ku and V. If a reaction involves two or more substrates, each must be evalu-... 

Mass transfer can alter the observed kinetic parameter of enzyme reactions. Hints of this are provided by non-linear Lineweaver-Burk plots (or other linearization methods), non-linear Arrhenius plots, or differing Ku values for native and immobilized enzymes. Different expressions have been developed for the description of apparent Michaelis constants under the influence of external mass transfer limitations by Homby (1968) [Eq. (5.69)], Kobayashi (1971), [Eq. (5.70)], and Schuler (1972) [Eq. (5.71)]. 

In the determination of steady state reaction kinetic constants of enzyme-substrate reactions, FABMS also provides some very unique capabilities. Since these studies are best performed in the absence of glycerol in the reaction mixture, the preferred method is that which analyzes aliquots which are removed from a batch reaction at timed intervals. Quantitation of the reactants and products of interest is essential. When using internal standards, generally, the closer in mass the ion of interest is to that of the internal standard, the better is the quantitative accuracy. Using these techniques in the determination of kinetic constants of trypsin with several peptide substrates, it was found that these constants could be easily measured (8). FABMS was used to follow the decrease in the reactant substrate and/or the increase in the products with time and with varying concentrations of substrate. Rates of reactions were calculated from these data for each of the several substrate concentrations used and from the Lineweaver-Burk plot, the values of Km and Vmax are obtained. 

Allopurinol is a drug given to gout sufferers. It inhibits an enzyme of the purine degradation pathway called xanthine oxidase by acting in the capacity of a substrate. However, its product, oxidized allopurinol, is not able to leave the active site of the enzyme, thus blocking it. Is there a name for this type of substrate What inhibition kinetics would you observe with a Lineweaver-Burk plot, and why ... 

An inhibition of an Mg2+ ATPase on corn root plasma membrane was also observed. Kinetic data on aluminum inhibition present a competitive pattern, as demonstrated by the Lineweaver-Burk plot with an apparent inhibition constant (K,) of 40 pM [44]. These results were obtained at pH 6.6. The authors suggested that the inhibition may be a result of either the formation of an inefficient substrate (Al-ATP) or an interaction directly with the enzyme structure. 

The rate of hydrolysis of 3H-phenyl-cocaine in the presence and absence of each monoclonal antibody as a function of substrate concentration was determined. Production of radiolabeled benzoic acid at time points corresponding to < 5% reaction extent provided initial rates. A saturation kinetics and a linear Lineweaver-Burk plot for each artificial enzyme were plotted. The first-order rate constants (kcat) and Michaelis constants (Km) of selected antibodies are provided in Table 2. 

The inhibitor constants, Kj, may be determined by measuring the variation of v with [S] at different concentrations of the inhibitor. Lineweaver—Burk plots can then be used to find K/ from measurements made in the presence, and absence, of inhibitor (see Fig. 2 and Table I). The kinetics of the inhibition of such enzymes as alpha-amylase, hefo-amylase, and phosphorylase have been studied, and inhibitor constants evaluated. 

Figure 8.36. A Double-Reciprocal or Lineweaver-Burk Plot. A double-reciprocal plot of enzyme kinetics is generated... 

Fig. 3. p-Glucosidase inhibition shown by Lineweaver-Burk plot (reproduced from [2]). Lineweaver-Burk plot of kinetic data from peak 2 cellobiase ((3-glucosidase) at several product inhibitor levels. This is an example of noncompetitive inhibition where the product is not only completing for binding in the active site but also binding to a secondary site on the enzyme that alters the enzyme catalytic ability... 

The answer is b. (Murray, pp 48-73. Scriver, pp 4571-4636. Sack, pp 3-17. Wilson, pp 287-317.) When an enzyme obeys classic Michaelis-Menten kinetics as seen in the figure presented in question 154, the Michaelis constant (Km) and the maximal rate (V ax) can be readily derived. By plotting a reciprocal of the Michaelis-Menten equation, a straight-line Lineweaver-Burk plot is produced. The y intercept is l/Vmax, while the x intercept is —l/K . Thus, a reciprocal of these absolute values yields Vjnax and Kn. 

A plot of l/rp against 1/CA would give a straight line with an intercept of 1/ Vmax. The line crosses the %-axis at 1 /Km, as shown in Figure 3.6a. Although the Lineweaver-Burk plot is widely used to evaluate the kinetic parameters of enzyme reactions, its accuracy is affected greatly by the accuracy of data at low substrate concentrations. 

Figure 8.15 Lineweaver-Burke plot of enzyme kinetic data in which Mv is plotted against 1/[S).

D23.4 Refer to eqns 23.26 and 23.27, which are the analogues of the Michaelis-Menten and Lineweaver-Burk equations (23.21 and 23,22), as well as to Figure 23.13, There are three major modes of inhibition that give rise to distinctly different kinetic behavior (Figure 23.13), In competitive inhibition the inhibitor binds only to the active site of the enzyme and thereby inhibits the attachment of the substrate. This condition corresponds to a > 1 and a = 1 (because ESI does not form). The slope of the Lineweaver-Burk plot increases by a factor of a relative to the slope for data on the uninhibited enzyme (a = a = I), The y-intercept does not change as a result of competitive inhibition, In uncompetitive inhibition, the inhibitor binds to a site of the enzyme that is removed from the active site, but only if the substrate is already present. The inhibition occurs because ESI reduces the concentration of ES, the active type of the complex, In this case a = 1 (because El does not form) and or > 1. The y-intercepl of the Lineweaver-Burk plot increases by a factor of a relative to they-intercept for data on the uninhibited enzyme, but the slope does not change. In non-competitive inhibition, the inhibitor binds to a site other than the active site, and its presence reduces the ability of the substrate to bind to the active site. Inhibition occurs at both the E and ES sites. This condition corresponds to a > I and a > I. Both the slope and y-intercept... 

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