Line weaver Burk plot

Included in the following table are some data points from a hypothetical enzyme kinetics study. Using a spreadsheet program with graphing abilities (such as Excel), generate a Line-weaver-Burk plot of the data points in the table. Determine the best-fit line for the data along with Vnax, ATm, and r2 (the square of the correlation coefficient of the line). Does this enzyme follow Michaelis-Menten kinetics Why or why not ... 

The effects of L on the appearance of the Kitz-Wilson plot is exactly analogous to the effect of an enzyme competitive inhibitor on a Line-weaver-Burk plot. 

Fig. 2.—Graphical Determination of the Maximum Velocity, V, and the Michaelis Constant, K . [(o) v against [S] (f>) t) against[S], Lineweaver—Burk plot (c) a Line-weaver—Burk plot for competitive inhibition (d) a Lineweaver—Burk plot for noncompetitive inhibition.]...

Line weaver—Burk plot, in enzyme kinetics, 289,292... 

Figure 4-12 Double reciprocal (l/v versus 1/[S]) Line weaver-Burk plot. The [S] range chosen is optimal for the determination of Km and "VTnaj. ...

The results given in Table 3.3 are plotted as shown in Figure 3.9. This Line-weaver-Burk plot shows that the mechanism is competitive inhibition. From the line for the data without the inhibitor, Km and Vmax are obtained as 0.98gmolm-3 and 9.1 mmol m-3 s 1, respectively. From the slopes of the lines, K is evaluated as 0.6 gmol m-3. 

The Eadie-Hofstee plot does a betterjob than the Line-weaver-Burke plot in evenly distributing the data points over the entire substrate concentration range, and can be a useful visual technique for ascertaining whether enzyme kinetics are typical (as shown) or atypical (see Figure 8.18, B and G). The Michaelis-Menten approach basically assumes that enzymes present a single binding site to each substrate. Estimates of V x of drug... 

To confirm that the imprinted recognition site was indeed the reactive center, reactions were conducted in the presence of the imprinting template, 28, to determine its ability to inhibit the polymer-catalyzed reaction. A series of aldol reactions were conducted with increasing concentrations of 28. Figure 6 shows a Line weaver-Burk plot (a) and a Dixon plot (b) illustrating the increase in concentration of 28 leads to the decrease in efficiency of the MIP P-17 for the catalysis of chalcone formation. The concentration-dependent inhibition of chalcone production by 28 implies the presence of a specific reaction center in the polymer matrix. 

In 1990, we reported [ 166] that CPT-11 inhibits acetylcholinesterase activity. In the paper, the rate of thiocholine (TCh) production from acetylthio-choline iodide (ATChl) by acetylcholinesterase was analyzed with Line-weaver-Burk plots (Figure 2.3). This kinetic analysis indicated that CPT-11 noncompetitively inhibited acetylcholinesterase. In this case, the apparent Michaelis constant (Km) and inhibition constant (Ki) ranged from 63 to 68 yUM and from 0.221 to 0.300 /iM, respectively (Table 2.3). The same analysis revealed that the Ki value of physostigmine (a potent acetylcholinesterase... 

Such an effect can be useful in the case of enzymatic substrate determinations (cf. 2.6.1.3). When inhibitor activity is absent, i. e. [I] = 0, Equation 2.72 is transformed into the Michaelis-Menten equation (Equation 2.41). The Line-weaver-Burk plot (Fig. 2.30a) shows that the intercept 1 /V with the ordinate is the same in the presence and in the absence of the inhibitor, i. e. the value of V is not affected although the slopes of the lines differ. This shows that the inhibitor can be fully dislodged by the substrate from the active site of the enzyme when the substrate is present in high concentration. In other words, inhibition can be overcome at high substrate concentrations (see application in Fig. 2.49). The inhibitor constant, Ki, can be calculated from the corresponding intercepts with the abscissa in Fig. 2.30a by calculating the value of from the abscissa intercept when [I] = 0. 

This equation is very similcir to the Michaelis-Menten equation for the uninhibited enzyme (eqn 8.1) uid is cdso cunenable to cuialysis by a version of the Line weaver-Burk plot ... 

In all cases, the efficiency of the inhibitor may be obtained by determining and from a control experiment with uninhibited enzyme and then repeating the experiment with a known concentration of inhibitor. From the slope and y-intercept of the Line weaver-Burk plot for the inhibited enzyme (eqn 8.9), the mode of inhibition, the values of a or a, and the values of fCj or K can be obtained. 

Double reciprocal plot (Benesi-Hildebrand or Line weaver-Burk) ... 

Michaelis-Menten saturation kinetics should occur only at low substrate concentrations, near 10 M. Using a spectrophotometric assay it is possible to observe statistically significant deviations as shown by the Line-weaver-Burke and Eadie plots in Figure 14 using DOPA as the substrate in the catecholase reaction. 

Competitive inhibitors do not change the value of Vmax> which is reached when sufficiently high concentrations of the substrate are present so as to completely displace the inhibitor. However, the affinity of the substrate for the enzyme appears to be decreased in the presence of a competitive inhibitor. This happens because the free enzyme E is not only in equilibrium with the enzyme-substrate complex E. S, but also with the enzyme-inhibitor complex E. L Competitive inhibitors increase the apparent of the substrate by a factor of (1 + The evaluation of the kinetics is again greatly facilitated by the conversion of Equation 17.15 into a linear form using Line-weaver-Burk, Eadie-Hofstee, or Hanes-Woolf plots, as shown in Fig. 17.7. 

Lineweaver—Burk Method.8 The Michaelis-Menten equation may be algebraically rearranged to Eq. 2.22, yielding a third linear plot, called the Line-weaver-Burk or double-reciprocal plot ... 

To confirm their competitive, anticytokinin nature, we used the method of Line weaver and Burk [14]. The results of the treatment on the c-hexyl compound, one of the most active members of the class, is shown in Fig. 4, where the reciprocal of the growth response was plotted against the reciprocal of the concentration of added kinetin. That the resultant set of straight lines has a common intercept suggests that the 5-triazines share the site of action with cytokinins. 

Fig. 3. The reciprocal of the substrate concentration plotted against the reciprocal of the velocity of an enzymatic reaction according to the method of Line-weaver and Burk.

The initial catalysis rates were obtained by fixing concentration of polymer 23C and changing the substrate concentration. Michaelis-Meten kinetics for the polymer was confirmed by plotting the double reciprocal form of Line-weaver and Burk (1/v vs 1/s) (Fig. 13), which gave K and V ,ax- The A < at was obtained from V ax (fccat=Vmax/[E]o). assuming that one polymer molecule had one active center, since the polymer with Mn of lower than 4000 showed no significant catalytic activities. k<-at was found to be 6.0 x 10 h for 23C, which was 670-fold higher than the 9.12x 10" h" of the uncatalyzed reaction [98]. 

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