Eadie-Hofstee graph

Use the data in Table 2.2 to plot Michaelis-Menten, Lineweaver-Burke and Eadie-Hofstee graphs to determine Km and Vm DC values. Answers are given at the end of the chapter. 

Test your IT skills. Try creating Excel spreadsheets based on the Michaelis-Menten equation (Equation 2.9) and its variants (Equations 2.10 and 2.11). Insert into your spreadsheets your own values for JCm, Vmax, [S], [I] and A) and use Excel to plot Michaelis-Menten, Lineweaver-Burke and Eadie-Hofstee graphs. 

Uncompetitive inhibition occurs when the inhibitor binds with ES but not with E (1 /Ki = 0). The manifestation of this type of inhibition is an Eadie-Hofstee graph with lines intersecting on the rate/[S] axis (Fig. 11.13B). The general case where the parameters of Scheme 11.20 are finite and Km and kcat k cat is called mixed inhibition, and the lines of the Eadie-Hofstee plots do not intersect on either axis. 

Enzyme kinetics Michaelis constant, symbol iCm maximum velocity of an enzyme catalysed reaction, Vm DC inhibitor constant, symbol X Michaelis-Menten equation and graph in the absence and the presence of inhibitors. Lineweaver-Burke and Eadie-Hofstee plots. 

Historically, data have been transformed to facilitate plotting on linear plots such as Lineweaver-Burk (1/y versus 1/[S]), Hanes-Woolf ([S]/y versus [S]), or Eadie-Hofstee (v/[S] versus y). However, with the present availability of affordable nonlinear regression and graphing software packages such as GraphPad Prism,... 

Figure 9-6 Reciprocal plots used to analyze kinetics of two-substrate enzymes. (A) Plot of 1 / against 1 / [A] for a series of different concentrations of the second substrate B. (B) A secondary plot in which the intercepts from graph A are plotted against 1/ [B], (C) Secondary plot in which the slopes from graph A have been plotted against 1 / [B]. The figures have been drawn for the case that Kmp = 10 3 M, Kun, = 2 Km, and K B = KeqAKmB (Eq. 9-46) = KmJ 200 and [A] and [B] are in emits of moles per liter. Eadie-Hofstee plots of v / [A] vs vf at constant [B] can also be used as the primary plots. The student can easily convert Eq. 9-44 to the proper form analogous to Eq. 9-21.

In addition to being easier to fit than the hyperbolic Michaelis-Menten equation, Lineweaver-Burk graphs clearly show differences between types of enzyme inhibitors. This will be discussed in Section 4.5. However, Lineweaver-Burk equations have their own distinct issues. Nonlinear data, possibly indicating cooperative multiunit enzymes or allosteric effects, often seem nearly linear when graphed according to a Lineweaver-Burk equation. Said another way, the Lineweaver-Burk equation forces nonlinear data into a linear relationship. Variations of the Lineweaver-Burk equation that are not double reciprocal relationships include the Eadie-Hofstee equation7 (V vs. V7[S]) (Equation 4.14) and the Hanes-Woolf equation8 ([S]/V vs. [S]) (Equation 4.15). Both are... 

Which of these plots should be used To generally understand the behavior of enzymes, use the simple graph of initial velocity against substrate concentration. The linearized forms are useful for calculation of ATM and Fmax. The Lineweaver-Burke plot is useful for distinguishing between types of inhibition (Chapter 8). The Eadie-Hofstee plot is better than the Lineweaver-Burke plot at picking up deviations from the Michaelis-Menten equation. 

This graph gives a direct readout of Km. Calculation of V mx can be done from the KJ Vmax intercept. The Hanes plot avoids the problem of the Eadie-Hofstee plot of having velocity (dependent) data influencing the data on the horizontal (independent) axis. 

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