Double reciprocal plot competitive inhibition

Double reciprocal plots distinguish between competitive and noncompetitive inhibitors and simpbfy evaluation of inhibition constants Aj. v, is determined at several substrate concentrations both in the presence and in the absence of inhibitor. For classic competitive inhibition, the lines that connect the experimental data points meet at they axis (Figure 8-9). Since they intercept is equal to IIV, this pattern indicates that wben 1/[S] approaches 0, Vj is independent of the presence of inhibitor. Note, however, that the intercept on the X axis does vary with inhibitor concentration—and that since is smaller than HK, (the apparent... 

At very low substrate concentration ([S] approaches zero), the enzyme is mostly present as E. Since an uncompetitive inhibitor does not combine with E, the inhibitor has no effect on the velocity and no effect on Vmsa/Km (the slope of the double-reciprocal plot). In this case, termed uncompetitive, the slopes of the double-reciprocal plots are independent of inhibitor concentration and only the intercepts are affected. A series of parallel lines results when different inhibitor concentrations are used. This type of inhibition is often observed for enzymes that catalyze the reaction between two substrates. Often an inhibitor that is competitive against one of the substrates is found to give uncompetitive inhibition when the other substrate is varied. The inhibitor does combine at the active site but does not prevent the binding of one of the substrates (and vice versa). 

This type of inhibition differs from that exhibited by classical competitive inhibitors, because the substrate can still bind to the El complex and the EIS complex can go on to form product (albeit at a slower rate) without the inhibitor being released from the binding site. While standard double-reciprocal plots of partial competitive inhibitors will be linear (except for some steady-state, i.e., non-rapid-equilibrium, cases), secondary slope replots will be nonlinear. See Nonlinear Inhibition... 

For non-rapid-equilibrium cases (i.e., steady-state cases) the enzyme rate expression is much more complex, containing terms with [A] and with [I]. Depending on the relative magnitude of those terms in the initial rate expression, there may be nonlinearity in the standard double-reciprocal plot. In such cases, computer-based numerical analysis may be the only means for obtaining estimates of the magnitude of the kinetic parameters involving the partial inhibition. See Competitive Inhibition... 

Another action of phenothiazines is to compete with NADPH for the oxidase, an inference which was based on fulfillment of the criteria for competitive inhibition in double reciprocal plots of 1/[NADPH] vs. 1/rate of formation of O in the presence and absence of inhibitor It seems worth considering that all the effects of phenothiazines might be mediated through this effect and that the process of activation represents the presentation of substrate to the enzyme from which, in the resting state, the substrate is kept separate. 

The numbers listed were derived from the inhibition index values given by Ukita et al. (454)- The substrate was cy tidine 2 3 -phosphate, 8.8 mM. The Michaelis constant was reported to be 24 mM in the bicarbonate buffer 0.03 M, pH 7.6, 37°, that was used in all of the measurements. The inhibition constants were derived on the assumption of competitive inhibition in all cases. This type of inhibition was specifically shown for those compounds with a second number in parentheses. This latter number was obtained from multipoint double reciprocal plots in the usual manner. The agreement of the two... 

Competitive inhibition. A series of double-reciprocal plots (1/v versus 1/[S]) measured at different concentrations of the inhibitor (I) all intersect at the same point (on the ordinate. The slopes of the plots and the intercepts on the abscissa are simple, linear functions of [IJ/A j, where K, is the dissociation constant of the inhibitor-enzyme complex. 

One standard equation for competitive inhibition is given in Eq. (6). This equation shows that the presence of the inhibitor modifies the observed Km but not the observed Vm. A double reciprocal plot gives an x intercept of — 1 Km and a y intercept of 1/Vrn. 

The characteristics of the double reciprocal plots given by Equation (5.149), Equation (5.154), and Equation (5.155) determine what kind of enzyme inhibition may occur competitive, noncompetitive, or uncompetitive. In a given concentration of enzyme and inhibitor, the substrate concentration is changed and the double reciprocal plot of 1/V against 1/[A] is drawn. Figure 5.24a illustrates the double... 

FIGURE 5.24 The double reciprocal plots of (a) competitive inhibition, (b) noncompetitive inhibition, and (c) uncompetitive inhibition. [Graph reconstructed from data by Nnane et al., Br. J. Cancer, 83, 74 (2000).]... 

Figure 5.9 Double-reciprocal plots showing different types of inhibition. (A) Competitive inhibition [Equation (5.28)J (B) noncompetitive inhibition [Equation (5.29)] (C) uncompetitive inhibition (Equation 5.30). Km and Vmax are estimated from the slopes of uninhibited reactions, and K, from the slopes and/or intercepts of the inhibited reactions.

Fig. 13.4. DGBP competitively inhibits GGDPS with respect to FPP. In vitro GGDPS assays were performed utilizing various concentrations of FPP substrate or DGBP using an established method [110]. A double reciprocal plot liV vs. lA) is shown.

Fig. 7.6 shows the double reciprocal plots for reactions run in the presence of several concentrations of I. The series of lines intersecting the y-axis at the same point is the diagnostic pattern for the competitive inhibition of a reaction. As in the uninhibited reaction the y-axis intercept is lA max- This is the same for all values of [I] but the x-axis intercepts are the different values of-1/K vi(app) each concentration of I. The slopes of the lines are proportional to K]yi(app) which, as shown in Eqn. 7.27, also contains the term, K, the dissociation constant for the M-I complex. These slopes follow Eqn. 7.28,... 

Double-reciprocal plots are especially useful for distinguishing between competitive, uncompetitive, and noncompetitive inhibitors. In competitive inhibition, the intercept on they-axis of the plot of I/Vq versus 1/fS] is the... 

Figure 8.20 Competitive inhibition illustrated on a double-reciprocal plot. A...

Inhibition can be analysed visually by double reciprocal plots (Figure 5.12). In the case of competitive inhibition, 1/v versus 1/[S] plots at various values of P] give a series of lines all intersecting at 1 /[S] = 0, 1/v = 1/ Fmax, whereas in the case of anticompetitive inhibition, the 1/v versus 1/[S] plots at various values of P] are all parallel. The fascination of double reciprocal plots was such that a further form of inhibition, where the lines all intersected at 1/v = 0, 1/[S] = — 1/ Am was defined and confusingly termed non-competitive inhibition the physical situation, rarely encountered in practice, was that inhibitor bound equally to E and ES. The rate law is that of eqn. (5.30). In practice, inhibition which is neither cleanly competitive nor cleanly uncompetitive is best described by the mixed inhibition eqn. (5.31). 

Figure 5.12 Double reciprocal plots, drawn to scale, for (a) competitive and (b) anticompetitive inhibition.

Double reciprocal plots for an inhibited enzyme. The black line shows the double reciprocal plot for an enzyme in the absence of inhibitor. The other lines show the effect of an uncompetitive inhibitor (gray), a noncompetitive inhibitor (light purple), or a competitive inhibitor (purple). 

Figure 6.38. Double reciprocal plot for competitive inhibition.

FIGURE 8.37 Competitive inhibition illustrated on a doublereciprocal plot. A double-reciprocal plot of enzyme kinetics in the presence ( ) and absence ( .-) of a competitive inhibitor illustrates that the inhibitor has no effect on l/max but increases Km-... 

Double-reciprocal plots are especially useful for distinguishing between competitive and noncompetitive inhibitors. In competitive inhibition, the intercept on they -axis of the plot of 1/Vq versus 1/[S] is the same in the presence and in the absence of inhibitor, although the slope is increased (Figure 8.37). That the intercept is unchanged is because a competitive inhibitor does not alter At a sufficiently high concentration, virtually all the... 

The first potential site for NO binding and subsequent inhibition of NOS activity is the arginine binding site. This site could be excluded, however, because a double-reciprocal plot of substrate concentration versus reaction rate in the presence of increasing concentrations of the NO donor, SNAP (30 and 100 /xM), indicated that NO inhibited NOS by mechanisms that are not competitive with L-arginine. Likewise, SOD decreased the V ax without altering the for L-arginine. 

Figure 6.10 Double-reciprocal plot for simple competitive inhibition...

ACTIVE FIGURE 6.12 A Lineweaver-Burk double-reciprocal plot of enzyme kinetics for competitive inhibition. Sign in at www.thomsonedu.com/iogin to see an interactive version of this figure. 

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