Noncompetitive inhibition double reciprocal plot

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... 

For simple noncompetitive inhibition, E and EI possess identical affinity for substrate, and the EIS complex generates product at a negligible rate (Figure 8-10). More complex noncompetitive inhibition occurs when binding of the inhibitor does affect the apparent affinity of the enzyme for substrate, causing the tines to intercept in either the third or fourth quadrants of a double reciprocal plot (not shown). 

Rule 1. Upon obtaining a double-reciprocal plot of 1/v vx. 1/[A] (where [A] is the initial substrate concentration and V is the initial velocity) at varying concentrations of the inhibitor (I), if the vertical intercept varies with the concentration of the reversible inhibitor, then the inhibitor can bind to an enzyme form that does not bind the varied substrate. For example, for the simple Uni Uni mechanism (E + A EX E -P P), a noncompetitive or uncompetitive inhibitor (both of which exhibit changes in the vertical intercept at varying concentrations of the inhibitor), I binds to EX, a form of the enzyme that does not bind free A. In such cases, saturation with the varied substrate will not completely reverse the inhibition. 

A limiting case of noncompetitive inhibition, characterized by f/iird-quadrant convergence of double-reciprocal plots of 1/v versus 1/[S] in the absence and presence of several constant levels of the inhibitor. 

Consider the standard Uni Uni mechanism (E + A EX E + P). A noncompetitive inhibitor, I, can bind reversibly to either the free enzyme (E) to form an El complex (having a dissociation constant K s), or to the central complex (EX) to form the EXl ternary complex (having a dissociation constant Xu). Both the slope and vertical intercept of the standard double-reciprocal plot (1/v vx. 1/[A]) are affected by the presence of the inhibitor. If the secondary replots of the slopes and the intercepts (thus, slopes or vertical intercepts vx [I]) are linear (See Nonlinear Inhibition), then the values of those dissociation constants can be obtained from these replots. If Kis = Xu, then a plot of 1/v vx 1/[A] at different constant concentrations of the inhibitor will have a common intersection point on the horizontal axis (if not. See Mixed-Type Inhibition). Note that the above analysis assumes that the inhibitor binds in a rapid equilibrium fashion. If steady-state binding conditions are present, then nonlinearity may occur, depending on the magnitude of the [I] and [A] terms in the rate expression. See also Mixed Type Inhibition... 

Figure 9-12 Double reciprocal plots for two cases of noncompetitive inhibition.

Noncompetitive inhibition. The double-reciprocal plots pass through different points on the ordinate, but intersect at the same point (— l/Km) on the abscissa. The slopes and the intercepts on the ordinate are linear functions of [I]/AT,. 

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.

Figure 11-11 Double reciprocal plots of noncompetitive inhibition.

Figure 8.38. Noncompetitive Inhibition Illustrated on a Double-Reciprocal Plot. A double-reciprocal plot of enzyme kinetics in the presence ( and absence of a noncompetitive inhibitor shows thatiT is unaltered and...

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... 

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 6.43. Double reciprocal plot for noncompetitive inhibition.

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... 

An important feature of enzymes is that their active sites can often be occupied by, or react with, molecules other than the substrate, leading to inhibition of enzyme activity. Several inhibition mechanisms are known, but it is necessary only to distinguish between irreversible and reversible inhibition. Irreversible inhibition arises when the inhibitor molecule I dissociates very slowly or not at all from the enzyme active site. The best-known examples occur when I reacts covalently with a critical residue in the active site. Inhibition of cholinesterase enzymes by the reaction of organo-phosphorus compounds with a serine residue is a case in point. This type of inhibition is said to be noncompetitive—enzyme activity cannot be restored by addition of excess substrate. So although addition of I reduces V,n x, Km is unaffected. The double-reciprocal plot in such cases has the same. v-axis intercept as the plot for the uninhibited enzyme, but greater slope. 

Fig. 3. Double reciprocal plots of a) competitive inhibition, where introduction of the inhibitor produces changes exclusively in the substrate affinity constant (Km) and b) noncompetitive inhibition, where inhibition is observed as a decrease in the maximum velocity of the enzyme catalyzed (Vm x) reaction...

When the inhibition of HIV-1 RT by E-EBU-dM was further analyzed with varying concentrations of substrate and compound, double-reciprocal plots showed that this compound inhibits competitively with respect to dlTP and noncompetitively with respect to dGTP (data not shown). The ATm of HIV-1 RT for dTTP and dGTP was 27 and 7.7 pM, respectively. The Ki of the enzyme for E-EBU-dM with dTTP as substrate was 0.42 pM. Some benzodiazepine (TIBO) derivatives (47)50 reported recendy behave quite similarly to HEPT derivatives. Considering their common specificity for HIV-1, the HEPT and TIBO derivatives might work through a similar mechanism of action. However, the TIBO derivatives apparently differ from the HEPT analogues in that they do not act as competitive inhibitors of HIV-1 RT with respect to dTTP. 

To clarify the mode of inhibition, the reaction was analyzed by the method of Lineweaver and Burk (1934) at two concentrations of aldehyde reagents and five concentrations of cytochrome c. The data were plotted by the double reciprocal procedure and the inhibition was found to be both competitive and noncompetitive in type. 

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