Substrate concentration competitive inhibition

Ret er.stble inhibition, in contrast with irreversible inhibition, is acterized by a rapid dissociation of the enzyme-inhibitor complex. In the type of reversible inhibition called competitive inhibition, an enzyme can bind substrate (forming an ES complex) or inhibitor 1) but not both (ESI). The competitive inhibitor often resembles the substrate and binds to the active site of the enzyme (Figure 8.15). The substrate is thereby prevented from binding to the same active site. A competitive inhibitor dimmishes the rate oj catalysis by reducing the pro-por/ion of enzyme molecules bound to a substrate. At any given inhibitor concentration, competitive inhibition can be relieved by increasing... 

Pharmacodynamic-based toxic effects are those where there is altered responsiveness of the target site perhaps due to variations in the receptor. For example, individual variation in the response to digitoxin means that some patients suffer toxic effects after a therapeutic dose (see below Chapter 7). The inhibition of enzymes, blockade of receptors or changes in membrane permeability which underlie these types of effects often rely on reversible interactions. These are dependent on the concentration of the toxic compound at the site of action, and possibly the concentration of an endogenous substrate if competitive inhibition is involved. Therefore, with the loss of the toxic compound from the body, by the processes of metabolism and excretion, the concentration at the site of action falls and the normal function of the receptor or enzyme returns. This is in direct contrast to the type of toxic effect in which a cellular structure or macromolecule is permanently damaged, altered or destroyed by a toxic compound. In some cases, however, irreversible inhibition of an enzyme may occur, which if not fatal for the organism will require the synthesis of new enzyme, as is the case with organophosphorus compounds which inhibit cholinesterases. 

Competitive inhibitors are inhibitors which have an effect on the but not on the V of an enzyme-catalysed reaction. The V is unchanged because the number of functional active sites is not altered but a greater substrate concentration is required to achieve the maximum utilization of the sites. Consequently, the for the substrate increases. Competitive inhibition may be overcome by the addition of more substrate to the enzyme reaction mixture. Competitive inhibitors often bear a structural similarity to the substrate and compete with the substrate for the active sites of the enzyme, i.e. they are isosteric. However, competitive inhibitors are not necessarily structurally analogous to the substrate, e.g. salicylate inhibition of 3-phospho-glycerate kinase, and may bind to a site distinct from the active site, e.g. L-isoleucine inhibition of threonine deaminase from Escherichia coli. The classical example of competitive inhibition is the action of malonate on succinate dehydrogenase (Figure 6.9) which advanced the elucidation of the... 

Reversible inhibition is characterized by an equilibrium between the enzyme and the inhibitor, defined by an equilibrium constant, Ki, which represents the affinity of the inhibitor fra- its enzyme. Reversible inhibitors are classed into three groups according to their effect on die enzymatic site (Figure 4.4). The inhibition is "competitive" when the inhibitor and the substrate are in competition for the same site, and the degree of inhibition decreases as the substrate concentration increases. When the degree of inhibition increases with the substrate concentration, the inhibition is called "uncompetitive". Finally, if the degree of inhibition is independent of the substrate concentration, then the inhibition is said to be "non-competitive . 

Substrate and product inhibitions analyses involved considerations of competitive, uncompetitive, non-competitive and mixed inhibition models. The kinetic studies of the enantiomeric hydrolysis reaction in the membrane reactor included inhibition effects by substrate (ibuprofen ester) and product (2-ethoxyethanol) while varying substrate concentration (5-50 mmol-I ). The initial reaction rate obtained from experimental data was used in the primary (Hanes-Woolf plot) and secondary plots (1/Vmax versus inhibitor concentration), which gave estimates of substrate inhibition (K[s) and product inhibition constants (A jp). The inhibitor constant (K[s or K[v) is a measure of enzyme-inhibitor affinity. It is the dissociation constant of the enzyme-inhibitor complex. 

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... 

We now consider the case of a competitive inhibitor which has been added to the above reaction at the fixed concentration of 40 mM [15]. The following initial velocities of the competitively inhibited Michaelis-Menten process are observed at the same substrate concentrations as above ... 

Facilitated transport combines some properties of both mechanisms discussed above. This type of transport is carrier mediated so that there is substrate specificity, a transport maximum, and competitive inhibition. However, facilitated transport is not energy-dependent and is unable to transport a substrate against a concentration gradient. 

The inhibition modality for a slow binding inhibitor is easily determined from the effects of substrate concentration on the value of k0bs at any fixed inhibitor concentration (Tian and Tsou, 1982 Copeland, 2000). For a competitive inhibitor the value of fcobs will diminish hyperbolically with increasing substrate concentration according to Equation (6.15) ... 

Although kinetic evidence for prior equilibrium inclusion was not obtained, competitive inhibition by cyclohexanol and apparent substrate specificity once again provide strong support for this mechanism. Since the rate of the catalytic reaction is strictly proportional to the concentration of the ionized hydroxamate function (kinetic and spectrophotometric p/Cas are identical within experimental error and are equal to 8.5), the reaction probably proceeds by a nucleophilic mechanism to produce an acyl intermediate. Although acyl derivatives of N-alkylhydroxamic acids are exceptionally labile in aqueous solution, deacylation is nevertheless the ratedetermining step of the overall hydrolysis (Gruhn and Bender, 1969). 

The hallmarks of competitive inhibition are that ymax is not affected by adding the inhibitor and the plots intersect on the y axis. A high concentration of substrate prevents the inhibitor from exerting its effect. 

An inhibitor can have different effects on the velocity when the substrate concentration is varied. If the inhibitor and substrate compete for the same form of the enzyme, the inhibition is COMPETITIVE. If not, the inhibition is either NONCOMPETITIVE or UNCOMPETITIVE depending on whether or not the inhibitor can affect the velocity at low substrate concentrations. 

At low concentrations of substrate ([S] < Km), the enzyme is predominantly in the E form. The competitive inhibitor can combine with E, so the presense of the inhibitor decreases the velocity when the substrate concentration is low. At low substrate concentration ([S] < Km), the velocity is just Vmay IKm. Since the inhibitor decreases the velocity and the velocity at low substrate concentration is proportional to Vmax/Km, the presence of the inhibitor affects the slopes of the Lineweaver-Burk plots the slope is just the reciprocal of Vmax/Km. Increasing the inhibitor concentration causes Km/Vmax to increase. The characteristic pattern of competitive inhibition can then be rationalized if you simply remember that a competitive inhibitor combines only with E. 

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). 

Uncompetitive inhibition is extremely rare in nature, and can arise when the inhibitor binds to the enzyme-substrate complex, rather than to the free enzyme, as in competitive inhibition [111]. In uncompetitive inhibition, an increase in the concentration of the inhibitor requires a disproportionately large increase in the concentration of the substrate to maintain the same metabolic turnover. 

As discussed above, the degree of inhibition is indicated by the ratio of k3/k and defines an inhibitor constant (Kj) [Eq. (3.19)], whose value reports the dissociation of the enzyme-inhibitor complex (El) [Eq. (3.20)]. Deriving the equation for competitive inhibition under steady-state conditions leads to Eq. (3.21). Reciprocal plots of 1/v versus 1/5 (Lineweaver-Burk plots) as a function of various inhibitor concentrations readily reveal competitive inhibition and define their characteristic properties (Fig. 3.5). Notice that Vmax does not change. Irrespective of how much competitive inhibitor is present, its effect can be overcome by adding a sufficient amount of substrate, i.e., substrate can be added until Vmax is reached. Also notice that K i does change with inhibitor concentration therefore the Km that is measured in the presence of inhibitor is an apparent Km- The true KM can only be obtained in the absence of inhibitor. 

A reciprocal plot of the effect of varying concentrations of a noncompetitive inhibitor on enzyme-catalyzed substrate turnover readily reveals the nature and characteristics of this type of inhibition (Fig. 3.6). Notice that in this case, the properties that characterize noncompetitive inhibition are virtually opposite of those that characterize competitive inhibition. With a noncompetitive inhibitor Emax does change but KM is constant. 

For example, experimental data might reveal that a novel enzyme inhibitor causes a concentration-dependent increase in Km, with no effect on and with Lineweaver-Burk plots indicative of competitive inhibition. Flowever, even at very high inhibitor concentrations and very low substrate concentrations, it is observed that the degree of inhibition levels off when some 60% of activity still remains. Furthermore, it has been confirmed that only one enzyme is present, and all appropriate blank rates have been accounted for. It is clear that full competitive inhibition cannot account for such observations because complete inhibition can be attained at infinitely high concentrations of a full competitive inhibitor. Thus, it is likely that the inhibitor binds to the enzyme at an allosteric site. 

Full and partial competitive inhibitory mechanisms, (a) Reaction scheme for full competitive inhibition indicates binding of substrate and inhibitor to a common site, (b) Lineweaver-Burk plot for full competitive inhibition reveals a common intercept with the 1/v axis and an increase in slope to infinity at infinitely high inhibitor concentrations. In this example, Ki = 3 pM. (c) Replot of Lineweaver-Burk slopes from (b) is linear, confirming a full inhibitory mechanism, (d) Reaction scheme for partial competitive inhibition indicates binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor affects the affinity of enzyme for substrate and the presence of substrate affects the affinity of enzyme for inhibitor, both by a factor a. (e) Lineweaver-Burk plot for partial competitive inhibition reveals a common intercept with the 1/v axis and an increase in slope to a finite value at infinitely high inhibitor concentrations. In this example, Ki = 3 pM and = 4. (f) Replot of Lineweaver-Burk slopes from (e) is hyperbolic, confirming a partial inhibitory mechanism... 

Plots devised by Dixon to determine K, for tight-binding inhibitors, (a) A primary plot of v versus total inhibitor present ([/Id yields a concave line. In this example, [S] = 3 x Km and thus v = 67% of Straight lines drawn from Vo (when [/It = 0) through points corresponding to Vq/2, Vq/3, etc. intersect with the x-axis at points separated by a distance /Cj app/ when inhibition is competitive. When inhibition is noncompetitive, intersection points are separated by a distance equivalent to K. The positions of lines for n = 1 and n = 0 can then be deduced and the total enzyme concentration, [EJt, can be determined from the distance between the origin and the intersection point of the n = 0 line on the x-axis. If inhibition is competitive, this experiment is repeated at several different substrate concentrations such that a value for K, app is obtained at each substrate concentration. (b) Values for app are replotted versus [S], and the y-intercept yields a value for /Cj. If inhibition is noncompetitive, this replot is not necessary (see text)... 

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