Pure competitive inhibition

Equation (9.40) for pure competitive inhibition predicts that the slope of a Lineweaver-Burk plot will change with changes in inhibitor concentration, but the intercept on the l/u0 axis (1/Vmax) will not. Thus, the above data are consistent with the inhibitor (I) acting as a pure competitive inhibitor. 

The slope of the reciprocal plot in the presence of a pure noncompetitive inhibitor is a linear function of [I] as shown earlier for pure competitive inhibition. The 1/v-axis intercept (1/Vroax,) is also a linear function of [I] as shown below. 

Competitive inhibitors bind at the substrate binding site, i.e. they compete with the substrate for the active site. In pure competitive inhibition, the inhibitor is assumed to bind to the free enzyme but not to the enzyme-substrate (ES) complex. 

This equation corresponds to that of mixed inhibition if K A). However, if K( = K, then pure competitive inhibition is the result. Thus it can be seen that, mechanistically speaking, pure noncompetitive inhibition is a special case of mixed inhibition. 

Fig. 5-17 Lineweaver-Burk piot 1/Vq versus 1/[S]q for (a) pure competitive inhibition and (b) pure noncompe-tive inhibition.

Table 3. Inhibition by a mixture of two exclusive inhibitors Case 1. Pure competitive inhibition by two exclusive inhibitors EX...

Case 5. Pure competitive inhibition by two nonexclusive inhfttitors... 

Competitive inhibitors bind at the substrate-binding site, that is, they compete with the substrate for the active site. In pure competitive inhibition, the inhibitor is assumed to bind to the free enzyme but not to the enzyme-substrate (ES) complex. The enzyme is unable to bind both S and I at the same time and in competitive inhibition, the enzyme-inhibitor complex El does not react with substrate S. Competitive inhibitors often resemble structurally the substrate. As an example, we can mention malonate, which is an inhibitor for dehydrogenation of succinate of an enzyme-succinic dehydrogenase and resembles the structure of succinate (Fig. 6.36)... 

K, which necessitates use of high specific activity radioactive toxin (at or above 10 Ci/mmol). Work in progress indicates that all type-1 brevetoxins inhibit tritiated PbTx-3 binding in a purely competitive manner, whereas the type-2 brevetoxins inhibit in a mixed competition manner at higher concentrations (Figure 7). 

Hj-receptor antagonists reversibly and competitively inhibit histamine action on H2 receptors. They are pure antagonists since they do not affect Hj receptors, j3-adrenorecep-tors, or muscarinic receptors. 

Toxicity. Atractyloside, a diterpenoid glycoside that occurs naturally in plants, may be present at levels as high as 600 mg/kg of dried plant material. Gonsumption of plants containing atractyloside or carboxyatractyl-oside has caused fatal renal proximal tubule necrosis and/or centrilobular hepatic necrosis in man and farm animals. Although pure atractyloside and crude plant extracts disrupt carbohydrate homeostasis and induce similar pathophysiological lesions in the kidney and liver, it is also possible that the toxicity of atractyloside may be confounded by the presence of other natural constituents in plants. Atractyloside competitively inhibits the adenine nucleoside... 

A non-competitive inhibitor causes an apparent fall in the amount of enzyme present, irrespective of the concentration of the substrate. In purely non-competitive inhibition, Km does not alter. 

Table-TV shows the effect ot fhe LMW fraction on the activity of some of these enzymes in vitro. Maltase, lactase and invertase were competitively inhibited at a concentration of 10 mg/ ml. When the effectsof a range of concentrations (2.5-20 mg/ml) of the LMW fraction were studied, it was revealed that the inhibition was not of the pure competitive type. Table V shows the effect of the HMW fraction. Low concentrations had to be used in the assays, as the intense brown color of this fraction interfered with the spectrophotometric measurements. In spite of this a strong competitive inhibition of lactase and of invertase was found. Maltase was also inhibited, and, to a lesser extent, even trehalase. a-Amylase from saliva was not affected at the concentration tested.

Non-competitive inhibitors bind reversibly to an allosteric site (see Appendix 7) on the enzyme. In pure non-competitive inhibition, the binding of the inhibitor to the enzyme does not influence the binding of the substrate to the enzyme. However, this situation is uncommon, and the binding of the inhibitor usually causes conformational changes in the structure of the enzyme, which in turn affects the binding of the substrate to the enzyme. This is known as mixed noncompetitive inhibition. The fact that the inhibitor does not bind to the active site of the enzyme means that the structure of the substrate cannot be used as the basis of designing new drugs that act in this manner to inhibit enzyme action. 

Allopurinol [al oh PURE i nole] is a purine analog. It reduces the production of uric acid by competitively inhibiting the last two steps in uric acid biosynthesis, which are catalyzed by xanthine oxidase (see Figure 39.14). [Note Uric acid is less water-soluble than its precursors. When xanthine oxidase is inhibited, the circulating purine derivatives (xanthine and hypoxanthine) are more soluble and therefore are less likely to precipitate]. 

In addition to the above, there is mixed inhibition, which exists if i increases or decreases as substrate concentration increases, but not to the same extent as for the pure competitive or anticompetitive cases,... 

The mechanism of inhibition is defined by the relative values of Ais and Ay, which are respectively the inhibition constants at [ATP] Km and >> Km.2S 29 Inhibition constants are measures of potency, because they equal the free inhibitor concentration when the rate is reduced by 50%. The mechanism is competitive if inhibition tends to zero when ATP is saturating dATP] Km). This mechanism is seen if Ais Ay. Conversely, the mechanism is uncompetitive if inhibition tends to zero when [ATP] Km, because As Ay. Inhibition is noncompetitive when it occurs both at [ATP] Km and [ATP] Am. Pure noncompetitive inhibition (Ais = Ay) arises when potency is independent of ATP-concentration. Mixed noncompetitive inhibition (Als A Ay) occurs if there is a tendency towards competitive or uncompetitive. 

Figure 4.6 Apparent inhibition constant as a function of ATP-concentration relative to Km for competitive inhibition (solid line), pure noncompetitive inhibition (dashed line) and uncompetitive inhibition (dotted line). The inhibition constant is 5 nM for each mechanism.

Figure 4.6 shows the relationship between K and [A TP] for various mechanisms of inhibition. For a competitive inhibitor, when ATP is present much below its Km value, K approximates to the inhibition constant, is. At [A TP] = Km, K = 2Idetermining potency and selectivity (See Section 4.4.2.1.). The selectivity at physiological [ATP] is influenced by which is an intrinsic property of the enzyme determining the relative ease of competitive inhibition. Uncompetitive inhibition may be barely detectable when [A TP] Km, because K Ki KJ[ATP. When [ATP] = Km, K = 2Kih and at [ATP] Km, K Ka. For pure noncompetitive inhibitors, K = K at all concentrations of ATP. It is noteworthy that when [ATP] = Km, K is between 1 and 2-times the value of the inhibition constant for each of these mechanisms. 

Lineweaver-Burk plots provide a good illustration of competitive inhibition and pure noncompetitive inhibition (Fig. 9.18). In competitive inhibition, plots of 1/v vs 1/[S] at a series of inhibitor concentrations intersect on the ordinate. Thus, at infinite substrate concentration, or 1/[S] = 0, there is no effect of the inhibitor. In pure noncompetitive inhibition, the inhibitor decreases the velocity even when [S] has been extrapolated to an infinite concentration. However, if the inhibitor has no effect on the binding of the substrate, the is the same for every concentration of inhibitor, and the lines intersect on the abcissa. 

Fig. 9.18. Lineweaver-Burk plots of competitive and pure noncompetitive inhibition. A. lAi versus 1/[S] in the presence of a competitive inhibitor. The competitive inhibitor alters the intersection on the abscissa. The new intersection is 1/K , p (also called 1/K ). A compietitive inhibitor does not affect B. 1/Vj versus 1/[S] in the presence of a pure noncompetitive inhibitor. The noncompetitive inhibitor alters the intersection on the ordinate, Wmax.app W niax> But docs not offcct 1/K j. A pure noncompetitive inhibitor binds to E and ES with the same affinity. If the inhibitor has different affinities for E and ES, the lines will intersect above or below the abscissa, and the noncompetitive inhibitor will change both the and the V, .

To derive the Lineweaver-Burk equations, we proceed by taking the reciprocals of each side of Eqs. (5.25), (5.29), and (5.32). The corresponding graphs of 1/vq versus l/[S]o have various characteristic changes in slopes and intercepts as [I] is varied. Competitive inhibition gives lines that all intersect on the ordinate. Pure noncompetitive inhibition (in which K, = K,) gives lines that all intersect on the abscissa. For anti- or uncompetitive inhibition, the telltale feature is that the set of lines are all parallel to each other. For mixed inhibition [K K in Eq. (5.32)], both the slopes and the intercepts on the ordinate and abscissa differ for different values of [I] see Fig. 5-31. 

HT inhibits the binding of both radio ligands purely competitively i.e. increase in Kj at constant B ... 

Bacteria use/ -aminobenzoic acid only for conversion to 7.8-dihydrofolic acid (Griffin and Brown, 1964). Thus, E, coli condenses j -aminobenzoic acid (and, alternatively, -aminobenzoylglutamic acid) with 2-amino-4-oxo-6-hydroxy-methyl-7,8-dihydropteridine 9.21) (as the 6-pyrophosphate) to give dihydro-pteroic acid (and alternatively, dihydrofolic acid) (Jaenicke and Chan, 1960). The sulfonamides competitively inhibit the isolated enzyme dihydrofolate the-tase which catalyses these steps (G. Brown, 1962). From Lactobacillus plantamm two enzymes responsible for this synthesis have been isolated in a pure state (Shiota et al., 1969a). The first of these catalyses the esterification of the pteridine 9.21) to its pyrophosphoryl derivative. The second is Brown s dihydrofolate synthetase. This second enzyme has also been isolated from several strains of Pneumococcus, found to have a mol. wt. of 90000, and to need ATP and Mg " as coenzymes (Ortiz, 1970). 

The inhibition of malate dehydrogenase is of interest in that for the forward reaction to oxaloacetate it is purely competitive whereas in the reverse direction in the presence of NADH, it is mixed. Of interest too is the use of NADH as the hydrogen donor in the determination of peroxidase because this enzyme was inhibited competitively by competition... 

Pure 0. are nonreducing, r.s. 60-80, thermostable, meltable and there is no tendency for browning reactions. The competitive inhibition of glucan formation from sucrose in the oral region by Streptococcus mutans in the presence of o. is the reason for its application as a noncariogenic sweetener in Japan since 1980. 

In many ecosystems, plants tend to pattern themselves as pure stands or as individuals spaced in rather specific densities or configurations. Many desert species show obvious zones of inhibition around which few, if any, alien species are able to invade. These patterns often cannot be adequately explained by competition alone, and are probably caused by a combination of factors including allelopathy. The phenomenon happens with herbaceous plants as well as woody shrubs and trees. 

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