Enzyme kinetics noncompetitive

Uncompetitive antagonism, form of inhibition (originally defined for enzyme kinetics) in which both the maximal asymptotic value of the response and the equilibrium dissociation constant of the activator (i.e., agonist) are reduced by the antagonist. This differs from noncompetitive antagonism where the affinity of the receptor for the activating drug is not altered. Uncompetitive effects can occur due to allosteric modulation of receptor activity by an allosteric modulator (see Chapter 6.4). 

A Lineweaver-Burk plot of enzyme kinetics in the presence and absence of a noncompetitive inhibitor is shown in Figure E5.5. Umax in the presence of a noncompetitive inhibitor is decreased, but KM is unaffected. The effect of a competitive inhibitor on the direct linear plot is shown in Figure E5.6. 

Inhibition kinetics are included in the second category of assay applications. An earlier discussion outlined the kinetic differentiation between competitive and noncompetitive inhibition. The same experimental conditions that pertain to evaluation of Ku and Vmax hold for A) estimation. A constant level of inhibitor is added to each assay, but the substrate concentration is varied as for Ku determination. In summary, a study of enzyme kinetics is approached by measuring initial reaction velocities under conditions where only one factor (substrate, enzyme, cofactor) is varied and all others are held constant. 

FIGURE 4.14 Effect of a pure noncompetitive, reversible inhibitor on enzyme kinetics... 

Other inhibition models of NH4 and NO3 uptake have been based on enzyme kinetics (e.g., noncompetitive inhibition Frost and Franzen (1992)). Whether or not the analogy is strictly applicable, this would appear to have a more sound biological basis than the Wroblewski (1977) exponential function. Yajnik and Sharada (2003) defined general equations for a two-nutrient interaction that can be reduced to hyperbolic inhibition such as that employed by Frost and Franzen (1992) and Parker (1993). 

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...

In textbooks dealing with enzyme kinetics, it is customary to distinguish four types of reversible inhibitions (i) competitive (ii) noncompetitive (iii) uncompetitive and, (iv) mixed inhibition. Competitive inhibition, e.g., given by the product which retains an affinity for the active site, is very common. Non-competitive inhibition, however, is very rarely encountered, if at all. Uncompetitive inhibition, i.e. where the inhibitor binds to the enzyme-substrate complex but not to the free enzyme, occurs also quite often, as does the mixed inhibition, which is a combination of competitive and uncompetitive inhibitions. The simple Michaelis-Menten equation can still be used, but with a modified Ema, or i.e. ... 

The answer is c. (Murray, pp 48-73. Scriver, pp 4571-4636. Sack, pp 3-17. Wilson, pp 287-317.) Allosteric enzymes, unlike simpler enzymes, do not obey Michaelis-Menten kinetics. Often, one active site of an allosteric enzyme molecule can positively affect another active site in the same molecule. This leads to cooperativity and sigmoidal enzyme kinetics in a plot of [S] versus V The terms competitive inhibition and noncompetitive inhibition apply to Michaelis-Menten kinetics and not to allosteric enzymes. 

Inhibitors structurally related to the substrate may be bound to the enzyme active center and compete with the substrate (competitive inhibition). If the inhibitor is not only bound to the enzyme but also to the enzyme-substrate complex, the active center is usually deformed and its function is thus impaired in this case the substrate and the inhibitor do not compete with each other (noncompetitive inhibition). Competitive and noncompetitive inhibition effect the enzyme kinetics differently. A competitive inhibitor does not change but increases. Km (Fig. 25a) in contrast, noncompetitive inhibition results in an unchanged Km and an increased vmax (Fig. 25b). Some enzymes, e.g. invertase, are inhibited by high product concentration (product inhibition). 

ACTIVE FIGURE 6.13 A Lineweaver-Burk plot of enzyme kinetics for noncompetitive inhibition. Sign in at www.thomsonedu.com/iogin to expiore an interactive version of this figure. 

For each of the four types of inhibition of a Michaelis-Menten enzyme [competitive, Eq. (5.25) noncompetitive and mixed Eq. (5.29) and uncompetitive, Eq. (5.32)], derive the corresponding Lineweaver-Burk equations [Eqs. (5.26), and (5.30), respectively] and draw the characteristic plots that are the basis for the rapid visnal identification of which type of inhibition apphes when analyzing enzyme kinetic data. 

Pure noncompetitive inhibition (decrease in V ax with no change in K ) is seldom observed in enzyme kinetics studies, except in the case of very small inhibitors, such as protons, metal ions, and small anions. For noncompetitive... 

Fig. 4.18 Global effectiveness factor (mean integral value) of an immobilized enzyme with noncompetitive product inhibition kinetics in a spherical particle as a function of bulk substrate concentration and Thiele modulus (K/K2 = 1 substrate conversion = 0.9)...

Nitrogenase catalyzes the reduction of many substrates (Table IV) and there are several lines of evidence to suggest that these are reduced at different sites or by different forms of the enzyme. Kinetic studies of interactions between pairs of substrates (Hwang ef al., J973 Rivera-Ortiz and Burris, 1975) show that Ng inhibits acetylene reduction competitively whereas the reverse inhibition is noncompetitive azide and acetylene are mutually noncompetitive and azide and cyanide inhibit each other competitively. HCN inhibits Nj reduction competitively whereas Nj inhibits HCN reduction in an undetermined manner. N2O inhibits Nj reduction competitively and acetylene reduction noncompetitively. ATP-dependent evolution by nitrogenase is inhibited by all the other reducible substrates and CO inhibits all substrate reductions except H2 evolution in a noncompetitive manner. Table V summarizes these results. The simplest interpretation of these results is that the N2-reducing site reduces N2 and possibly N2O but no other substrate azide, HCN, and methyl isocyanide are reduced at the same site... 

Such is the case in certain reactions catalyzed by enzymes. The type of inhibition considered thus far can be called competitive inhibition the inhibitor competes with the reactant (called substrate S in enzyme kinetics) for the same active centers. But there also exists a different kind of inhibition called noncompetitive inhibition. A noncompetitive inhibitor D is one that combines with the enzyme E at a site which is different from that which combines with the substrate S. The complex ED between enzyme and inhibitor is then still able to combine further with a substrate molecule but the tertiary complex EDS thus formed is unreactive. If the rate-determining step of the reaction is the decomposition of the complex between enzyme E and substrate S, the sequence with noncompetitive inhibition can be represented as ... 

Competitive and noncompetitive inhibitions change enzyme kinetics differently. A competitive inhibitor does not change Vmax (maximum velocity) but increases K a noncompetitive inhibitor decreases Vmax and fCm remains unchanged. 

Figure 5.10. Demonstration of the four basic types of inhibitions of enzyme kinetics in Lineweaver-Burk plots (see Fig. 4.24c) (a) competitive, (b) noncompetitive, (c) uncompetitive, and (d) substrate inhibition. The parameters and can be estimated from the intercepts and slope of the line with p — 0, where p = inhibitor concentration.

Figure 6.24. Parameter estimation from integral reactors (DCSTR and CPFR) in the case of simple enzyme kinetics (a), substrate inhibition (b), and competitive (c) and noncompetitive product inhibition (d), according to Levenspiel (1979).

Interaction matrix this matrix is suggested to identify the different interactions that can exist between compounds and enzymes in the process. In this case, the reaction structure defined in the previous step is useful to visuahze and classify those relationships that can happen with a higher degree of probabihty. Similar ideas about the interaction between compounds can be found in the scientific literature or from experimental experience in the laboratory. In order to build the matrix, the compounds involved in the process (i.e., substrates, intermediates, by-products, products, etc.) are arranged in rows (i.e.. A, B, C,...), and the enzymes E ) are arranged in columns (for i = 1, 2, 3,...). In this way, the matrix is filled defining the relationship between each compound and enzyme in turn, that is, (S) for substrate, (P) for product, (I) for inhibitor, or (X) when there is no interaction between one compound and one enzyme. This compiled information is extremely useful to make decisions about the relevant terms or kinetic parameters that must be added or removed from the reaction rate expressions and process model. The position of the new term/parameter in the final expression is defined by the enzyme kinetic mechanism which shows how the compound inhibits the enzyme, for example, competitive, uncompetitive, noncompetitive, or mixed inhibition. 

FIGURE 8.11 A study of o-diphenol oxidase with catechol substrate including competitive (PHBA) and noncompetitive (phenylthiourea) inhibitors. (From Kimball, J., Enzyme kinetics, http / users.rcn.com/ jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html. With permission.)... 

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

To refer to the kinetics of allosteric inhibition as competitive or noncompetitive with substrate carries misleading mechanistic implications. We refer instead to two classes of regulated enzymes K-series and V-se-ries enzymes. For K-series allosteric enzymes, the substrate saturation kinetics are competitive in the sense that is raised without an effect on V. For V-series allosteric enzymes, the allosteric inhibitor lowers... 

Because noncompetitive inhibitors bind to both the free enzyme and the ES complex, or subsequent species in the reaction pathway, we would expect these molecules to exert a kinetic effect on the E + S —> ES" process, thus effecting the apparent values of both VmdX/KM (influenced by both the K and al, terms) and Vmax (influenced by the aK term). This is reflected in the velocity equation for noncompetitive inhibition ... 

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