Allosteric modulator kinetics

In general, the kinetics of most allosteric modulators have been shown to be faster than the kinetics of binding of the tracer ligand. This is an initial assumption for this experimental approach. Under these circumstances, the rate of dissociation of the tracer ligand (pA t) n the presence of the allosteric ligand is given by [11, 12]... 

FIGURE 4.13 Effect of the allosteric modulator 5-(N-ethyl-N-isopropyl)-amyloride (EPA) on the kinetics dissociation of [3H] yohimbine from c/j-adrenoceptors, (a) Receptor occupancy of [3H] yohimbine with time in the absence (filled circles) and presence (open circles) of EPA 0.03 mM, 0.1 mM (filled triangles), 0.3 mM (open squares), 1 mM (filled squares), and 3 mM (open triangles), (b) Regression of observed rate constant for offset of concentration of [3H] yohimbine in the presence of various concentrations of EPA on concentrations of EPA (abscissae in mM on a logarithmic scale). Data redrawn from [13]. 

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). 

Allosteric enzymes do not follow the Michaelis-Menten kinetic relationships between substrate concentration Fmax and Km because their kinetic behaviour is greatly altered by variations in the concentration of the allosteric modulator. Generally, homotrophic enzymes show sigmoidal behaviour with reference to the substrate concentration, rather than the rectangular hyperbolae shown in classical Michaelis-Menten kinetics. Thus, to increase the rate of reaction from 10 per cent to 90 per cent of maximum requires an 81-fold increase in substrate concentration, as shown in Fig. 5.34a. Positive cooperativity is the term used to describe the substrate concentration-activity curve which is sigmoidal an increase in the rate from 10 to 90 per cent requires only a nine-fold increase in substrate concentration (Fig. 5.346). Negative cooperativity is used to describe the flattening of the plot (Fig. 5.34c) and requires requires over 6000-fold increase to increase the rate from 10 to 90 per cent of maximum rate. 

The regulation of enzymes by metabolites leads to the concept of allostenc regulation. Allosteric means other structure. Allosteric modulators can bind at a site other than the active site in question and cause activation or inhibition. These modulators can include the substrate itself, which binds at another active site in a multi-subunit enzyme. In fact, allosterically modulated enzymes almost always have a complex quaternary structure (multiple subunits) and exhibit non-Michaelis-Menten kinetics. 

AChE]. Potential links for the countermeasmes atropine, a muscarinic receptor antagonist, and diazepam, a positive allosteric modulator of the G ABAa receptor and NMDA antagonists, are shown without detailed kinetic interactions. 

The fact that the aliosterically preferred conformation may be relatively rare in the library of conformations available to the receptor may have kinetic implications. Specifically, if the binding site for the modulator appears only when the preferred conformation is formed spontaneously, then complete conversion to alios terically modified receptor may require a relatively long period of equilibration. For example, the allosteric p38 MAP kinase inhibitor BIRB 796 binds to a conformation of MAP kinase requiring movement of a Phe residue by 10 angstroms (so-called out conformation). The association rate for this modulator is 8.5 x 105 M-1 s-1, 50 times slower than that required for other inhibitors (4.3 x 107 M 1 s-1). The result is that while other inhibitors reach equilibrium within 30 minutes, BIRB 376 requires 2 full hours of equilibration time [8],... 

The activity of allosteric enzymes is adjusted by reversible binding of a specific modulator to a regulatory site. Modulators may be the substrate itself or some other metabolite, and the effect of the modulator may be inhibitory or stimulatory. The kinetic behavior of allosteric enzymes reflects cooperative interactions among enzyme subunits. 

Some allosteric enzymes are also classified by the way in which they are affected by the binding of a modulator some affect the value of Km without affecting that of Vmtx- They are classed as K-series enzymes while others, which affect Fmax without affecting Km, are called M-series enzymes. Figure 5.35 shows the characteristic kinetic patterns observed for K-series and M-series enzymes. There are, of course, exceptions to these two extremes of kinetic behaviour. 

The allosteric kinetic effects of ATCase are shown in Figure 7-6. The interaction of substrates with the enzyme is cooperative (an example of homotropic cooperativity), as indicated by the sigmoidal shapes of the v versus [S] plots, CTP being an inhibitor and ATP an activator. These modulators compete for the same regulatory site and modulate the affinity of the enzyme for its... 

Aside from the inordinately dominant light of molecular genetics, the new wave in biochemistry today is, what has come to be called, metabolic control analysis (MCA) (Comish-Bowden and Cardenas, 1990). The impetus behind this wave is the desire to achieve a holistic view of the control of metabolic systems, with emphasis on the notion of system. The classical, singular focus on individual, feedback-modulated (e.g., allosteric), rate-limiting enzymes entails a naive and myopic view of metabolic regulation. It has become increasingly evident that control of metabolic pathways is distributive, rather than localized to one reaction. MCA places a given enzyme reaction into the kinetic context of the network of substrate-product connections, effector relationships, etc., as supposedly exist in situ, it shows that control of fluxes, metabolite concentrations, inter alia, is a systemic function and not an inherent property of individual enzymes. Such... 

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