Enzyme Inhibition Kinetics

ESI Study of enzyme kinetics, inhibition, multiple reaction monitoring Norris ef al. [52]... 

Usually initial rates are measured in enzyme kinetics so as to avoid problems arising from kinetic complications such as product inhibition. 

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). 

The above rate equation is in agreement with that reported by Madhav and Ching [3]. Tliis rapid equilibrium treatment is a simple approach that allows the transformations of all complexes in terms of [E, [5], Kls and Kjp, which only deal with equilibrium expressions for the binding of the substrate to the enzyme. In the absence of inhibition, the enzyme kinetics are reduced to the simplest Michaelis-Menten model, as shown in Figure 5.21. The rate equation for the Michaelis-Menten model is given in ordinary textbooks and is as follows 11... 

The reaction rate for this enzyme kinetics example is expressed by the Michaelis-Menten equation and with product inhibition. 

In the presence of sucrose alone as the single substrate, initial reaction rates follow Michaelis-Menten kinetics up to 200 mM sucrose concentration, but the enzyme is inhibited by higher concentrations of substrate.30 The inhibitor constant for sucrose is 730 mM. This inhibition can be overcome by the addition of acceptors.31,32 The enzyme activity is significantly enhanced, and stabilized, by the presence of dextran, and by calcium ions. 

Substrates may affect enzyme kinetics either by activation or by inhibition. Substrate activation may be observed if the enzyme has two (or more) binding sites, and substrate binding at one site enhances the alfinity of the substrate for the other site(s). The result is a highly active ternary complex, consisting of the enzyme and two substrate molecules, which subsequently dissociates to generate the product. Substrate inhibition may occur in a similar way, except that the ternary complex is nonreactive. We consider first, by means of an example, inhibition by a single substrate, and second, inhibition by multiple substrates. 

MS is lower than that of M the system is in the regime of substrate saturation addition of more S does not lead to a rate increase. The behaviour of the reaction rate in case B is typical of enzymes and in biochemistry this is referred to as Michaelis-Menten kinetics. The success of the application of the Michaelis-Menten kinetics in biochemistry is based on the fact that indeed only two reactions are involved the complexation of the substrate in the pocket of the enzyme and the actual conversion of the substrate. Usually the exchange of the substrate in the binding pocket is very fast and thus we can ignore the term k2[H2] in the denominator. Complications arise if the product binds to the binding site of the enzyme, product inhibition, and more complex kinetics result. 

Thereafter, a reference text such as Enzyme Kinetics (Segel, 1993) should be consulted to determine whether or not the proposed mechanism has been described and characterized previously. For the example given, it would be found that the proposed mechanism corresponds to a system referred to as partial competitive inhibition, and an equation is provided which can be applied to the experimental data. If the data can be fitted successfully by applying the equation through nonlinear regression, the proposed mechanism would be supported further secondary graphing approaches to confirm the mechanism are also provided in texts such as Enzyme Kinetics, and values could be obtained for the various associated constants. If the data cannot be fitted successfully, the proposed reaction scheme should be revisited and altered appropriately, and the whole process repeated. 

MULTISUBSTRATE SYSTEMS. Wong and Hanes were probably among the first to suggest that alternative substrates may be useful in mechanistic studies. Fromm s laboratory was the first to use and extend the theory of alternative substrate inhibition to address specific questions about multisubstrate enzyme kinetic mechanisms. Huang demonstrated the advantages of a constant ratio approach when dealing with alternative substrate kinetics. 

While requiring the availability of competitive inhibitors for each of the substrates, Fromm s use of competitive inhibitors to distinguish multisubstrate enzyme kinetic pathways represents the most powerful initial rate method. See Alternative Substrate Inhibition... 

Di- and trifluoromethyl ketones inhibit a great number of esterases and proteases with often very high inhibition constants (cf. Chapter 7). Although the fluorinated ketone is covalently bonded to the nucleophilic residue of the enzyme, the inhibition is reversible, as the inhibitor could be displaced by another nucleophile. The covalent nature of the interactions as well as the tetrahedral structure of the adducts have been demonstrated by kinetic studies, by NMR experiments, and by the X-ray diffraction of the enzyme-substrate complexes. ... 

A number of cases are known in which the properties of an enzyme are markedly altered by interaction with a membrane. Of course, in some cases the normal function of an enzyme is destroyed when it is removed from the membrane. For example, the mitochondrial coupling factor cannot synthesize ATP when removed from the membrane, since coupling to a proton gradient is required. The portion of the coupling factor that is easily solubilized (F,) is an ATPase. The steady-state kinetic properties of this solubilized ATPase are appreciably changed when it is reconstituted with mitochondrial membranes The turnover numbers and pH dependencies are different the solubilized enzyme is strongly inhibited by ADP, whereas the reconstituted enzyme is not and the reconstituted enzyme is inhibited by oligomycin, whereas the solubilized enzyme is not. 

Since the four yeast PGK inhibitors are commercially available it was logical to test them for T. brucei PGK inhibition. The first three compounds were active in the millimolar range. However, SPADNS exhibited a K of 10.0 pMin these in preliminary tests [88]. Moreover, when assayed against a commercially available rabbit muscle PGK, SPADNS had no influence on the enzyme kinetics up to a concentration of 250 pM[88], In conclusion, SPADNS appears to be an excellent lead because of its potency and selectivity. Crystallographic experiments to determine its binding mode to I brucei VGK are underway. 

Inhibition may be incorporated into the mechanism of micellar catalysis in the same way it is handled in enzyme kinetics. Representing the inhibitor by /, we can revise Reaction (G) as follows ... 

The oxidation of acetaldehyde to acetic acid has been studied with NAD-linked ALDH purified from human, rat and Syrian hamster liver (Klyosov et al., 1996). The mitochondrial enzymes from these species have very similar kinetic properties, whereas human cytosolic ALDHl has a value of about 180 pM, compared with 15 pM and 12 pM for rats and hamsters, respectively. Apparently, in human liver, only mitochondrial ALDH oxidizes acetaldehyde at physiological concentrations, whereas both mitochondrial and cytosolic ALDHs of rodents can participate in acetaldehyde metabolism. The rodent cytosolic ALDHs are at least 10 times more sensitive that the human enzyme to inhibition by disulfiram. 

Reversible Inhibition One common type of reversible inhibition is called competitive (Fig. 6-15a). A competitive inhibitor competes with the substrate for the active site of an enzyme. While the inhibitor (I) occupies the active site it prevents binding of the substrate to the enzyme. Many competitive inhibitors are compounds that resemble the substrate and combine with the enzyme to form an El complex, but without leading to catalysis. Even fleeting combinations of this type will reduce the efficiency of the enzyme. By taking into account the molecular geometry of inhibitors that resemble the substrate, we can reach conclusions about which parts of the normal substrate bind to the enzyme. Competitive inhibition can be analyzed quantitatively by steady-state kinetics. In the presence of a competitive inhibitor, the Michaelis-Menten equation (Eqn 6-9) becomes... 

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