Enzyme kinetics competitive

In this chapter we described the thermodynamics of enzyme-inhibitor interactions and defined three potential modes of reversible binding of inhibitors to enzyme molecules. Competitive inhibitors bind to the free enzyme form in direct competition with substrate molecules. Noncompetitive inhibitors bind to both the free enzyme and to the ES complex or subsequent enzyme forms that are populated during catalysis. Uncompetitive inhibitors bind exclusively to the ES complex or to subsequent enzyme forms. We saw that one can distinguish among these inhibition modes by their effects on the apparent values of the steady state kinetic parameters Umax, Km, and VmdX/KM. We further saw that for bisubstrate reactions, the inhibition modality depends on the reaction mechanism used by the enzyme. Finally, we described how one may use the dissociation constant for inhibition (Kh o.K or both) to best evaluate the relative affinity of different inhibitors for ones target enzyme, and thus drive compound optimization through medicinal chemistry efforts. 

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. 

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... 

Enzyme kinetic studies of inhibitor are very important for considering as a therapeutic agent. It is interesting to note that isoprenoid-substituted flavonoids having non-steroidal structures are potent un-competitive inhibitors of 5a-reductase. So, it would be expected that the isoprenoid-substituted flavonoid derivative would be an interesting lead compounds for testosterone 5a-reductase inhibitor. 

NACs in a laboratory column system containing aquifer material from the banks of a river-groundwater infiltration site (Fig. 14.11). The columns were run under ferrogenic conditions. Note that zero-order kinetics suggests that the reactive sites were always saturated such as encountered in enzyme kinetics at saturation (Box 12.2). In this system, all model compounds as well as other NACs including again TNT, ADNTs, and DANTs (data not shown, see Hofstetter et al., 1999) reacted at virtually the same rate. However, when present in mixtures, the compounds showed competition for the reactive sites. A competition quotient, Qc (competition with the reference compound 4-C1-NB present at about equal concentrations) was determined for all model compounds ... 

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... 

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. 

Many problems involving competitive reaction kinetics may be treated by invoking the steady-state assumption within the digital simulation this has been done in at least two instances [29-34]. The first of these involves the development of a model for enzyme catalysis in the amperometric enzyme electrode [29-31]. In this model, the enzyme E is considered to be immobilized in a diffusion medium covering an electrode that is operated at a fixed potential such that the product (P) of enzyme catalysis is electroactive under diffusion-controlled conditions. (This model has also served as the basis for the simulation of the voltammetric response of the enzyme electrode [35].) The substrate (S) diffuses through the medium that contains the immobilized enzyme and is catalyzed to form P by straightforward enzyme kinetics ... 

An expression describing enzyme kinetics in the presence of a competitive inhibitor can be derived straightforwardly. Consider the reaction that we treated previously. 

Because of its cyclic nature, this process presents analogies with molecular catalysis it may be considered as physical catalysis operating a change in location, a translocation, on the substrate, like chemical catalysis operates a transformation into products. The carrier is the transport catalyst which strongly increases the rate of passage of the substrate with respect to free diffusion and shows enzyme-like features (saturation kinetics, competition and inhibition phenomena, etc.). The active species is the carrier-substrate supermolecule. The transport of substrate Sj may be coupled to the flow of a second species S2 in the same (symport) or opposite antiport) direction. 

This mechanism is important for compounds that lack sufficient lipid solubility to move rapidly across the membrane by simple diffusion. A membrane-associated protein is usually involved, specificity, competitive inhibition, and the saturation phenomenon and their kinetics are best described by Michaelis-Menton enzyme kinetic models. Membrane penetration by this mechanism is more rapid than simple diffusion and, in the case of active transport, may proceed beyond the point where concentrations are equal on both... 

FIGURE 4.12 Effect of a competitive, reversible inhibitor on enzyme kinetics... 

For a detailed review of simple to complex enzyme kinetics, a book by Segel (21) is recommended. Most P450 oxidations show hyperbolic saturation kinetics and competitive inhibition between substrates. Therefore, both Km values and drug interactions can be predicted from inhibition studies. Competitive inhibition suggests that the enzymes have a single binding site and only one substrate can bind at any one time. For the inhibition of substrate A by substrate B to be competitive, the following must be observed ... 

Reversible inhibition can be competitive or non-competitive. Competitive inhibitors bind to the active site and compete with the substrate for binding to the enzyme. However this means that increasing the S concentration will progressively outcompete the inhibitor. Accordingly a Lineweaver—Burk analysis of enzyme kinetic data obtained in the presence or absence of a competitive inhibitor will yield the same Fmax (at infinite S concentration) but the Am in the presence of the inhibitor (A, ) will be higher (poorer binding) than the Am measured in the absence of competitive inhibitor. Knowing the inhibitor concentration [I] one can calculate the A) from the relation ... 

The non-competitive and uncompetitive modes of inhibition described above are special cases that in practice arise very rarely in these simple forms. In reality, the situation is usually more complex in that inhibitors bind with differing affinities to the free and substrate-bound forms of the enzyme, and also the ternary EIS complex may be able to undergo catalysis, albeit at a lower rate. These circumstances define what is called mixed inhibition, which is less easy to characterise since the kinetic behaviour and equations are much more complex. The reader is referred to Cor-nish-Bowden (1995) for a comprehensive and authoritative account of this and other aspects of enzyme kinetics. 

Figure 8.37. Competitive Inhibitiou Illustrated on a Double-Reciprocal Plot. A double-reciprocal plot of enzyme kinetics in the presence ( - - ) and absence of a competitive inhibitor illustrates that the inhibitor has no effect...

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