Inhibition uncompetitive

Uncompetitive inhibitors bind with the ES complex, affecting both the apparent Km and the apparent VW of an enzymatic reaction. Their behavior is approximated by Eq. 2.42  

In this case, Lineweaver-Burk plots (Fig. 2.15) yield a series of parallel lines (in the case of one-substrate enzymes), in which the y intercept is equal to the reciprocal of the apparent Vmax, 1/Vmax,app = 1 + while the x intercept is the 

Uncompetitive inhibition results when both the substrate and the inhibitor adsorb on the same site and the resulting complex is unreactive. The equilibria associated with this type of inhibition is shown in Eqn. 7.36. 

The reciprocal form of the rate expression for this equation is given by Eqn. 7.37. 

The plots of 1/v versus 1/(S] at various concentrations of I are shown in Fig. 7.9. The slopes of the lines are not modified by either [I] or K so the lines for the plots associated with different concentrations of 1 are parallel to the line from the uninhibited reaction. The y-axis intercepts are lA max(app) nd the x-axis intercepts are 1/K v (app). max(app) rewritten in linear form as in 

1/K]vi(app), the x-axis intercepts in Fig. 7.9, is given in Eqn. 7.30. The replot of 1/Kivi(app) versus [I] also has a slope of 1/K x Wmax x-axis intercept of -K. Tne y-axis intercept, though, is l/Km. Thus, values for Kj and V ,a can be obtained directly with determined by using Eqn. 7.31. 

From the above discussion it can be seen that a competitive inhibitor has no effect on V ,aj but does influence K yj(app). Non-competitive inhibition, however, results in a decrease in but no change in while uncompetitive inhibition produces a decrease in both V ,a and Kiyj. 

Uncompetitive inhibition occurs when the inhibitor binds to the enzyme-substrate complex instead of the enzyme, resulting in a decrease and values. Equation 4.36 shows the mechanism and Equation 4.37 shows the reaction rate expression  

Determination of the inhibition kinetic parameter follows the same approach as simple Michaelis-Menten kinetics, discussed earlier, using the linearization approach. Thus, it is better to express Equations 4.33, 4.35, and 4.37 in terms of apparent parameters, as shown in Equation 4.38. This is compatible with a simple Michaelis-Menten relationship. 

Supercritical Fluids Technology in Lipase Catalyzed Processes 

Apparent Kinetic Parameter for Different Inhibition Mechanisms Mechanism 

FIGURE 4.3 Graphical representation of Burke double reciprocal plots for (a) without inhibition, (b) competitive inhibition, (c) noncompetitive inhibition, and (d) uncompetitive inhibition. 

In this type of reversible inhibition, a compound interacts with the enzyme-substrate complex at a site other than the active site. 

This results in an apparent decrease in both Vmax and Ks. The apparent increase in affinity of enzyme for substrate (i.e., a decrease in Ks) is due to unproductive substrate binding, resulting in a decrease in free enzyme 

The model of uncompetitive inhibition describes a case in which the inhibitor combines reversibly with the enzyme-substrate complex after it forms. Further, it is assumed that this complex is so stable that it does not lead to formation of the expected product. If this were not so, this case would reduce to a special case of noncompetitive inhibition. The formation of the inactive complex, ESI, is written as 

Following the procedures analogous to those used in describing the other types of inhibition, we can derive the equation 

FIGURE 6.10 A Lineweaver-Burk plot for the case of uncompetitive inhibition at three concentrations of inhibitor. 

From the discussion presented, it should be apparent that enzyme inhibition is an important but compHcating aspect of the study of the kinetics of enzyme-catalyzed reactions. Although it will not be discussed in detail, another type of inhibition occurs when a product forms a stable complex with the enzyme. This leads to a decrease in the rate of the reaction not only because some substrate is constmied, but also because of the decrease in the effective concentration of the enzyme. For additional details on enzyme inhibition, consult the references Usted at the end of this chapter. 

Substances which only bind the ES complex and not the free enzyme E are uncompetitive inhibitors  

Compared to Eq. (30) the rate equation for uncompetitive inhibitors includes the same term for the equilibrium of decomposition of the ESI complex into E, S and I, but no term for an equilibrium of an El complex. 

One special form of uncompetitive inhibition is substrate inhibition. Here a second substrate molecule binds at the ES complex resulting in an inactive ESS complex. This form of inhibition is often found and will be discussed below (see acylase kinetics, Fig. 7-20 A). 

As with the other types of inhibition, equations have been derived that permit estimation of the inhibition constant as well as and Unax for uncompetitive inhibition (Eq. 4.19). 

That both catalytic (Vmax) and specific ( m) effects are noted in uncompetitive inhibition is noted in the following equations. Both and are reduced by the same factor (Eqs. 4.20 and 4.21)  

In the simplest monosubstrate case, an uncompetitive inhibitor would bind reversibly to the enzyme-substrate complex yielding an inactive EAI complex the inhibitor does not bind to the free enzyme. The kinetic model for this type of inhibition would be 

However, this case is not a common one. A very rare example is the reaction catalyzed by isocitrate dehydrogenase. In this reaction a-ketoglutarate shows a strong uncompetitive substrate inhibition versus NADPH or CO2, which does not result from combination with the enzyme-NADP complex. Presumably, it occurs by imine formation with a lysine in the central complex. The closure of the active site exposes this lysine, which must have a low enough ipKa to form an imine at neutral pH. In support of this model, oxalyl-glycine, a mimic of a-ketoglutarate that binds with equal affinity, does not show the effect (Grissom Cleland, 1988). 

This kinetic model is the usual description of an uncompetitive inhibition in biochemical textbooks, again usually without specifying that it is a rare case. 

As a mle, an uncompetitive inhibition occurs only if there are more than one substrate or product (Huang, 1990). For example, an uncompetitive inhibition will take place in a Rapid Equilibrium Order bisubstrate reaction, when an inhibitor competes with B while A is the variable substrate. Thus, the equilibria shown below describe an ordered bisubstrate system in which an inhibitor competes with B but does not bind to free enzyme. 

In this case, I wiU be an uncompetitive inhibitor with respect to A and a competitive inhibitor with respect to B (Fig. 3). 

Here the inhibitor has no affinity for the enzyme itself and thus does not c pete with the substrate for the enzyme instead it ties up the enzyme-subs complex by forming an inhibitor-enzyme-substrate complex. (I E S) w is inactive. In uncompetitive inhibition, the inhibitor reversibly ties enzyme-substrate complex after it has been formed. 

As with competitive inhibition, two additional reaction steps are addr the Michaelis-Menten kinetics for uncompetitive inhibition as shown in i tion Steps 4 and 5. 

Raie luiv fot Starting with equation for rate of formation of product. Equation (7-34), 

Reaction Mechanisms. Pathweys, Bioreacilons, and Bioreactors Chap. 7 

Starting with the equation for the rate of formation of product. Equation (9-34), and then applying the pseudo-steady-state hypothesis to the intermediate (I E S), we arrive at the rate law for uncompetitive inhibition 

The intermediate st are shown in the Chapter 9 Summary Notes on the DVD-ROM and on the Web. Rearranging Equation (9-40) 

Reaction Mechanisms. Pathways, Bioreactions, and Bioreactors Chapter 9 

In this case the inhibitor reacts only with enzyme-substrate complex  

Rearranging Equation 2.76 into an equation for a straight line, the reaction rate becomes  

The double reciprocal plot (Fig. 2.30c) shows that in the presence of an uncompetitive inhibitor, both the maximum velocity, V, and Km are changed but not the ratio of Km/V. Hence the slopes of the lines are equal and in the presence of increasing amounts of inhibitor, the lines plotted are parallel. Uncompetitive inhibition is rarely found in single-substrate reactions. It occurs more often in two-substrate reactions. 

In conclusion, it can be stated that the three types of reversible inhibition are kinetically distinguishable by plots of reaction rate versus substrate concentration using the procedure developed by Lineweaver and Burk (Fig. 2.30). 

---

Like a noncompetitive inhibitor, an uncompetitive inhibitor does not compete with the substrate since it binds to the enzyme—substrate complex but not to the free enzyme. Uncompetitive inhibition... 

The three most common types of inhibitors in enzymatic reactions are competitive, non-competitive, and uncompetitive. Competitive inliibition occurs when tlie substrate and inhibitor have similar molecules that compete for the identical site on the enzyme. Non-competitive inhibition results in enzymes containing at least two different types of sites. The inhibitor attaches to only one type of site and the substrate only to the other. Uncompetitive inhibition occurs when the inhibitor deactivates the enzyme substrate complex. The effect of an inhibitor is determined by measuring the enzyme velocity at various... 

Enzyme reaction kinetics were modelled on the basis of rapid equilibrium assumption. Rapid equilibrium condition (also known as quasi-equilibrium) assumes that only the early components of the reaction are at equilibrium.8-10 In rapid equilibrium conditions, the enzyme (E), substrate (S) and enzyme-substrate (ES), the central complex equilibrate rapidly compared with the dissociation rate of ES into E and product (P ). The combined inhibition effects by 2-ethoxyethanol as a non-competitive inhibitor and (S)-ibuprofen ester as an uncompetitive inhibition resulted in an overall mechanism, shown in Figure 5.20. 

P-site ligands inhibit adenylyl cyclases by a noncompetitive, dead-end- (post-transition-state) mechanism (cf. Fig. 6). Typically this is observed when reactions are conducted with Mn2+ or Mg2+ on forskolin- or hormone-activated adenylyl cyclases. However, under- some circumstances, uncompetitive inhibition has been noted. This is typically observed with enzyme that has been stably activated with GTPyS, with Mg2+ as cation. That this is the mechanism of P-site inhibition was most clearly demonstrated with expressed chimeric adenylyl cyclase studied by the reverse reaction. Under these conditions, inhibition by 2 -d-3 -AMP was competitive with cAMP. That is, the P-site is not a site per se, but rather an enzyme configuration and these ligands bind to the post-transition-state configuration from which product has left, but before the enzyme cycles to accept new substrate. Consequently, as post-transition-state inhibitors, P-site ligands are remarkably potent and specific inhibitors of adenylyl cyclases and have been used in many studies of tissue and cell function to suppress cAMP formation. 

Figure 3.2 Cartoon representations of the three major forms of reversible inhibitor interactions with enzymes (A) competitive inhibition (B) noncompetitive inhibition (C) uncompetitive inhibition. Source-. From Copeland (2000).

An inhibitor that binds exclusively to the ES complex, or a subsequent species, with little or no affinity for the free enzyme is referred to as uncompetitive. Inhibitors of this modality require the prior formation of the ES complex for binding and inhibition. Hence these inhibitors affect the steps in catalysis subsequent to initial substrate binding that is, they affect the ES —> ES1 step. One might then expect that these inhibitors would exclusively affect the apparent value of Vm and not influence the value of KM. This, however, is incorrect. Recall, as illustrated in Figure 3.1, that the formation of the ESI ternary complex represents a thermodynamic cycle between the ES, El, and ESI states. Hence the augmentation of the affinity of an uncompetitive inhibitor that accompanies ES complex formation must be balanced by an equal augmentation of substrate affinity for the El complex. The result of this is that the apparent values of both Vmax and Ku decrease with increasing concentrations of an uncompetitive inhibitor (Table 3.3). The velocity equation for uncompetitive inhibition is as follows ... 

Comparing Equations (3.1), (3.3), and (3.7), it is easy to recognize that competitive and uncompetitive inhibition are merely special cases of the more general case of... 

The modality of compounds that inhibit enzymes catalyzing bisubstrate reactions will differ with respect to the two substrates of the reaction, and the pattern of inhibition will depend on the reaction mechanism of the enzyme. Thus, when we use terms like competitive, noncompetitive, or uncompetitive inhibition, we must... 

For uncompetitive inhibition, the value of kobs will increase as a rectangular hyperbola with increasing substrate concentrations according to Equation (6.17) ... 

And for tight binding uncompetitive inhibition, the relationship is given by... 

A much more useful classification of inhibitors can be made on the basis of the mechanisms by which they act. Competitive inhibitors combine, with the enzyme at the same site as the substrate does, thus blocking the first step in the sequence. Noncompetitive inhibitors combine with the enzyme at some other site to give a complex that can still combine with the substrate, but the resultant ternary complex is unreactive. Uncompetitive inhibition results when the inhibitor and substrate combine with enzyme forms as in the following mechanism. 

These three classes of inhibition can be distinguished by virtue of the effect of variations in inhibitor concentration on the slopes and intercepts of reciprocal plots. For competitive inhibition only the slope varies. For uncompetitive inhibition only the intercept varies, while for noncompetitive inhibition both the slope and the intercept vary. 

At very low substrate concentration ([S] approaches zero), the enzyme is mostly present as E. Since an uncompetitive inhibitor does not combine with E, the inhibitor has no effect on the velocity and no effect on Vmsa/Km (the slope of the double-reciprocal plot). In this case, termed uncompetitive, the slopes of the double-reciprocal plots are independent of inhibitor concentration and only the intercepts are affected. A series of parallel lines results when different inhibitor concentrations are used. This type of inhibition is often observed for enzymes that catalyze the reaction between two substrates. Often an inhibitor that is competitive against one of the substrates is found to give uncompetitive inhibition when the other substrate is varied. The inhibitor does combine at the active site but does not prevent the binding of one of the substrates (and vice versa). 

Uncompetitive inhibition is extremely rare in nature, and can arise when the inhibitor binds to the enzyme-substrate complex, rather than to the free enzyme, as in competitive inhibition [111]. In uncompetitive inhibition, an increase in the concentration of the inhibitor requires a disproportionately large increase in the concentration of the substrate to maintain the same metabolic turnover. 

In uncompetitive inhibition, the inhibitor combines with enzyme-substrate complex to form an uncreative complex (InES) as follows ... 

It can be observed that when InES is not at all formed, the above rate law reduces as equation (6.56), while if InE is not formed at all, the rate law reduces to that for uncompetitive inhibition as equation (6.58). 

The inhibition can be interpreted as an increase of the Michaelis constant KM. In the case of uncompetitive inhibition (Fig. 9B), the binding of the substrate to the enzyme is not affected. However, the [ES] complex becomes inactive upon binding of the inhibitor Using Kj —> oo, the corresponding rate equation is... 

---