Dead-end inhibitors

The example of methotrexate points out that the inhibition modality of dead end inhibitors, with respect to a specific substrate, will depend on the reaction mechanism of the target enzyme. Thus a complete understanding of inhibition mechanism requires an understanding of the underlying reaction mechanism of the target enzyme. A comprehensive discussion of these issues has been provided by Segel (1975). Table 3.6 summarizes the pattern of dead-end inhibition observed for competitive inhibitors of one substrate in the common bisubstrate reaction mecha-... 

Based on the results of the preceding section, potential inhibitors to the enzymes involved, G6PDH and GR, were searched for among the compounds that participate in the experimental system considered. It was found that GR is inhibited by G6P, the substrate of the other reaction. To determine the inhibition constant, G6P was considered as a dead-end inhibitor [146] that... 

Exchange-inert complexes of Cr(III) with nucleotide ligands are very stable toward hydrolysis. Such complexes have proven to be extremely useful as chirality probes in that different coordination isomers can be prepared and separated These nucleotide complexes have also proved useful as dead-end inhibitors of enzyme-catalyzed reactions. Because Cr(lII) is paramagnetic, distances can be measured by measuring the effects of Cr(lll) on the NMR signals of nearby atoms when the Cr(lll)-nucleotide complex binds to the surface of a mac-romolecule. See Exchange-Inert Complexes... 

The determination of kinetic mechanisms requires more than just initial velocity patterns, and inhibition studies are usually required. Several types of inhibitors are useful. The products are substrates in the reverse reaction and thus have some affinity for the enzyme and will give inhibition unless their inhibition constants exceed their solubility. Dead-end inhibitors are molecules that play musical chairs with the substrates for open portions of the active site but do not react. Substrates may act as dead-end inhibitors by combining at points in the mechanism where they are not intended and thus cause substrate inhibition. The inhibition patterns caused by these inhibitors are useful in distinguishing between different kinetic mechanisms. 

Noncompetitive inhibitions result from combination of the inhibitor with an enzyme form other than the one the substrate combines with, and one that is present at both high and low levels of the substrate. An example is a dead-end inhibitor resembling the first substrate in an ordered mechanism. It is competitive versus A, but noncompetitive versus B, because B cannot prevent the binding of the inhibitor to free enzyme. In a random mechanism, an inhibitor binding at one site is noncompetitive versus a substrate binding at another site. 

Competitive and noncompetitive inhibitions are the most common types, especially for product inhibitors. The first product (P) released in an ordered mechanism, for example, gives noncompetitive inhibition versus either substrate A or B as the result of partially reversing the reaction. This result can occur at either low or high levels of substrate, and thus V/K as well as V is affected. A dead-end inhibitor combining with EQ in the same fashion, however, gives uncompetitive inhibition because it cannot reverse the reaction. 

Substrate inhibition is caused by the substrate acting as a dead-end inhibitor, which is most common in the nonphysio-logical direction of the reaction. When the concentration of the substrate giving the inhibition is varied, the rate equation is... 

Use of Dead-End Inhibitors to Determine Rate-Limiting Steps... 

The use of dead-end inhibitors to deduce kinetic mechanisms was covered in Volume 11, but two points deserve further mention. 

A dead-end inhibitor will often have affinity for both EA and EQ in an ordered mechanism and thus gives noncompetitive patterns in both directions. As long as it only causes inhibition by combining with EA and EQ, however, one can simply compare the K s value in one direction with the ATu value in the other as done above. Since one has data in both directions of the reaction, one learns the relative rates of the steps in both directions. This is a powerful technique that should be applied more often. 

Inhibition by substrates that also act as dead-end inhibitors was discussed in Volume II. In an ordered mechanism, however, addition of an inhibitor that mimics the first substrate may permit binding of the second substrate. In such a situation the presence of the inhibitor will induce substrate inhibition by the second substrate, as long as the first substrate addition is not in rapid equilibrium ... 

One of the first SULTlAl inhibitors identified in the rat liver was 2, 6-dichloro-4-nitrophenol (DCNP) (Mulder and Scholtens, 1977). DCNP is a dead-end inhibitor, and exhibits low IC50 values toward SULTlAl and SULT 1 A3 (Seah and Wong, 1994). Hydroxylated polychlorinated biphenyls (HPCBs) are potent inhibitors of recombinant human SULTIEI. HPCBs exhibit low micromolar IC50 values toward thyroid hormones (Schuur et al., 1998). Several dietary chemicals such as quercetin, curcumin, and flavones are known to inhibit SULTs. Some commonly used drugs that inhibit SULTlAl and SULT1A3 activity include NSAIDs such as mefenamic acid, naproxen, and salicylic acid. 

A variety of kinetic experiments is used to deduce this information. The algebraic form of the rate equation as a function of substrate concentrations limits the kinetic mechanism, wh inhibition patterns for products or dead-end inhibitors versus the various substrates pin it down, and often help to determine the rate-limiting step. Isotope exchange and partitioning studies complete the analysis of kinetic mechanism. The chemical mechanism is deduced by studying the pH variation of the kinetic parameters, which identifies the acid-base catalysts, and necessary protonation states of the substrate for binding and catalysis, and by certain kinetic isotope effect studies. 

The distribution equations describe the distribution of enzyme among various possible forms. These distribution equations when multiplied by Eo give the steady-state concentrations of various enzyme forms. The distribution equations are very complex with bisubstrate and trisubstrate reactions. They have some inherent interest by themselves, however, and are useful in deriving rate equations for reactions in the presence of dead-end inhibitors (Chapter 11), and equations for rates of isotopic exchange (Chapter 16). 

The kinetics of substrates, products, and alternate products define the form of the rate equation, and are certainly necessary to deduce the kinetic mechanism. However, they are often not sufficient to do this unequivocally and other kinetic approaches are necessary, especially when reaction can be studied in only one direction. One of the most useful approaches in such cases involves the use of dead-end inhibitors (Cleland, 1970, 1979, 1990). 

Dead-end inhibitor is a compound that reacts with one or more enzyme forms to yield a complex that cannot participate in the reaction. These nonreacting... 

The velocity equation in the presence of a dead-end inhibitor can be derived in the usual manner by the King-Altman method. However, if we know the velocity equation for the uninhibited reaction, then we can easily write the new velocity equation as modified by the inhibitor, without going through an entire derivation. The effect of a dead-end inhibitor is to multiply certain terms in the denominator of the uninhibited velocity equation by the factor F (F = i -t-//Xj), ths fractional concentration of an inhibitor. The terms multiplied by F, are those representing the enzyme form, or enzyme forms, combining with the inhibitor. Then, the Kj represents the dissociation constant of the specific enzyme form-inhibitor complex. 

To see how velocity equations are affected by a dead-end inhibitor, let us illustrate the above rule with several examples. 

Dead-end Inhibition in a Steady-State Ordered Bi Bi System In this system, a frequent case is when a dead-end inhibitor combines with EA... 

One of the most useful functions of dead-end inhibitors is in verifying ordered addition of substrates in cases where product inhibition studies cannot or do not give an unequivocal answer. This is one of the main reasons why the apphcation of dead-end inhibitors gained the popularity in enzyme kinetics (Leskovac et al, 1998, 1999). For this purpose, Fromm (1979) has developed a set of mles which are helpful in the identification of various mechanisms with the aid of dead-end inhibitors (Table 2). 

Mechanism Dead-end inhibitor 1 plot competitive with substrate A 1 plot B... 

Consider the pure noncompetitive inhibition by two nonexclusive inhibitors, depicted by Eq. (5.35). If there are two dead-end inhibitors I and X, the kinetic question is whether an EIX complex forms, and if so, whether the dissociation constant of I from EIX is the same as from El (and similarly for X from EIX and EX). To answer this, the substrate concentration is held constant, the concentration of the two inhibitors is varied, and initial velocities are determined (Yonetani Theorell, 1965 Yonetani, 1982). 

At higher concentrations, the substrates will often act as dead-end inhibitors, particularly when a reaction is being studied in the nonphysiological direction substrate inhibition does not normally occur at physiological substrate concentrations. To the kineticist, however, substrate inhibitions are one of the best diagnostic tools for studying mechanisms, and their importance cannot be overemphasized (Cleland, 1970, 1977, 1979 Fromm 1975). 

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