Reversible binding equilibria

As we described in Chapter 3, the binding of reversible inhibitors to enzymes is an equilibrium process that can be defined in terms of the common thermodynamic parameters of dissociation constant and free energy of binding. As with any binding reaction, the dissociation constant can only be measured accurately after equilibrium has been established fully measurements made prior to the full establishment of equilibrium will not reflect the true affinity of the complex. In Appendix 1 we review the basic principles and equations of biochemical kinetics. For reversible binding equilibrium the amount of complex formed over time is given by the equation... 

The interaction between free thyroid hormones and their binding proteins (TBPs) conforms to a reversible binding equilibrium that is described by the following mass action relationship ... 

Figure 11. Allosteric regulation A conformational change of the active site of an enzyme induced by reversible binding of an effector molecule (A). The model of Monod, Wyman, and Changeux (B) Cooperativity in the MWC is induced by a shift of the equilibrium between the T and R state upon binding of the receptor. Note that the sequential dissociation constants Kr and KR do not change. The T and R states of the enzyme differ in their catalytic properties for substrates. Both plots are adapted from Ref. 140. See color insert.

The basis for this technique lies in the competition between the test antigen and a labelled antigen for the available binding sites on a fixed amount of antibody. While the binding sites are traditionally associated with an antibody, any source of specific reversible binding sites may be used to create an assay in this format. Examples of such are specific transport proteins such as thyroxine-binding globulin and certain cellular receptors such as opiate or benzodiazepine receptors. Under these circumstances the equilibrium mixture may be represented thus ... 

Here, we present a formulation suitable for reversible binding of a Ugand and a protein to form a binary complex. The two-state kinetic model involves the interacting molecular species L and P under equilibrium conditions, exchanging between their respective free states and the bound state L P according to Scheme 1. 

The thermodynamics of solute interaction with nonpolar ligates of the stationary phase will be treated later in this chapter within the framework of the solvophobic theory 107-108). According to this theoretical approach the equilibrium constant for the reversible binding of a given eluite to the hydrocarbonaceous ligates at fixed eluent properties and temperature can be approximated by the relationship... 

In general, the reversible binding of a protein (P) to a ligand (L) can be described by a simple equilibrium expression ... 

The process of reversible inhibition is described by an equilibrium interaction between enzyme and inhibitor. Most inhibition processes can be classified as competitive or noncompetitive, depending on how the inhibitor impairs enzyme action. A competitive inhibitor is usually similar in structure to the substrate and is capable of reversible binding to the enzyme active site. In contrast to the substrate molecule, the inhibitor molecule cannot undergo chemical transformation to a product however, it does interfere with substrate binding. A noncompetitive inhibitor does not bind in the active site of an enzyme but binds at some other region of the enzyme molecule. Upon binding of the noncompetitive inhibitor, the enzyme is reversibly converted to a nonfunctional conformational state, and the substrate, which is fully capable of binding to the active site, is not converted to product. 

Thus, every El complex reduces the amount of enzyme available for catalysis, regardless of where the inhibitor binds. As shown in Eq. (2.60), inhibition is a reversible process. The degree of reversibility depends on the ratio k k i or in other words, on the inhibitor binding equilibrium constant, K . 

Equilibrium data for the oxygen absorption have been deduced, and it is claimed that, for example, [MnI2(PBu3)] will reversibly absorb and desorb completely one mole of dioxygen per mole of complex for more than 400 times, if the reaction is carried out in solution at — 20 °C. In the solid state only the thiocyanato compounds of formulation [Mn(NCS)2(PR3)] (where R = alkyl) reversibly bind oxygen.192... 

The means by which NAD affects the oxidation of NADH is still uncertain. The evidence for two pyridine nucleotide binding sites is not compelling. The alternative explanation that NAD functions by reversing the equilibrium between EHj and 4-electron-reduced enzyme (EH4) is shown in Eq. (9). There is some kinetic evidence for a dead end complex... 

Drugs bind to different proteins, such as albumin, globulins (e.g., al-acid glycoprotein AAG and trans-cortin), and lipoprotein in plasma, and to tissue proteins. The reversible binding of a drug to macromolecules such as proteins is an equilibrium process. The relationship is described by the law of mass action ... 

The curvature seen for reversible inhibition [Fig. 2.12 (a)] indicates that an inhibitor-binding equilibrium precedes the conversion of substrate to product. Three types of reversible inhibition may be distinguished. (1) Competitive inhibition occurs when the degree of inhibition decreases as substrate concentration increases and Vmax is unaffected. (2) Noncompetitive inhibition exists when the degree of inhibition does not vary with substrate concentration, and Km is unaffected. (3) Uncompetitive inhibition exists if the degree of inhibition increases as substrate concentration increases both Vmax and Km are affected. Uncompetitive inhibition is often thought of as a mixture of competitive and noncompetitive behavior. 

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