Kinetic binding equilibrium

L. G. Reevaluating equilibrium and kinetic binding parameters for lipophilic drugs based on a structural model for drug interaction with biological membranes. 

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... 

Mason, R. R Rhodes, D. G. Herbette, L. G., Reevaluating equilibrium and kinetic binding parameters for lipophilic drugs based on a structural model for drug interaction with bilogical membranes, J. Med. Chem. 34, 869-877 (1991). 

The most dramatic rate retardations of proton transfers have been observed when the acidic or basic site is contained within a molecular cavity. The first kinetic and equilibrium studies of the protonation of such a basic site were made with large ring bicyclic diamines [72] (Simmons and Park, 1968 Park and Simmons, 1968a). It was also observed (Park and Simmons, 1968b) that chloride ion could be trapped inside the diprotonated amines. The binding of metal ions and small molecules by macrocyclic compounds is now a well-known phenomenon (Pedersen, 1967, 1978 Lehn, 1978). In the first studies of proton encapsulation, equilibrium and kinetic measurements were made with several macrobicyclic diamines [72] using an nmr technique. 

The appropriate definition of nonspecific binding is essential prior to characterization of the kinetic and equilibrium properties of the binding interaction. As a rule, nonspecific binding can be defined using a concentration of the unlabelled ligand that is 100 times its Ka value for the sites of interest. Failure to appropriately define nonspecific binding will invalidate the determination of the binding parameters. 

Enolization and ketonization kinetics and equilibrium constants have been reported for phenylacetylpyridines (85a), and their enol tautomers (85b), together with estimates of the stability of a third type of tautomer, the zwitterion (85c). The latter provides a nitrogen protonation route for the keto-enol tautomerization. The two alternative acid-catalysed routes for enolization, i.e. O- versus Af-protonation, are assessed in terms of pK differences, and of equilibrium proton-activating factors which measure the C-H acidifying effects of the binding of a proton catalyst at oxygen or at nitrogen. 

There are numerous advantages to the FAC approach that differentiate it from many forms of bioassay - MS-dependent or otherwise. The FAC method offers thermodynamic and kinetic binding data from the breakthrough curves. As with the classical application of the FA method, the quality of the data is superb relative to other chromatographic or electrophoretic methods [9, 10]. It is an equilibrium method, as opposed to systems that rely upon the separation of bound from unbound, and this forms the basis of its accuracy. 

Kinetic and equilibrium data are available for complex formation between iron(III) and 4-Me0C6H4C(S)N(0 )H, a system studied in relation to the possibility that some natural siderophores may bind iron through a thiohydroxamate moiety. The Fe " " complex of this... 

A potential limitation encountered when one seeks to characterize the kinetic binding order of certain rapid equilibrium enzyme-catalyzed reactions containing specific abortive complexes. Frieden pointed out that initial rate kinetics alone were limited in the ability to distinguish a rapid equilibrium random Bi Bi mechanism from a rapid equilibrium ordered Bi Bi mechanism if the ordered mechanism could also form the EB and EP abortive complexes. Isotope exchange at equilibrium experiments would also be ineffective. However, such a dilemma would be a problem only for those rapid equilibrium enzymes having fccat values less than 30-50 sec h For those rapid equilibrium systems in which kcat is small, Frieden s dilemma necessitates the use of procedures other than standard initial rate kinetics. 

LZ Avila, Y-H Chu, EC Blossey, GM Whitesides. Use of affinity capillary electrophoresis to determine kinetic and equilibrium constants for binding of arylsulfonamides to bovine carbonic anhydrase. J Med Chem 36 126-133,... 

The basic requirement for any kinetic or equilibrium study is the availability of a convenient assay for monitoring the concentrations of reagents. In this chapter, we introduce the more common methods, emphasizing their practical uses and limitations rather than giving an in-depth or exhaustive survey, and we outline how binding and kinetic data are analyzed. 

The binding kinetics and equilibrium constants for five different (A/T)4 DNA sites have been studied in detail, showing that the binding kinetics are complex (Breusegem et al. 2002). Further information as to Hoechst binding by DNA has been obtained by fluorescence spectroscopy (Adhikary et al. 2003) and, to some extent, by atomic force microscopy (Zareie et al. 1998). 

A drug that binds reversibly to a protein, as shown in Figure 1A, displays hyperbolic saturation kinetics. At equilibrium, the fraction bound is as described by Eq. (1), where Kh = kyi/ku, ES is the enzyme-substrate complex and E, is the total enzyme ... 

If two different substrates bind simultaneously to the active site, then the standard Michaelis-Menten equations and competitive inhibition kinetics do not apply. Instead it is necessary to base the kinetic analyses on a more complex kinetic scheme. The scheme in Figure 6 is a simplified representation of a substrate and an effector binding to an enzyme, with the assumption that product release is fast. In Figure 6, S is the substrate and B is the effector molecule. Product can be formed from both the ES and ESB complexes. If the rates of product formation are slow relative to the binding equilibrium, we can consider each substrate independently (i.e., we do not include the formation of the effector metabolites from EB and ESB in the kinetic derivations). This results in the following relatively simple equation for the velocity ... 

Table 2 Kinetic and equilibrium parameters for O2 and CO binding in model heme complexes ...

Here we describe studies of the interaction of interleukin-6 (IL-6) with a soluble form of its cell surface receptor (sIL-6R). A procedure utilising a competition approach is presented which allows the determination of the equilibrium constant in solution thus avoiding any potential problems associated with deviation in kinetic characteristics upon surface immobilisation. In addition, binding characteristics of stable monomeric and dimeric forms of IL-6 are presented to demonstrate both the drastic influence of solute multivalency on kinetic and equilibrium properties and the importance of auxiliary techniques such as analytical ultracentrifugation for the interpretation of SPR data. 

Bradfield CA, Kende AS, Poland A. 1988. Kinetic and equilibrium studies of Ah receptor-ligand binding use of [125I]2-iodo-7,8-dibromodibenzo-p-dioxin. Molec. Pharmacol. 34 229-37... 

Figure 14.10. Elution profile of RNase as a function of free [5 -TMP], using an affinity column with immobilized 5 -TMP. The concentrations of 5 -TMP in the mobile phase were 5.0 x 10 4Af, 4.0 x 10 4Af, 3.0 x 10 4Af,2.0 x 10 4M, 1.0 x 10 4M, and 7.5 x 10 5M for the earliest to the latest eluted fractions, respectively. The inset shows the plot according to Eq. 14.24 that was used to determine the association constant for the RNase-5 -TMP binding reaction.11 [Reprinted, with permission, from B. M. Dunn and I. M. Chaiken, Biochemistry 14 (No. 11), 1975, 2343-2349. Evaluation of Quantitative Affinity Chromatography by Comparison with Kinetic and Equilibrium Dialysis Methods for the Analysis of Nucleotide Binding to Staphylococcal Nuclease . 1975 by American Chemical Society.]...

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