Conformational selection or induced fit: A flux description of reaction mechanism

Abstract

The mechanism of ligand binding coupled to conformational changes in macromolecules has recently attracted considerable interest. The 2 limiting cases are the ''induced fit'' mechanism (binding first) or ''conformational selection'' (conformational change first). Described here are the criteria by which the sequence of events can be determined quantitatively. The relative importance of the 2 pathways is determined not by comparing rate constants (a common misconception) but instead by comparing the flux through each pathway. The simple rules for calculating flux in multistep mechanisms are described and then applied to 2 examples from the literature, neither of which has previously been analyzed using the concept of flux. The first example is the mechanism of conformational change in the binding of NADPH to dihydrofolate reductase. The second example is the mechanism of flavodoxin folding coupled to binding of its cofactor, flavin mononucleotide. In both cases, the mechanism switches from being dominated by the conformational selection pathway at low ligand concentration to induced fit at high ligand concentration. Over a wide range of conditions, a significant fraction of the flux occurs through both pathways. Such a mixed mechanism likely will be discovered for many cases of coupled conformational change and ligand binding when kinetic data are analyzed by using a fluxbased approach.kinetics ͉ binding ͉ folding ͉ coupled equilibria ͉ mechanism T he binding of ligands by macromolecules is crucial to a multitude of physiological processes. These include the cascade of reactions accompanying hormone binding to receptors (1, 2), enzyme reactions initiated by the binding of substrates to the enzyme (3, 4), gene regulation by the binding of molecules to DNA (5) and RNA (6), and the folding of unstructured proteins to produce biologically active molecules (7,8). In virtually all cases, the binding of ligands and conformational changes go hand in hand. Consequently, considerable effort has been expended in assessing the detailed mechanism by which ligand binding and conformational changes are coupled (3,4,9). Two limiting mechanisms are generally considered: (i) ''conformational selection,'' whereby the ligand selectively binds to a form of the macromolecule that is present only in small amounts, eventually converting the macromolecule to the ligand-bound conformation; and (ii) ''induced fit,'' (9) whereby ligand binds to the predominant free conformation followed by a conformational change in the macromolecule to give the preferred ligandbound conformation.The 2 limiting mechanisms can be written as:Conformational Selection: P weak º P tightInduced Fit: P weak ϩ L º P weak ⅐LIn these equations, P weak and P tight represent the tightly and weakly binding conformations of the protein (or other macromolecule), and L is the ligand. In point of fact, these 2 limiting mechanisms can be distinguished by kinetic measurements of the rate of the conformational change. In the simplest case, which often prevails, the ligand-bind...

Protein dynamics and function from solution state NMR spectroscopy

“…Flux analyses have shown that ligand and protein concentrations are central in determining whether conformational selection or induced fit pathways dominate. It is in fact possible for a conformational selection model to dominate at low substrate concentrations but to be replaced by induced-fit binding at higher substrate concentrations, as was demonstrated to be the case for DHFR (Hammes et al 2009). A generic problem with the conformational selection model is that active states of enzymes may be unavailable to substrates due to steric occlusion (Sullivan & Holyoak, 2008).…”

Single Enzyme Studies Reveal the Existence of Discrete Functional States for Monomeric Enzymes and How They Are “Selected” upon Allosteric Regulation

“…3,38,40−45 The same mechanism was successfully borrowed for monomeric enzymes to explain substrate mediated conformational redistributions along the reaction coordinate. 2,5,25,46 Regulated enzymes, however, in addition to their reaction coordinate, have a conformational-regulatory coordinate. A major structural rearrangement has to occur for the enzyme to adopt the active conformational state, where the active site is accessible and catalysis may occur.…”

Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model

“…The opposite scenario, of rapid transitions between non-binding and binding conformations together with the ability to form binding contacts even in the unbound state, would tend to favour induced fit. [22][23][24] Experiments have yielded valuable results on many aspects of IDP structure and function. The most powerful methods are single molecule FRET experiments, which can provide information on the overall dimensions 25 and dynamics 26 of unbound IDPs, and NMR experiments which give more localised and higher resolution structural data 27,28 and also mechanistic insights.…”