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...

Understanding the molecular mechanisms of enzyme catalysis and allosteric regulation has been a primary goal of biochemistry for many years. The dynamics of these processes, approached through a variety of kinetic methods, are discussed. The results obtained for many different enzymes suggest that multiple intermediates and conformations are general characteristics of the catalytic process and allosteric regulation. Ribonuclease, dihydrofolate reductase, chymotrypsin, aspartate aminotransferase, and aspartate transcarbamoylase are considered as specific examples. Typical and maximum rates of conformational changes and catalysis are also discussed, based on results obtained from model systems. The nature and rates of interconversion of the intermediates, along with structural information, can be used as the bases for understanding the incredible catalytic efficiency of enzymes. Potential roles of conformational changes in the catalytic process are discussed in terms of static and environmental effects, and in terms of dynamic coupling within the enzyme-substrate complex.

This brief review discusses our current understanding of the molecular basis of enzyme catalysis. A historical development is presented, beginning with steady state kinetics and progressing through modern fast reaction methods, NMR, and single molecule fluorescence techniques. Experimental results are summarized for ribonuclease, aspartate aminotransferase, and especially dihydrofolate reductase (DHFR). Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all enzyme mechanisms. In the case of DHFR, theoretical investigations have provided detailed information about the movement of atoms within the enzyme-substrate complex as the reaction proceeds along the collective reaction coordinate for hydride transfer. A general mechanism is presented for enzyme catalysis that includes multiple intermediates and a complex, multidimensional standard free energy surface. Protein flexibility, diverse protein conformations, and cooperative conformational changes are important features of this model.

The concept is developed that enzyme mechanisms should be viewed as "catalytic networks" with multiple conformations that occur serially and in parallel in the mechanism. These coupled ensembles of conformations require a multi-dimensional standard free-energy surface that is very "rugged", containing multiple minima and transition states. Experimental and theoretical evidence is presented to support this concept.