The Callender Lab

A first step in the catalytic cycle is to bind substrate to form the Michaelis complex. The efficiency of extricating ligands from solution to the binding pocket is very high, is often very specific to a particular ligand, and sometimes occurs with diffusion limited, or even faster, speeds. It has been suggested that conformational gating is a mechanism for enzyme-substrate binding specificity in some cases. We have a limited view of the dynamic binding of ligands to proteins. Most of our knowledge is based on static structural pictures of a protein comparing the empty protein with the protein-ligand complex and from thermodynamic studies of binding. In these structural pictures, the bound ligand is partially or completely isolated from contact with solvent. In forming the Michaelis complex, the binding pocket is substantially rearranged: protein flaps or loops often close over the bound ligand, the binding pocket is desolvated, and catalytically important residues are brought into contact with the bound substrate. These molecular motions can involve substantial portions of the protein. The dynamics of how proteins bind ligands has been little studied and is very obscure. We can propose several models that take into account formation of protein-ligand complexes preceeding the Michaelis complex. There may be a small number of sparsely populated intermediate states from unbound ligand to the Michaelis complex. Or, non-specific ligand-protein binding occurs followed by diffusion on the protein surface to the binding site. The time-scales of motions involved in the binding of ligand are expected to be from picoseconds to milliseconds and even longer.

An overlay of TIM in the loop open and loop closed (with bound substrate in CPK) conformations. The mobile loop is shown in dark yellow (open) and red (closed), and motion of the indole ring of Trp168 and the active site residue Glu165 shown as stick diagrams. The active site loop descends down to engulf the substrate and position some of the catalytic residues. The loop's Trp indole ring rotates about 50 degrees between the two conformation; its fluorescence emission was used as a marker for the loop motion.

Our study (1) on the binding of substrate and substrate surrogates to triosephosphate isomerase (TIM) used laser induced T-jump kinetics approaches (2-4) to perturb binding equilibria and determine events in the 100 ns to 10 ms time range. The figure shows an overlay of TIM in the loop open and loop closed conformations determined by crystallography studies. The T-jump studies were able to probe quite completely loop kinetics and thermodynamics. We found that the loop remained closed over the catalytic site during the entire time required for chemistry to occur. The loop opening rate and its thermodynamics closely matched TIM's kcat, on the microsecond time scale. We also found that the binding pathway involved, at least, the formation of one encounter complex, present in small amounts at equilibrium (and so invisible to static studies) and produced at diffusion limited rates. Remarkably, much of the binding enthalpy is realized in this encounter complex. All this is qualitatively in agreement with what is known about triosephosphate isomerase, which is an exceptionally efficient enzyme. It can be surmised from TIM's protein structure that loop dynamics in TIM probably involve a rigid motion of the protein residues that make up the loop; hence, a simple model of binding emerging from the kinetic data seems reasonable.

The rate of the observed signal relaxation, kobs, in response to a T-jump for the substrate mimic G3P ligated TIM system is plotted for various final jump temperatures and free enzyme [E] and ligand [L] concentrations. A fit to the plot of kobs versus the sum of the free ligand and free enzyme concentration using a kinetic model involving the fast formation of an encounter complex between G3P and TIM followed by the closure of the loop yields the values of the rates of loop opening and closing, kopen and kclosed, and several thermodynamic parameters.

1. "Active Site Loop Motion in Triosephosphate Isomerase: T-jump relaxation spectroscopy of thermal activation", Ruel Desamero, Sharon Rozovsky, Nick Zhadin, Ann McDermott, Robert Callender, Biochemistry 42, 2941-2951 (2003)
2. "Probing Protein Dynamics Using Temperature Jump Relaxation Spectroscopy", Robert Callender, and R. Brian Dyer, Current Opinion in Structural Biology 12, 628-633 (2002).
3. "The Dynamics of Protein Ligand Binding on Multiple Time Scales: NADH Binding to Lactate Dehydrogenase", Hong Deng, Nick Zhadin, and Robert Callender, Biochemistry 40, 3767-3773 (2001).
4. "Infrared Studies of Fast Events in Protein Folding", R. Brian Dyer, Feng Gai, William H. Woodruff, Rudolf Gilmanshin, and Robert Callender, Accs. Chem. Res. 31, 709-716 (1998).