The Dynamical Nature of Substrate Binding to Enzymes

Lactate Dehydrogenase

Binding Dynamics

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.

Although it is clear that atomic motion must take place or else proteins can not function, probing atomic motion in proteins is difficult, however, both theoretically as well as experimentally; we know very little quantitatively about this problem. New approaches are needed to study protein dynamics. We have examine the atomic motion involved in the binding of substrate to an enzyme, lactate dehydrogenase (LDH), using a new and promising approach to define atomic motion in proteins over a large time range (10^-10 to 10⁺² s): laser-induced temperature-jump relaxation methods to perturb the reaction equilibrium and isotope-edited IR spectroscopy to follow the accompanying structural relaxation dynamics.

Fig. 1. A cartoon of the active site of lactate dehydrogenase showing the relative arrangement of reacting groups (not to scale). The substrate pyruvate is shown; the ‑CH₃ group is replaced by ‑NH₂ to form oxamate. The hydride transfer is indicated by the bold arrow, hydrogen transfer by light arrow.

Fig. 2. A ribbon diagram of the structure of LDH in the vicinity of the active site. The protein is shown in grey with the 'mobile' loop shown in purple and its two Arg residues shown in red; the loop closes over the binding pocket after both NADH and substrate are bound and Arg109 of the mobile loop comes in contact with bound substrate. NADH is shown with the its adenosine (yellow), pyrophosphate (blue), and nicotinamide (green) moieties. Bound substrate is not shown, but its location is located at the end of the nicotinamide moiety of NADH.

Lactate Dehydrogenase (LDH)

LDH is an efficient enzyme that catalyzes the direct transfer of a hydride ion from the pro-R face of the reduced nicotinamide group of NADH to the C2 carbon of pyruvate producing NAD⁺ and the alcohol lactate, accelerating the solution reaction by some fourteen orders of magnitude (Fig. 1). Binding of the pyruvate substrate in LDH is ordered and follows the formation of the LDH/NADH binary complex. The unreactive substrate surrogate, oxamate, is employed in our study so that the observed kinetics are due solely to binding and not to chemical events.

The basic experimental design is to subject the chemical system:

to a rapid change in temperature (induced by irradiating the protein solution with a pulse of near IR light; T-jumps of 20 degrees C are typical). The relaxation of the system is monitored by a spectroscopic probe and the kinetic steps along with their microscopic rate constants are determined. Using the fluorescence of the nicotinamde ring of NADH as an indicator of structural transformation, we have determined (1) that oxamate binds in a multistep process (at 20 degrees C):

It appears that the various key components of the catalytically competent architecture are brought together as separate events, with the formation of strong hydrogen bonding between active site His195 and substrate early in binding and the closure of the catalytically necessary protein surface loop over the bound substrate as the final event of the binding process. This loop remains closed during the entire period that chemistry takes place for native substrates; however, motions of other key molecular groups bringing the complex in and out of catalytic competence appear to occur on faster times scales. The on-enzyme Kd's, (the ratios of the microscopic rate constants for each unimolecular step), are not far from one. It appears that substantial, ca. 10-15%, transient melting of the protein takes place to permit substrate access to the protein binding site. The nature of activating the various steps in the binding process seems to be one overall involving substantial entropic changes.

While the NADH emission is an effective probe for following the kinetic steps, we turn to IR spectroscopy to provide specific structural attributes of the transient forms (2). One excellent marker for structure is the oxamate's C=O stretch frequency. Upon binding to the protein, this bond is highly polarized, with concomitant downshift in its stretch frequency, as the key catalytic groups of the active site, His195 (located within the binding pocket) and Arg109 (located on a 10 residue surface loop which closes over the binding pocket), form hydrogen bonds with the C=O moiety. These groups stabilize the polar transition state of the reaction by electrostatic interactions and are responsible for about half of the catalytic power of LDH (six orders of magnitude in rate enhancement).

Fig. 3. Transient fluorescence at 450 nm (excitation 360 nm) (Top) and IR isotope edited absorption (¹³C₂ – ¹²C₂) at 1606 cm^‑1 (Bottom) of LDH/NADH-oxamate solutions (600/620/700 mN) after laser T-jump to T_final = 38 ºC. Bi-exponential fits overlay the data and the fitting results are listed.

T-jump isotope edited IR transient studies were carried out at 1606 cm^‑1 since this frequency is a marker band for the major population of the LDH/NADH•oxamate complex and since interfering protein IR background is minimal at this frequency. Fig. 3 shows the results along with T-jump fluorescence data under identical conditions. Two relaxation times are observed in both the IR and fluorescence traces. At the high concentrations used in this experiment, it is expected that very small signals would be observed from the bimolecular formation of the encounter complex. Furthermore, previous measurements have shown that most of the NADH fluorescence is highly quenched before the final step (loop closure) of substrate binding; thus the small positive amplitude process in the fluorescence transient (~500 s^-1) corresponds to the loop opening following the T-jump (i.e., the loss of the LDH^{3(loopClosed)}/NADH-oxamate species in the reaction scheme). The faster relaxation process (~7000 s^‑1) is therefore assigned to the loss of the LDH^2(loopOpen)/ NADH-oxamate species.

The IR data show that the hydrogen bonds between the protein and the substrate mimic C=O moiety are formed in separate events. Clearly, loss of the C=O--Arg109 interaction dominates the slower relaxation process (~500 s^‑1) since loop closure brings Arg109 into the active site pocket in contact with substrate on this time scale and the IR transient of Fig. 2 shows changes in the C=O stretch frequency with the same rate. The faster relaxation process (~7000 s^‑1) must therefore correspond to the loss of the C=O--His195 contact. Consistent with these assignments, the relative amplitudes of the two phases of the IR transient are inverted compared to the fluorescence amplitudes. Transient loss of IR absorbance at 1606 cm^-1 primarily monitors the loss of the C=O--Arg109 interaction (large amplitude slow phase; loop opening). The loss of the C=O--His195 interaction is weakly detected at this frequency (small amplitude fast phase) because its characteristic IR band is at higher frequency (1622 cm^‑1), but is broad and upshifts strongly upon breaking the H-bonding interaction.

In summary, following the time-dependent perturbation of the oxamate C=O bond using isotope edited IR spectroscopy has resolved the individual steps in the polarization of this bond, revealing how the catalytically competent Michaelis complex is formed. This approach yields unique insight into the coupling of protein dynamics (loop and other atomic motions) to substrate binding and activation.

(1) "Structural Transformations in the Dynamics of Michaelis Complex Formation in Lactate Dehydrogenase", Sebastian McClendon, Dung M. Vu, Keith Clinch, Robert Callender, and R. Brian Dyer, Biophysical J. Letter 89, L07-L09 (2005).

(2) "The Approach to the Michaelis Complex in Lactate Dehydrogenase: the substrate binding pathway", Sebastian McClendon, Nick Zhadin, and Robert Callender, Biophysical J., in the press (2005).