Functional roles of enzyme dynamics in accelerating active site chemistry: Emerging techniques and changing concepts

Abstract

With the growing acceptance of the contribution of protein conformational ensembles to enzyme catalysis and regulation, research in the field of protein dynamics has shifted toward an understanding of the atomistic properties of protein dynamical networks and the mechanisms and time scales that control such behavior. A full description of an enzymatic reaction coordinate is expected to extend beyond the active site and include site-specific networks that communicate with the protein/water interface. Advances in experimental tools for the spatial resolution of thermal activation pathways are being complemented by biophysical methods for visualizing dynamics in real time. An emerging multidimensional model integrates the impacts of bound substrate/effector on the distribution of protein substates that are in rapid equilibration near room temperature with reaction-specific protein embedded heat transfer conduits.

Figure 1

Conformational selection occurs in many biological processes including substrate binding, allosteric activation, directed evolution and thermal adaptation. Conformational ensemble redistribution has been detected in many systems (dihydrofolate reductase (DHFR) [50], Kemp eliminase [52,53], and tryptophan synthase [49]) using different techniques including X-ray crystallography, NMR, and computational simulations. For the above example, the impact of perturbation is shown to alter both the number of accessible protein sub-states and their relative populations (where green represents the most stable state).

Figure 2

Collision theory states that for a chemical reaction to occur in condensed phase, the reacting particles must collide with one another. The rate of the reaction depends on the frequency and direction of collisions. For such temperature dependent reactions, thermal energy is expected from all directions in the solvent bath via isotropic thermal energy transfer (Left). As a comparison, enzymes are anisotropic structures, with active sites for chemical reactivity that are generally protected from direct collisions with solvent, implicating the protein scaffold as the basis for controlled heat transfer from the solvent bath to an enzyme active site (Right). Mechanistically, it is likely that enzymes will have evolved throughout time to construct privileged thermal conduits for efficient conduction of heat from the solvent to the active site that facilitate catalysis.

Figure 3

1. Procedures for uncovering thermal transfer pathways using temperature dependent hydrogen deuterium exchange coupled to mass spectrometry (TDHDX) using data from mADA as an example. (1) HDX samples are digested into small peptides and analyzed by mass spectrometry to detect deuteron incorporation. Most peptides will manifest at least 2 types of patterns: temperature independent plots (left panel, mapped in gold for the two peptides of this type in mADA) with the example shown undergoing rapid exchange for 4/14 amides and no further detectable change in deuteron uptake throughout the experimental period; and temperature dependent plots (right panel, mapped in cyan for the 10 peptides of this type in mADA) and where temperature dependency is apparent throughout the time course. 2. Peptides with apparent temperature dependent trends are fitted to a three-exponential equation (see Ref [86,87] for details) providing rate constants for the different regimes of HDX exchange. Using weighted average rate constants, Arrhenius plots can be generated to calculate the observed activation energy for HDX (Ea_HDX) for each peptide. Accordingly, corresponding parameters are analyzed for mutants of interest and compared to the WT protein behavior. 3. using ΔEa_HDX as a proxy, regions within a protein that show functionally relevant changes in protein flexibility are mapped onto the protein structure, to reveal possible thermal energy transfer pathways for efficient enzyme catalysis. Emerging models for enzyme catalysis invoke a redistribution of protein substates upon binding of substrate (Figure 1) that acts in concert with efficient heat transfer via embedded thermal conduit (Figure 2) to enhance the probability of reaction barrier crossings.