A Biophysical Perspective on Enzyme Catalysis

Abstract

Even after a century of investigation, our understanding of how enzymes work remains far from complete. In particular, several factors that enable enzymes to achieve high catalytic efficiencies remain only poorly understood. A number of theories have been developed, which propose or reaffirm that enzymes work as structural scaffolds, serving to bring together and properly orient the participants so that the reaction can proceed; therefore, leading to enzymes being viewed as only passive participants in the catalyzed reaction. A growing body of evidence shows that enzymes are not rigid structures but are constantly undergoing a wide range of internal motions and conformational fluctuations. In this Perspective, on the basis of studies from our group, we discuss the emerging biophysical model of enzyme catalysis that provides a detailed understanding of the interconnection among internal protein motions, conformational substates, enzyme mechanisms, and the catalytic efficiency of enzymes. For a number of enzymes, networks of conserved residues that extend from the surface of the enzyme all the way to the active site have been discovered. These networks are hypothesized to serve as pathways of energy transfer that enables thermodynamical coupling of the surrounding solvent with enzyme catalysis and play a role in promoting enzyme function. Additionally, the role of enzyme structure and electrostatic effects has been well acknowledged for quite some time. Collectively, the recent knowledge gained about enzyme mechanisms suggests that the conventional paradigm of enzyme structure encoding function is incomplete and needs to be extended to structure encodes dynamics, and together these enzyme features encode function including catalytic rate acceleration.

Figure 1:. The inter-connection between protein structure, dynamics and function has significance for improving catalytic efficiency of enzymes.

The active-site region provides environment with optimal electrostatics to promote the catalyzed reaction. Several enzymes contain conserved networks of residues that connect surface regions to the active-site. These networks provide thermo-dynamical coupling between the hydration-shell and bulk solvent, and the catalyzed reaction. The discovery of these enzyme energy networks allows new strategies for increasing the catalytic efficiency through enhancing the energy flow through these networks and optimization through mutations.

Figure 2:. Various types of internal protein motions.

(A) Fast motions. Each black dot indicates a unique conformation. (B) The conformational fluctuations correspond to large scale movements within protein domains or entire protein. These types of motions allow sampling large areas of conformational landscape that could span over multiple minima. (C) Structural changes are induced by events such as binding (or release) of other molecules. Shown here is the enzyme adenylate kinase in the apo form (left) and adenosine triphosphate (ATP) bound (right).

Figure 3:. Identified network of promoting motions/vibrations in three different enzyme folds,

cyclophilin A (CypA), dihydrofolate reductase (DHFR) and ribonuclease A (RNaseA).The flexible surface loop regions with high flexibility show the presence of residues with long side-chains and are interconnected to the active-site through preserved hydrogen bonds. Experimental studies have confirmed presence of these networks.^{, –} Reproduced from Ramanathan A et al. (2011),PLoS Biol 9(11): e1001193.

Figure 4:. A hypothesized model for the conformational and energetic coupling between enzyme and solvent.

Random motions of solvent molecules slave the motions of surface residues through collisions. These fast motions drive the conformational fluctuations of the interior regions of the protein. These intermediate motions eventually drive the slow conformational fluctuations, some of which promote the catalytic reaction. The hierarchy of protein motions enables the transfer of thermo-dynamical energy from the surface regions to the active-site.

Figure 5:. Internal protein motions and conformational fluctuations allow sampling of conformational sub-states.

Protein motions at fast time-scales allow sampling within a sub-state, while fluctuations at long time-scales allow access to different sub-states. Some of these higher energy states may contain function promoting structure and dynamical features. Any factors that change the sampling of the higher energy conformational will change access to these functionally important states and therefore change the rate of the reaction.