Protein dynamics and enzyme catalysis: Insights from simulations

Abstract

The role of protein dynamics in enzyme catalysis is one of the most active and controversial areas in enzymology today. Some researchers claim that protein dynamics are at the heart of enzyme catalytic efficiency, while others state that dynamics make no significant contribution to catalysis. What is the biochemist – or student – to make of the ferocious arguments in this area? Protein dynamics are complex and fascinating, as molecular dynamics simulations and experiments have shown. The essential question is: do these complex motions have functional significance? In particular, how do they affect or relate to chemical reactions within enzymes, and how are chemical and conformational changes coupled together? Biomolecular simulations can analyse enzyme reactions and dynamics in atomic detail, beyond that achievable in experiments: accurate atomistic modelling has an essential part to play in clarifying these issues. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Introduction

Proteins show complex internal motions, as biomolecular simulations have helped to reveal [1], [2], [3]. Protein dynamics are, in many cases, believed to be related to their biological functions. This can include changes from one conformation to another, or the ability to undergo particular motions or fluctuations as part of a biomolecular mechanism. Even for simple, small proteins, subtle structural changes can have important functional consequences. For example, myoglobin, a simple binding protein and the first protein to have its structure solved by X-ray crystallography [4], is known to exist in two distinct states, generally termed T (tense) and R (relaxed) [5]. The most important biological role of myoglobin is as a carrier of dioxygen, which binds to the iron in the haem functional group; carbon monoxide also binds with higher affinity, hence the well-known potential (as for haemoglobin) for poisoning. The two states, T and R, correspond to the unligated and ligated myoglobin, respectively. The differences in binding affinity observed for the different states of myoglobin are thought to be due to structural changes accompanying the binding [6] of a ligand to the iron centre of the porphyrin ring as myoglobin relaxes between the ligated and unligated tertiary structures. As a ligand dissociates from the iron, the iron atom moves ~ 0.3 Å out of the porphyrin plane and the haem distorts from planarity. Concerted motion of the rest of myoglobin completes the transition between the T and R states [7], [8]. Strickland et al. [9] performed quantum mechanics/molecular mechanics (QM/MM) calculations on myoglobin to investigate the bond energy of the Fe–CO bond and its effect on the tertiary structure. Crystal structures of the T and R states were used to initiate MM molecular dynamics (MD) simulations from which 11 conformations of myoglobin (from the two simulations) were selected, resulting in 22 structures for QM/MM calculations. Bond dissociation energies (BDE) (with the QM region treated with the hybrid B3LYP functional with a flexible basis set) were calculated for each structure and averaged to give a Δ^‡E_overall of 8.1 kcal mol^− 1, in good agreement with the experimental value of 10.8 kcal mol^− 1. The QM region alone gives a BDE of 13.9 kcal mol^− 1; therefore, the surrounding myoglobin environment destabilizes the Fe–CO bond by 5.8 kcal mol^− 1. It was observed that this weakening is greater in the T state where Δ^‡E is 2.6 kcal mol^− 1 greater than in the R state showing that the two states are functionally different.

Apparently, even in this ‘simple’ binding protein, relatively small changes in structure are important to function, in this case reducing the difference in binding affinity between CO and O₂. Should we therefore expect that structural changes play a central role in enzyme activity? It is at this point that clear definitions are necessary. It is well known, of course, that many enzymes undergo large conformational changes during their catalytic cycles (e.g., associated with substrate binding and product release) and that these changes are indeed critical to enzyme activity [10]. These conformational changes are often driven by chemical changes at an active site, though the mechanisms of the coupling of chemical and conformational changes are by no means well understood. More controversial is the widespread proposal that protein motions contribute to catalysis by accelerating the reaction rates of chemical steps. These two effects must be considered separately. For the latter, for the effects of dynamics to be shown to be relevant to catalysis, it would be necessary to show that comparable motions are not present in non-catalytic (or less active) equivalent reacting systems, and that their presence or absence alone significantly alters the reaction rate.