Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
ReviewProtein dynamics and enzyme catalysis: Insights from simulations☆
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‡Eoverall 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.
Section snippets
Protein dynamics and enzyme conformational changes
Structures determined by X-ray crystallography for biological macromolecules are the usual starting points for biomolecular simulations. Such structures have transformed the understanding of proteins, but it is important to remember that they are a best fit to data which usually contain significant time and spatial averaging and are interpretations of dynamic systems and should not be considered as a single molecular representation [11]. Crystal structures are models built and refined to fit to
Dynamics and catalysis
It is clear that conformational changes are part of the catalytic cycles of many enzymes, and the ability of many, if not all, enzymes to fluctuate and efficiently change from one conformation to another is an essential part of their overall catalytic efficiency, which has likely been selected by evolution. The dynamic nature of proteins, with multiple closely related structures of similar energy, and small energy barriers separating these minima, is probably integral to these properties [3],
Conformational effects in catalysis
Chorismate mutase is an ideal model system for the investigation of enzyme catalysis as it is the only known example of a biocatalysed pericyclic reaction that occurs via the same mechanism in solution. This allows for direct comparisons to be made between the reactions in the enzyme and in solution (and also potentially in other environments). The reaction catalysed by this enzyme has been widely studied by experimental [125], [126], [127] and computational [128], [129], [130], [131], [132],
Quantum tunnelling and protein dynamics
Transfer of hydrogen (H) (as a proton (H+), hydride (H−) or hydrogen atom) is an important step in many enzyme-catalysed reactions. Replacement of the transferring particle by a heavier isotope of hydrogen (tritium (T) or deuterium (D)), or isotopic substitution elsewhere (e.g., at other positions of the substrate) can lead to measurable changes in reaction rate (i.e., a kinetic isotope effect (KIE)). A number of experimental studies of enzyme kinetics have shown that in some cases, the
Conclusion
The question of the relationship of protein dynamics to enzyme catalysis continues to be hotly debated. Biomolecular simulations will be essential in resolving these debates. Simulations can provide atomically detailed analysis of enzyme-catalysed reactions, and analysis of catalysis, beyond what is achievable experimentally for enzymes. There are good indications that transition state theory is a good basic framework for understanding and modelling enzyme-catalysed reactions [95], [99], [106],
Acknowledgements
AJM is an EPSRC Leadership Fellow and (with KER) thanks the EPSRC for support. AJM and KER thank their co-workers in the work described here. JDM (with AJM) thanks the BBSRC for funding.
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This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.