Atomic resolution experimental interrogation of hydride quantum tunnelling in enzyme reaction chemistry

Enzymes are phenomenal catalysts accelerating reactions by as much as 10^21 compared with the rate of the non-catalyzed reaction. In all living things, these enzymes are specialized protein molecules that catalyze biochemical reactions for carrying out specific biological functions. Over a number of years, chemists and biochemists alike have attempted to harness this catalytic potential of enzyme systems to accelerate reactions that do not normally occur in Nature. This exploitation of enzymes as 'designer' catalysts requires in-depth and quantitative understanding of the physical chemistry of enzyme action. The drive to understand the origin of the power of enzyme catalysis has led to the development of quantitative, physical models for enzyme catalysis - the most recent incorporating quantum phenomena such as 'tunnelling' - to explain rate accelerations by enzyme enzymes. This has been augmented by the elucidation of atomic structures of biological catalysts using structural biology methods such as X-ray crystallography and NMR spectroscopy. This has defined the 'structure determines function' paradigm for enzymes and the notion that biological catalysis can be driven, for example, by complementary interactions between substrate molecules (or high energy states thereof) and the protein. Despite these advances, our understanding of biological catalysis is very incomplete, and we are unable to account for several orders of magnitude of the catalytic power of enzymes using current physical models. A more recent focus has been on the role of protein motions or dynamics in driving biological catalysis. This invokes a flexible enzyme catalyst that, when in complex with a substrate, can explore a myriad of different structural states over a variety of different timescales (sub picosecond to seconds). The catalytic power of enzymes is linked to the dynamical properties of the protein, but structural biology methods provide only 'static' depictions of the catalyst, or at best provide a time averaged ensemble of structures that may, or may not, be important in catalysis. The major challenge to the field and one that will open up more effective exploitation of enzyme catalysts in general, is to provide improved theory and analysis of the link between dynamical change and rate acceleration. The paradigm has thus progressed to one in which 'dynamics determines function'. In this application, we propose novel structural biology approaches that will provide atomic level insight into those high energy structural sub-states of a biological catalyst that are populated only transiently (millisecond through to < picosecond). This knowledge will underpin the development of more detailed insight into catalytic processes in enzyme systems and will form a platform for the emergence of more rigorous theory that will ultimately facilitate the improved exploitation of enzymes.

The link between enzyme motions and catalysis is of fundamental importance to understanding how biological systems operate. The level of understanding of the link is currently developing quickly though the application of solution NMR methods to obtain higher resolution descriptions of enzyme motions, which provides a hitherto unobtainable level of experimental information with which to test, challenge and improve theoretical models. In the proposed study, we intend to develop - using a novel application of NMR spectroscopy linked to detailed theoretical and computational analysis - a comprehensive picture of the link between both high and low frequency enzyme motions and catalytic competence in archetypal hydride transfer enzymes. Using pentaerythritol tetranitrate reductase (PETNR) and morphinone reductase (MR), the new experimental data will be combined with the large body of underpinning kinetic data, in a cycle of experiment-led theory development. This is intended to lead to an in-depth appreciation of reaction chemistry and its coupling to protein motion - information that is crucial for understanding quantitatively biological catalysis and inhibitors/biocatalyst design. Our specific objectives are to: 1. determine solution structures of PETNR and ligand complexes relevant to the reductive and oxidative half-reactions of the catalytic cycle; 2. provide detailed analysis of low frequency dynamics of the system as a function of pressure; 3. investigate catalytically important high frequency dynamics as a function of pressure; 4. use NMR observations to develop further theoretical and computational approaches to understanding hydride transfer reactions. The methods developed here will provide new, important tools for structural mapping of other reactive configurations in enzyme systems.