Reaction-coupled dynamics in DHFR catalysis

Enzymes are efficient catalysts that achieve rate enhancements of up to 21 orders of magnitude relative to uncatalysed reactions. However, the precise causes of these remarkable rate enhancements are not fully understood.

Hydrogen transfer reactions are of fundamental importance in all biological processes. In order to understand the effects controlling the speed of these reactions, enzyme motions must be taken into account. In particular, the influence of fast motions that actively promote the reaction is a current hot topic in mechanistic studies of enzyme catalysis. Enzymes are large molecules, but we have shown that while long-range enzyme motions play important roles in the physical steps of the catalytic cycle (i.e. binding of substrates, release of products and global conformational changes), they have no effect on the actual chemical step. Our recent results have shown that fast, localised enzyme motions do play a role in the chemical step, but that this role is not the one traditionally proposed.

Now that we have a more thorough general understanding of how fast enzyme motions couple to the chemical step, we are able to focus our efforts towards a precise atomistic understanding of the motions involved. We will investigate the relationship between dynamics and enzymatic chemistry using the enzyme dihydrofolate reductase (DHFR). This enzyme is required in many essential biochemical processes including synthesis of DNA and amino acids. It is therefore a long established drug target and several inhibitors have been discovered and successfully developed as antibacterial, antimalarial and anti-tumour drugs. The increasing and inherently unavoidable problem of drug resistance together with the poor yield from screening programmes demands a rational approach to develop new inhibitors based on a thorough understanding of the mechanistic and dynamic details of the catalytic process.

Based on our extensive previous research, we will approach this in the following way:

- Selective isotopic labelling of the enzyme. Isotopic labelling is a powerful strategy for investigating the role of enzyme dynamics, as the dynamics are affected but other properties of the enzyme are not. We already have data for the fully labelled 'heavy' enzyme; now we seek to identify the specific portions of the enzyme involved. We can produce individual parts of the protein either labelled or unlabelled using bacterial culture methods, and chemically join the different regions together to form the full length, active enzyme. Alternatively, we can incorporate labels directly by feeding the culture with labelled amino acids or their biochemical precursors.
- Measuring the effect of selective isotopic labelling on the kinetics of the chemical reaction catalysed by the enzyme. We have shown that full labelling of the enzyme has a significant effect on the kinetics. However, it is likely that only certain parts of the enzyme cause this effect. By comparing various patterns of selective labelling against the results for the fully labelled enzyme, we will pinpoint the regions directly involved.
- Investigation of the effect of selective isotopic labelling on the dynamics of the enzyme. By incorporating specific labels at positions of interest, and varying the overall mass of the enzyme by random fractional labelling at other sites, we can determine the effect on the enzyme dynamics using magnetic resonance techniques. This will complement the kinetic studies and will provide a thorough investigation of the contributions of fast motions in the active enzyme complex.
Overall, this project will provide detailed insight into how dynamics and catalysis are linked in enzymatic reactions. It will eventually allow us to develop a model of catalysis that can explain the enormous efficiency of Nature's catalysts and should lead to the rational design of enzyme inhibitors with applications as anti-infective and anti-cancer agents.

Our recent work has uncovered the role of protein motions in catalysis of hydride transfer by dihydrofolate reductase from Escherichia coli (EcDHFR). Rather than directly promoting hydride tunnelling, fast motions within the Michaelis complex influence the proportion of barrier recrossing events during the reaction. Based on published data and extensive preliminary results we propose an approach that links measurements of the effect of enzyme mass on the catalysed chemistry with NMR investigation of the enzyme dynamics.

We will produce EcDHFR isotopically labelled in specific positions. The labelled enzymes will form the basis of measurements of 'enzyme kinetic isotope effects', the ratio of the rate constants for hydride transfer catalysed by the 'heavy' enzyme and the natural abundance enzyme. Comparison with our existing data for the fully labelled enzyme will allow us to pinpoint those residues important for the dynamics involved with recrossing. NMR based measurement of heavy atom isotope effects on the reaction catalysed by selectively labelled EcDHFR will provide broad atomic-level detail of the influence of enzyme vibrational modes on the catalysed chemistry.

We will also perform NMR investigations of the effect of enzyme mass on dynamics, using selectively labelled EcDHFR in complex with NADP+ and folate. Both picosecond-nanosecond dynamics and microsecond-millisecond conformational fluctuations will be investigated. These results will be compared to the results of the kinetic experiments to build a full picture of the role of fast protein dynamics in EcDHFR catalysis.

This work will provide detailed insight how enzyme dynamics are coupled to hydride transfer in the DHFR catalysed reaction, and in the longer run improve our ability to rationally design DHFR inhibitors. However, many of the results generated here will be of generic value and contribute to a deepened and potentially simplified understanding of enzyme catalysis.

This programme is of fundamental importance to our understanding of enzyme catalysis. For the first time we are in position to develop a fundamental understanding of biocatalysis of generic value with direct consequences for much of biosciences and chemistry and with clear future application. A wide range of academic researchers will benefit from this work, as detailed under 'academic beneficiaries'. In addition, the PDRAs and technician working on the project will gain excellent training, providing scientific knowledge and skills as well as wider transferable skills suitable for future employment in a range of sectors.

Industry will not benefit directly or immediately but the knowledge obtained here will be of generic value in the future. The proposed work is of fundamental importance to biosciences and chemistry communities, and will lead to the development of a deeper understanding of fundamental biocatalysis by helping to shed light on the mechanism by which the enormous catalytic rates typically observed in enzymatic reactions are achieved. Such fundamental research has obvious benefits both for the scientific community and society at large. A better understanding of enzyme catalysis has many applications for biotechnological work in the pharmaceutical and medical sector as well as in health care or agriculture and potentially in the longer term for bioenergy and climate change. It will also facilitate the de novo design and the redesign of enzymes, areas, which have attracted much attention but would profit enormously from a better understanding of enzyme catalysis.

Long-term benefits can be expected through the application of the knowledge and protocols developed by industry; new DHFR inhibitors can be conceived and produced, which may lead to new therapeutic agents. DHFR inhibitors are validated clinical agents against microbial infections and against cancers.

Similarly, the benefit to the UK economy will not be immediate or direct. However, commercialization of products and systems arising indirectly from this research as described above will improve both employment and tax revenue, with further downstream benefits to the wider public. The potential for novel pharmaceuticals arising from downstream application of this work provides a route to more subtle benefit to the economy, as improved healthcare will reduce losses in productivity. The general public will directly benefit from this research through the planned outreach activities, which will supply them with information on how publicly funded research in academia affects biotechnology, a topic of interest to many.

Although it is not anticipated that intellectual property will arise from this work, progress and findings will periodically be discussed with the Research and Consultancy Division (RACD) at Cardiff University to assess any unexpected outcomes in this area. RACD is well equipped to protect intellectual property, set up license arrangements and handle all aspects of commercial exploitation in support of this project.

In order that the results can be fully exploited by us and the wider scientific community, communication is vital. The work will be published in internationally leading, peer-reviewed journals. Results will be presented by the investigators and PDRAs at national and international conferences, at public lectures and at meetings with industrial and academic collaborators. This will be extended to the popular press when appropriate. It is worthy of note that the applicants have significant experience in science communication.