Protein-ligand coupled motions in DHFR catalysis

Enzymes are efficient catalysts that can achieve rate enhancements of up to 21 orders of magnitude relative to the uncatalysed reactions. However, despite many decades of experimentation, 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 that control the speed of these reactions, motions in the enzyme-substrate complex must be taken into account. The role that enzyme motions play in the physical steps of the catalysed reaction (i.e. binding of substrates, release of products and global conformational changes) is well established. However, the influence of such dynamic motions on the actual chemistry of an enzyme-catalysed reaction is less well defined. In particular, the influence of fast motions that actively promote the reaction is a current hot topic in mechanistic enzyme catalysis.

We will investigate the correlation between dynamics and enzymatic chemistry using the enzyme dihydrofolate reductase (DHFR). This enzyme is required in many essential biochemical processes including the 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:

1) Isotopic labelling of the reactant molecules using chemical and enzymatic processes. Especially our multi-enzyme syntheses (exploiting and mimicking biochemical pathways) allow the efficient labelling of specific positions in the molecules involved; such specific labelling is no easily achieved by conventional chemical methods. These isotope-labelled compounds are essential for the investigations described under 2) and 3).

2) Heavy atom kinetic isotope effects (KIE of carbon and nitrogen in this case), which report directly on local dynamic motions, have not been measured to date due to the complexity of preparation of the labelled compounds. Our strategy described above now allows straightforward access to these compounds. A combination of these heavy atom KIEs with our existing, comprehensive hydrogen KIE data using computational models will provide a detailed map of the transition state of the DHFR catalysed reaction.

3) The coupling of the dynamics of protein, substrate and cofactor will be investigated by nuclear magnetic resonance spectroscopy. The specific, 'tailor-made' labelling of all components of the reaction in combination with modern NMR techniques allows for the first time a thorough investigation of the contributions of fast motions in the active enzyme complex on the reaction.

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.

We propose to elucidate the fundamental mechanisms that operate to couple enzyme catalysis and protein dynamics, especially the influence of fast motions within the Michaelis complex on hydride transfer by the dihydrofolate reductases from the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. Based on firm published data and extensive preliminary results we propose an approach that links measurements of heavy atom kinetic isotope effects (KIEs) with structural and dynamic investigations and theoretical work.

We will use chemical and enzymatic syntheses to produce dihydrofolate and NADP(H) with 13C and 15N labels in specific positions. These labelled compounds will from the basis of measurements of heavy atom KIEs (13C and 15N). Such experiments also require incorporation of a remote radiolabel from ATP or glutamate respectively during the synthetic procedure. The combination of the heavy atom KIEs with existing, extensive hydrogen KIEs into a single model by computational methods such as Gaussian will lead to a model for the transition states of the DHFR catalysed reactions.

NMR experiments using 13C and 15N labelled substrates, cofactors and protein will be used to determine the coupling of the dynamics of the protein to the bound ligands. Both picosecond-nanosecond dynamics and microsecond-millisecond conformational fluctuations of NADP+ and folate in complex with EcDHFR and TmDHFR (which forms a stable model for the Michaelis complex) will be determined.

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

This programme is of fundamental importance to our understanding of enzyme catalysis. Ever since Summer isolated urease in 1926, scientists have been intrigued by and tried to understand the enormous catalytic power of enzymes. Now 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.
This is fundamental research with obvious benefits for the scientific community and society. The benefits for industry are not immediate or direct. However, in the age of functional genomics and structural proteomics where an ever-increasing number of protein structures are solved, the need to further our understanding of the fundamental principles by which Nature's catalysts operate is self-evident. The work proposed here will help shed light on the mechanism by which the enormous catalytic rates typically observed in enzymatic reactions are achieved. It will therefore 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 with 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. Progress and findings will periodically be discussed with the Research and Consultancy Division (RACD) at Cardiff University to assess when intellectual property needs to be protected. RACD is well equipped to protect intellectual property, set up license arrangements and handle all aspects of commercial exploitation in support of this project. Similarly the Research and Development Unit at University of Bristol have experience on all aspects of intellectual property and will assist as required.
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. Appropriate training will be given to the PDRAs in the preparation of papers, posters and oral presentations to ensure that, alongside their scientific knowledge and skills, they are developing a portfolio of wide transferable skills. It is worthy of note that the three applicants have significant experience in science communication.