Protein-ligand coupled motions in DHFR catalysis

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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.