Protein-ligand coupled motions in DHFR catalysis

Lead Research Organisation: University of Bristol
Department Name: Chemistry


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Technical Summary

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.

Planned Impact

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.


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Description We have shown that we can probe the conformation of enzymes under different solvent conditions and the sub-states stabilised under these different conditions has profound effects on the enzymes catalytic viability. The mechanisms underlying the extraordinary rate enhancements achieved by enzymes remain unclear. One possibility suggests that motions of various parts of enzymes are somehow coupled to the chemical step they catalyse. This work examined this hypothesis by protein isotope labelling; that is replacing the atoms of the protein with chemically identical, but heavier versions. This heavier version of an enzyme has slower motions and was used as a platform to examine the effect of motions by stopped-flow kinetic, circular dichroism and nuclear magnetic resonance techniques.

The results suggest that while large scale motions are not coupled to catalysis, protein motions are evolutionarily tuned; with enzymes from heat or cold adapted organisms showing compensatory changes. This suggests they must play some role in determining the 'fitness' of enzymes. Our work has not only shed light on a fundamental problem in the physical sciences, but may eventually lead to the design of artificial catalysts, which display activity in non-physiological environments, and of enzyme inhibitors with many benefits in health care and agriculture. A dedicated line of research aimed at longer-term translation of this fundamental research into products with an economic impact has been started.

A useful byproduct of the enzymatic synthesis of isotopically labelled cofactors developed for this work is the use of the same enzymes for a cofactor recycling system, able to regenerate expensive cofactors using inexpensive reagents potentially enabling bio-transformations that were previously uneconomical. This may be highly beneficial for fine (bio)chemical production.
Exploitation Route Principally in the design of better more efficient enzymes or enzymes able to work under vastly different conditions (high temperature, low temperatures).

This work suggests others might fruitfully investigate what effects protein motions may have on reactions that do not involve direct 'dynamical' coupling. This work may also eventually lead to the design of artificial catalysts, which display activity in non-physiological environments, and of enzyme inhibitors with many benefits in health care and agriculture. A dedicated line of research aimed at longer-term translation of this fundamental research into products with an economic impact has been started.
Sectors Agriculture

Food and Drink



including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

Description This is a fundamental study of enzyme conformation, dynamics and ligand binding that has generated several high-profile publications. Although focused on enzymes of fundamental importance, the principles of enzyme function that have emerged from this study have wider implications for the study of biocatalysts and protein-ligand interactions. The collaboration between Cardiff and Bristol supported by this grant has had several regional and national impacts. First it led directly to investment in the Cardiff NMR facility and the requisition of a 600 MHz NMR spectrometer that has supported life science and chemical research since its inception around 2010. The work has also led to new avenues exploring the effect of enzyme dimerization on stability and catalysis which has direct impact on the design of immobilized biocatalysts. The collaboration led to further interaction between Allemann, Crump and Mulholland (Bristol). Crump and Mulholland are now beginning to collaborate on enzyme heat capacity and dynamics and engaging with Industry (UCB) to explore how this impacts on protein-ligand interactions and drug-design with the aim of providing better in-silico predictions of ligand binding affinities.
First Year Of Impact 2020
Sector Chemicals,Healthcare
Impact Types Economic

Description School outreach activity 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The talk was delivered by our outreach teaching fellow Tim Harrison and this sparked interest from the students and makes important ties to these regional schools leading to school visits and lab demonstrations sited at Bristol University. Over the past few years this has become an enormous activity undertaken by the School of Chemistry in Bristol.

The initial talk to pre-GCSE students has now stimulated further dialogue with a teacher pursuing a Masters degree who is interested leading edge science involving aspects of Medicinal Chemistry which relate to this project.
Year(s) Of Engagement Activity 2014