Protein-ligand coupled motions in DHFR catalysis

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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.