Combined experimental and computational investigations of a nucleophilic displacement reaction with a hydride leaving group

All of biology - life itself - depends on enzymes. Enzymes are large, natural molecules that allow specific biochemical reactions to take place quickly, that is to say enzymes are natural catalysts. They are very good catalysts, but as yet we do not understand what it is that makes them such good natural chemists. We need to know how chemical reactions happen in enzymes, something that is very difficult to do by experiments alone. There are many reasons for studying enzymes and the reactions they catalyse: many drugs are enzyme inhibitors (they stop specific enzymes from working), so better understanding of enzymes will help in the design of new drugs. Better understanding of individual enzymes should also help understand and predict the effects of genetic variation, for example in understanding why some people may benefit from a particular drug, or may be at risk from a disease. Enzymes are also very good and environmentally friendly catalysts - knowing how they function should help in the design and development of new 'green' catalysts for forensic, synthetic, analytical and biotechnological applications. Enzymes also show great promise as 'molecular machines' in the emerging field of nanotechnology. We will carry out a collaborative project bringing together experimental biochemistry with advanced computer modelling methods to analyse in detail how a remarkable enzyme works. This enzyme catalyses an unusual reaction, and is used in industrial applications, but it could be improved by making it more efficient, which we hope to do by designing changes to it. We will predict the effects of changes to the enzyme (mutations) by modelling, and test our predictions experimentally. We will develop and apply new high-level modelling methods, capable of dealing accurately with these large and complex systems, and the chemical reactions they catalyse. Carrying out experiments and modelling together will help develop the methods, by testing them predictions, and will also help in interpreting biochemical results and planning new experiments (e.g. designing altered enzymes). We will focus on phosphite dehydrogenase, an enzyme that catalyses a chemically unique reaction that so far has eluded detailed mechanistic understanding. Current computer modelling methods are useful for studying some aspects of enzyme reactions - they offer the unique potential of making molecular 'movies' of how enzymes work - but have important limitations. For example, large size of enzymes, and the need for intensive calculations, means that current calculations are typically limited to approximate and often unreliable computational methods. Reliable predictions of enzyme catalytic mechanisms require more accurate techniques. We will extend high-level methods, previously validated in studies of chemical reactions of small molecules, to study reactions in enzymes. We will develop new, hybrid methods that can describe the energies of breaking and forming chemical bonds well, and analyse how the reaction is affected by the dynamics of the enzyme. This work will be carried out in collaboration with experimental studies. The experimental data will be essential input for the calculations. We will make predictions and compare with experiments on the same enzyme to test our theoretical methods, use molecular models to analyse and interpret experimental data and test hypotheses about the enzyme reaction mechanism. This collaboration will involve the transfer and exchange of methods, data, ideas and researchers between our labs. The new methods we develop will be made widely available, and should be very useful to biologists, biochemists and other researchers working on biological catalysis.