Interdisciplinary approaches to elucidating fundamental reactive processes in enzyme systems using novel fast reaction methods.

Lead Research Organisation: University of Manchester
Department Name: Life Sciences


The ability to study the kinetics of biological processes on the millisecond (and faster) timescale is essential for detailed understanding of enzyme catalysis and the underlying regulatory processes in the cell. My group has developed a leading and internationally recognised centre of excellence in transient kinetic methods applied to the study of enzyme mechanism, which also embraces strong interdisciplinary links with structural biology and computational chemistry. Of particularly note is our work on enzymic H-tunnelling and electron transfer in complex flavoproteins, quinoproteins and haem dependent systems. This work is instrument-intensive, and relies heavily on a variety of sophisticated kinetic and spectroscopic apparatus, which the applicant has assembled and used effectively over a number of years. The applicant has developed the infrastructure for fast reaction kinetics to the point where assembled facilities are unique within the UK and probably continental Europe, including stopped-flow instruments, temperature and pressure-jump apparatus, rapid quench instrumentation, a state-of-the-art and customised laser flash photolysis instrument, a suite of fluorimeters allowing fluorescence life-time and polarisation studies and SPR instrumentation. A number of these instruments are contained within strict anaerobic environments within custom-built glove boxes. A number of detailed and important mechanistic questions in the areas of biological hydrogen and electron transfer require further development of (unique) fast reaction methods. The applicant's research group has been at the fore of studying H transfer by quantum tunnelling mechanisms and also the control of biological electron transfer by conformational sampling/gating. Detailed analysis of these mechanisms now requires the further development of fast reaction methods to include photoactivated electron transfer, the study of magnetic field effects in radical pair reactions, high pressure stopped-flow and cryogenic stopped-flow methods. The purpose of the application is to provide support for the applicant to develop new approaches to fast reaction analysis of redox enzyme mechanism. The questions to be addressed include the role of protein dynamics in facilitating H-transfer by quantum tunnelling mechanisms. The timescales for these conjectured small-scale dynamical changes are fast (sub picsecond) and are difficult to analyse experimentally. I will use high pressure stopped-flow studies combined with studies of the temperature dependence of a reaction using conventional and stable isotope substituted substrates to investigate the highly controversial role of protein dynamics in H-tunnelling. I propose to probe for the existence of transient radical species in enzyme catalysed reactions by developing novel stopped-flow studies performed at varying magnetic field strength. Radical pairs are prevalent in 'paper' mechanisms for many redox enzymes, but evidence for the existence is not forthcoming owing to their fleeting existence. By developing 'magnetic' stopped-flow studies the kinetics of reactions involving radical pairs will be sensitive to weak magnetic fields. Laser IR temperature jump methods and laser photoactivation of electron transfer reactions will also be developed to address the challenging and regulatory aspects of biological electron transfer by conformational sampling and gating mechanisms. Our structural work has indicated that sampling is a main feature of inter and intraprotein electron transfer in complex electron transfer proteins, but an understanding of the kinetics of these processes requires the development of new fast reaction methods. A number of model systems suited to studies of the mechanisms of H-tunnelling and electron transfer by conformational sampling are the subject of the study. The work will have generic value in developing our understanding of fundamental reactive processes in biology.

Technical Summary

Enzymes are extremely efficient catalysts. They can achieve rate enhancements of up to 10^21 over the uncatalysed reaction rate. Our understanding of the physical basis of this catalytic power is challenging, and has involved intensive research efforts for over 100 years. Recent years have witnessed new and important activity in this area, and extended our theoretical understanding beyond the shortcomings of semi-classical transition state theory to include roles for protein 'motion', low barrier hydrogen bonds and quantum mechanical tunnelling. Studies form our group and those of others have demonstrated that H-transfer by quantum tunnelling is a feature of enzyme catalysed H-transfer. This has been demonstrated experimentally through studies of kinetic isotope effects with a number of enzymes, and supported by computational simulation. The challenge now is (i) to understand at the atomic level the mechanisms of H-transfer, and the role of protein dynamics in facilitating these reactions and (ii) to provide experimental evidence for the existence of very short-lived species in the reaction chemistry. Herein, I propose to develop state-of-the-art fast reaction kinetic approaches to analyse enzyme catalysed H-transfer. I will develop high pressure and high pressure/variable temperature stopped-flow kinetic studies to obtain new data to test models of vibrationally assisted H-tunnelling. Magnetic stopped-flow studies will be developed to detect short-lived radical pair intermediates. Flow-flash photolysis and laser t-jump approaches will be developed to study the reaction chemistry of P450 enzymes, to identify intermediates and map proton transfer pathways. The work is ambitious and technically very challenging. The new data will be used to support computational analysis of H-transfer reactions by QM/MM methods, and to test available models for H-tunnelling by vibrationally assisted mechanisms.


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Antonyuk SV (2011) Carbon monoxide poisoning is prevented by the energy costs of conformational changes in gas-binding haemproteins. in Proceedings of the National Academy of Sciences of the United States of America

Description The recipient of the fellowship has made leading and sustained contributions to knowledge of enzyme mechanisms, structure and catalysis, and especially in transforming our understanding of non-trivial quantum effects in enzyme catalysis. In pursuing these studies his research has been set at the interface of chemistry, biology and physics, involving studies of the complex mechanisms of enzymes, from the quantum through to macromolecular levels, and spanning femtosecond to second timescales.
Sectors Chemicals


including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology


Description This detailed understanding of biological catalysis is now helping to drive/accelerate rational enzyme design for applications in industrial biocatalysis/chemicals manufacture. The work has had major impact in the biological catalysis field by challenging the indiscriminate use of transition state theory for enzyme catalysis in relation to ubiquitous H-transfer reactions. The importance of nuclear tunneling to biological catalysis was not appreciated by the community at the start of his work, but now is broadly accepted as a result of the many detailed publications that the group has produced.
First Year Of Impact 2006
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Cultural

Description EMFBRT
Amount £208,792 (GBP)
Funding ID BFT 10/35 
Organisation The EMF Biological Research Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2010 
End 10/2014
Description EUF7 DirectFuel
Amount £3,700,000 (GBP)
Funding ID grant agreement no. 256808 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 09/2010 
End 09/2014
Description TgK 
Organisation TgK Scientific
Country United Kingdom 
Sector Private 
PI Contribution Provided expertise, research time and equipment for instrument development.
Collaborator Contribution Provided expertise, user training and equipment for instrument development. Sponsored 2 CASE students.
Impact Developed/built in collaboration high pressure stopped flow, stopped flow FTIR, freeze quench stopped flow, and stopped flow raman experiments.
Start Year 2006