Understanding enzyme-catalysed phosphoryl transfer

One of the most remarkable features of the chemistry of living organisms is the evolutionary development of phosphate esters to provide on one hand the extremely stable backbone for biopolymers that encode genetic information, DNA and RNA, while on the other hand providing the temporal and transient regulation of protein activity, largely under the control of protein kinases and protein phosphatases. In the meantime, phosphate esters are also utilised for the generation, distribution, and application of energy throughout the living systems by the manipulation of anhydrides of phosphoric acid and its esters, notably adenosine triphosphate. The solution to the paradox between the remarkable chemical stability of phosphate mono- and di-esters and their facile manipulation lays in the catalytic power of so-called phosphoryl transfer enzymes to make and break P-O-C and P-O-P bonds rapidly, which gives rise to some of the largest enzymatic rate accelerations yet identified. Despite this central position for phosphoryl transfer enzymes in biological systems, the source of catalytic power and how it is regulated is understood only at a relatively rudimentary level. One of the principal reasons for this is that it is difficult to observe the enzymes in the fleeting moments of catalysis, since the lifetimes of the relevant species are so short. The most informative experimental approaches to unravelling this conundrum are where a chemical that provides a close mimic of the fleeting populated species, but which is stable for far more extended period, is inserted into the enzyme. We have discovered a new type of inorganic species that performs this role far better than any previously identified for phosphoryl transfer enzymes, whereby magnesium and fluoride combine in the active site of the enzyme to make an excellent mimic of a phosphate group being transferred. This exquisite trap of the enzyme, which catches it as if in the act of transferring a phosphate group, enables us to measure how the enzyme is able to impart the enormous stabilisation required to make phosphoryl transfer occur at a rate that is useful for biological systems. We will analyse the trapped enzyme using a combination of experimental and computational methods that we have also developed, in a synergistic and interdisciplinary research programme. The development of this understanding will enable us to guide the evolution of compounds that modulate the activity of phosphoryl transfer enzymes, which are targets for therapeutic and biotechnological intervention in a broad range of important processes from heart disease and cancer to crop protection. The work matches the stated aims of BBSRC in moving towards a more mathematical and physical understanding of biological processes.

This application aims to make a step change in our understanding of how enzymes control the transfer of phosphate groups. Phosphoryl transfer reactions lie at the heart of every system in living organisms. This is no accident - phosphate esters are extremely kinetically stable, ensuring that spontaneous hydrolysis in the absence of enzymes is a rare event. However, this presents substantial challenges for the enzymes involved in phosphoryl transfer reactions, and the underlying rate accelerations achieved by these proteins include the largest determined for any enzyme. However, the source of such catalytic power, and how it is controlled, are insufficiently understood to provide reliable guidance as to how to interfere with these reactions selectively. We aim to exploit our recently developed, novel toolkit for the examination of near-transition states of phosphoryl transfer enzymes to establish how an archetypal enzyme controls and performs its catalysis. Using an integration of NMR (including 19F-derived data), X-ray, DFT and classical mechanics investigations of metal fluoride complexes of phosphoryl transfer enzymes, our objectives are to answer four key questions covering how they achieve such catalytic prowess, namely: What role does charge balance play in catalysis? How is high affinity for the transition state achieved selectively? How does the substrate activate the enzyme? How does the enzyme control the dynamics of transition state formation? We have chosen phosphoglucomutase as the principal study target on account of both its high tractability for the proposed study and preliminary data that have led to the proposal of new hypotheses about how catalysis is realised. We have also carried out similar preliminary investigations of PSP, alkaline phosphatase, PGK, F1-ATPase, RhoA/RhoGAP, and the protein kinases P38alpha, MKK6 and haspin. We will focus initially on establishing fundamental principles with PGM, and then apply these to the other enzymes.

All biological systems rely on enzymes for their constitution, and the manipulation of the activity of component enzymes is the primary means to translate knowledge of a system into an entity with significant impact. Correspondingly, improving means with which the activity of enzymes can be modulated specifically and controllably is a primary goal of a spectrum of industry ranging from agrochemicals to pharmaceuticals. Every biological system known has a reliance on the enzyme-mediated transfer of biomolecules to or from phosphate groups. Unsurprisingly, therefore, phosphoryl transfer enzymes have been a focus for a wide spectrum of the academic community and for almost every major company targeting biological systems. Despite very substantial investment and the importance of the targets, progress worldwide in the development of reagents that interfere with the activity of phosphoryl transfer enzymes has been patchy. One of the principal problems is that our understanding of the operational details of these enzymes is not nearly as sophisticated as is desirable to guide product development. We seek to make a step change in the level of understanding of the activity of phosphoryl transfer enzymes, which will provide a more firm foundation upon which to build a more comprehensive scientific understanding of catalysis by enzymes with obvious industrial and medicinal benefits. While the work that we propose is of a fundamental nature, the targets that we will use include entities that have been identified as some of the most desirable targets for therapeutic intervention in cancer and in heart disease. The work will be disseminated to as wide an audience as possible. The primary source of dissemination of the results derived from the proposed studies will be through publication in high impact scientific journals. We have a long track-recording in publishing our research findings in major international, non-specialist journals, aimed at a broad interest readership. In addition, we will continue to disseminate the work at international scientific conferences that are highly attended by both academic and industrial delegates. Furthermore, we will continue our excellent track record in depositing and supporting data in publically-accessible databases of biological data. Outside of these academic-focussed activities, we have been engaged in numerous direct activities involving industry and non-specialist audiences. Much of the proposed word stems from discussions with representatives of two major UK pharmaceutical companies and two leading biotechnology companies, all with substantial interests in phosphoryl transfer enzymes, and all of which would benefit considerably from an improved understanding of the activity and control of these enzymes. Hence, their encouragement to address these fundamental issues from an academic perspective. Furthermore, the applicants are also developing new links with a leading agrochemical companies. Furthermore, the work parallels activities of the Structural Genomics Consortium at Oxford, which is jointly funded by academia and industry, and with whom we are building links. In addition, to our group specific activities outlined above, The University of Sheffield is engaged in coordinated schemes that educate and raise public awareness about the scientific output of the Faculty of Science, ensuring that the technology and capabilities of our work obtains the maximum exposure to potential beneficiaries. The UoS has put in place comprehensive Knowledge Transfer schemes, which we are exploring currently to engage in a related joint venture with China. The lead applicant is also engaging with beneficiaries through his employment with the University of Manchester. Widespread dissemination of the technology throughout the biotechnology business sector will be sought through attendance at national and local events.