The influence of metal fluorides on the structure and dynamics of phosphoryl transfer enzymes

Lead Research Organisation: University of Sheffield
Department Name: Molecular Biology and Biotechnology


Living systems depend on enzymes to speed up a multitude of chemical reactions via catalysis. One of the most widespread reactions required involves the transfer of a phosphate group between different molecules - phosphate transfer reactions play a central role in, for example, providing energy to cells, the synthesis and breakdown of vital components, and communication systems within cells. Phosphate groups are extremely difficult to remove and so highly efficient catalysis is crucial for the control of these cellular processes. Although model studies have taught us much about the intrinsic chemistry required, our understanding of the origins of the enormous accelerations of reaction rates afforded by phophate transfer enzymes - from taking longer than the time for which the universe has been in existence to less than a second - are at a rudimentary level . Ideally, we would study a snapshot of an enzyme at the critical point of catalysis but this is unachievable since the lifetime of such species is too short. Hence, the current framework for understanding of how this catalysis occurs depends on studies involving chemicals that look like the species present at the critical point of catalysis but which trap the enzyme in this state. We have discovered that a phosphate transfer enzyme is capable of assembling magnesium fluoride in the site where catalysis occurs. Magnesium fluoride looks more similar to the species predicited to be the determinant of the catalytic rate than any other yet described, but is not normally observable in the absence of the enzyme. The enzyme is using its immense binding capabilities to stabilise magnesium fluoride. This provides us with strong evidence as to how the enzyme is catalysing the phosphate transfer reaction. We will compare how the protein behaves in terms of its structure and movement in the presence of magnesium fluoride compared with the previously best known analogue of phosphate transfer catalysis, aluminium fluoride. The triangular shape of the magnesium species fits far closer to the transfering phosphate group than the square aluminium species. We will also investigate whether previously reported triangular aluminium species are in fact magnesium species, and we will examine the different ways in which fluoride can bind to the enzyme. The conclusions should be broadly applicable to other phosphate transfer enzymes. An understanding of this process is vital to the design inhibitors of enzymes for use as therapeutic agents (drugs) and to technologies that use enzymes out of their biological context, for example bioremediation. It will also help the theoretical understanding of how important biological molecules work.

Technical Summary

Phosphoryl transfer reactions play a central role throughout living systems - in metabolism, regulation, energy housekeeping and signalling. As phosphate esters are extremely kinetically stable, highly efficient catalysis is crucial for the control of these cellular processes. Ideally, a snapshot of an enzyme in a high-energy state would be immensely useful, as it would allow the very interactions that bring about catalysis to be observed. But this is unrealistic given how elusive high-energy intermediates and transition states inevitably must be for phosphoryl transfer reactions. Hence, the current structural and energetic framework for understanding of how this catalysis occurs depends on studies of transition state analogs (TSAs) that bind tightly in an enzyme active site. The recent discovery of MgF3- TSA complexes provides the closest representation of a phosphoryl transfer transition state yet known, and we intend to exploit the MgF3-:substrate complexes of beta-PGM to understand how the enzyme utilises its binding capabilities to stabilise this species. We will use solution NMR methods to compare the structural and dynamic consequences of beta-PGM binding to MgF3- with the analogous complex AlF4-. We will also assess for a broad range of enzymes whether previously assigned AlF3 complexes are really MgF3- complexes, and if not why AlF3 is selected over AlF4-. Finally, we will determine the structural and dynamic consequences of competing modes of fluoride binding, including those observed during the binding of inhibitors.


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Baxter NJ (2010) Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF-3 rather than by phosphoranes. in Proceedings of the National Academy of Sciences of the United States of America

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Bowler M (2010) Why did Nature select phosphate for its dominant roles in biology? in New Journal of Chemistry

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Golicnik M (2009) Kinetic analysis of beta-phosphoglucomutase and its inhibition by magnesium fluoride. in Journal of the American Chemical Society

Description This project addressed fundamental questions relating to how enzymes deliver catalysis, in support of the scientific community learning how to manipulate enzymes to deliver an acceptable degree of catalysis for substrates of choice. The specific questions focused on how enzymes are organized to accommodate the transition state of the reaction that they catalyse and how they manage charge distribution in this state. The key findings were:
1. We established metal fluoride moieties as being the principal ingredient of arguably the best transition state analogue known for any enzyme system. We described how these moieties can be used to obtain unprecedented levels of detail of the active site of an enzyme in a state that is as close as possible to the one utilised during catalysis.
2. We determined the large extent to which electrostatics dominate the organisation of near-transition state conformations in phosphoryl transfer enzymes.
3. We showed how the balancing of charges in the vicinity of active sites is a defining feature of phosphoryl transfer enzymes, regardless of their function or their family of origin.
Exploitation Route These studies provide a firmer foundation upon which to accelerate progress in synthetic biology and enzyme-based biotechnology. The primary industrial beneficiaries of this work are in the biotechnological and pharmaceutical sectors.
1. This work will benefit researchers in the biotechnological industries by providing a firmer footing from which to reposition enzymes for industrial use. The specific issues to be addressed in the work packages are common issues with many enzymes and are often limiting factors in the efficiency of redesigned systems.
2. This work will benefit researchers in the pharmaceutical industry by providing a better understanding of the functioning of enzymes, thereby helping them to develop more specific, targeted drug treatments.

We have also deposited protein structures in the PDB database, ID=2wfa, 2wf5, 2wf6, 2wf7, 2wf8, 2wf9 and 2whe, and NMR assignments in the BMRB database, ID=7234, 7235, 15467, 16409, 17851 and 17852.
Sectors Chemicals,Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description The primary publications related to this study have been cited over 240 times (Mar 2016). The citations relate to groups with strong industrial connections in the biotechnological and pharmaceutical sectors.
First Year Of Impact 2006
Sector Chemicals,Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic