Award details

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

Reference	BB/E017541/1
Principal Investigator / Supervisor	Professor Jon Waltho
Co-Investigators / Co-Supervisors	Dr Nicola Baxter
Institution	University of Sheffield
Department	Molecular Biology and Biotechnology
Funding type	Research
Value (£)	400,271
Status	Completed
Type	Research Grant
Start date	01/03/2007
End date	31/08/2010
Duration	42 months

Abstract

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.

Summary

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.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Industrial Biotechnology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search