Abstract
AbstractWater is widely assumed to be essential for life 1, although the exact molecular basis of this requirement is unclear 2-4. Water facilitates protein motions 5-9 and although enzyme activity has been demonstrated at low hydrations in organic solvents 10-13, such non-aqueous solvents may allow the necessary motions for catalysis. To examine enzyme function in the absence of solvation and bypass diffusional constraints we have tested the ability of an esterase to catalyse alcoholysis as an anhydrous powder, using a closed reaction system in which the substrates and products of the enzyme reaction are gaseous 14-15, and where the water content can be well defined 16. At hydrations equivalent to 3 (&177;2) molecules of water per molecule of enzyme, activity is observed that is several orders of magnitude greater than non-enzymatic catalysis. Neutron spectroscopy indicates that the fast (&8804;nanosecond) global anharmonic dynamics of the anhydrous functional enzyme are heavily suppressed. The results indicate that neither hydration water nor the solvent-activated fast anharmonic dynamics are required for enzyme function. An implication of these results is that one of the essential requirements of water for life may lie with its role as a diffusion medium rather than any of its more specific properties.
Highlights
D: Australian Institute of Neutron Science and Engineering, Menai, Australia e: University of Wollongong, School of Chemistry and Centre for Medical Bioscience, Northfield Ave., Wollongong, Australia f: Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT, U.K
Water facilitates protein motions [5,6,7,8,9] and enzyme activity has been demonstrated at low hydrations in organic solvents [10,11,12,13], such non-aqueous solvents may allow the necessary motions for catalysis
The results indicate that neither hydration water nor the solvent-activated fast anharmonic dynamics are required for enzyme function
Summary
Pig liver esterase (PLE) (150 units/mg, EC 3.1.1.1) was obtained from Sigma, and further partially purified using Fast Flow Q Sepharose. The enzyme powder was dried over high grade P2O5 99.99 % (Aldrich). To reach different hydration levels, the equilibration time of the protein over P2O5 has been extended (one, two or four weeks) and the temperature changed eventually from room temperature to 65°C over two weeks drying for the second lowest hydration point
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