Water 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 (&#xb1;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 (&#x2264;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

Jeremy Smith

John Finney

Moeava Tehei

Murielle Lopez

Rachel Dunn

Roy Daniel

Vandana Kurkal-Siebert

English

Nature Precedings

1 
 Enzyme activity and dynamics in near-anhydrous 
conditions 
Murielle Lopez a, Vandana Kurkal-Siebert b, Rachel V. Dunn c, Moeava Tehei d, e, John 
L. Finney f, Jeremy C. Smith g and Roy M. Daniel a 
a: Department of Biological Sciences, University of Waikato, Hamilton, New Zealand 
b: BASF SE, GPK/M-G200, 67056 Ludwigshafen, Germany 
c: Manchester Interdisciplinary Biocentre, 131 Princess Street, M17DN Manchester, 
U.K. 
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. 
g: UT/ORNL Center for Molecular Biophysics, Oak Ridge TN-37831-6164, U.S.A. 
Water 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 (±2) molecules 
2 
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 (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. 
Pig liver esterase (PLE) was used here to catalyse alcoholysis reactions where 
water is neither a substrate nor a product. The acyl transfer between methyl butyrate and 
propanol, giving rise to methanol and propyl butyrate, was followed by headspace 
analysis and gas chromatography. Previous work has indicated that any hydration 
required for activity is below the monolayer coverage 10,15,17-18. For instance, PLE has 
been found to have hydrolytic activity at a hydration level of 0.03 grams of water per 
gram of dried enzyme  (h); i.e., about100 water molecules per molecule of protein. 
Thus, if we are to probe possible activity close to zero water content, accurate 
determination of very low levels of protein hydration is essential. Among the techniques 
now available 19, the isotopic labelling of water molecules and its quantification by mass 
spectrometry is one of the most sensitive 16 and is used here to quantify the average 
number of water molecules bound to PLE after extensive drying.  
Figure 1 shows that enzyme activity is observed at all hydration levels 
investigated. The lowest hydration achieved  (see the inset to Figure 1) is about 3 (±2) 
water molecules per molecule of protein. This hydration level may relate to the presence 
of internal water molecules that cannot be removed by the method we have used, but 
with current analytical methods this is difficult to verify experimentally, and there is a 
significant possibility that the enzyme is actually anhydrous at this reported hydration. 
3 
The hydration level at which activity is observed is very much lower than the 0.2 h (a 
mole ratio of over 600) conventionally taken to be necessary for enzyme activity, and 
represents a qualitatively lower hydration regime in which experimental enzyme activity 
and dynamics studies can be made. The first stage of any protein sorption isotherm 
consists of the hydration of the ionised groups at the protein surface, up to about 0.05 h 
20. The data here show that enzyme activity occurs and increases up to this level of PLE 
hydration. Although the enzyme rates are low, they are several orders of magnitude 
higher than the un-catalysed rate. At very low hydrations there is no clear correlation 
between activity and hydration, so although completely anhydrous enzyme may not 
have been achieved, enzyme activity at zero hydration seems likely. Surface water that 
interacts directly with the protein has been generally thought to play a major role in 
protein function 4. Since a water content as low as 3 ± 2 water molecules per molecule 
of PLE represents an insignificant coverage of the charged groups of the protein surface 
the evidence here indicates that surface hydration water is not essential for activity, 
although it may facilitate it. 
Figure 1: Enzyme activity with respect to propyl butyrate and methanol 
production in the gas phase, as a function of the protein hydration h (g of water/ 
g protein). The inset is a blow-up of the very low hydration region of the plot. 
 
 
PLE being active at hydration levels close to zero, any motions required for the 
onset of enzyme activity are not likely to be dependent on hydration. Although water 
seems to play a major role in protein dynamics, previous work on xylanase in 
cryosolvent has revealed that this enzyme may be active while apparently rigid 21. Thus, 
any correlation between enzyme hydration, dynamics and activity is still not clear 22-23. 
4 
To examine the fast motions of the enzyme, the average internal atomic mean-square 
displacement of PLE, u2 was determined by neutron scattering with the IN5 time-of-
flight spectrometer and the IN16 backscattering spectrometer at the Institut Laüe-
Langevin, Grenoble, France.  
In Figure 2 u2 is shown as a function of temperature for three different 
hydrations. The curve for the “fully hydrated” control, (0.5 h), exhibits a change in 
slope at ~220K – this is the so-called “dynamical transition” or “glass transition” of the 
protein, where the protein motions apparently pass out of the timescale window of the 
instrument 24-25. For the two other lower-hydration samples, the anharmonic motions 
that are reflected in the increased slope above the dynamical transition,  are strongly 
suppressed, consistent with their being largely solvent driven 7,26-27. These results are 
consistent with an interpretation that water decreases the energy barriers between local 
minima, as is required for the onset of diffusive motions of the protein atoms 7,28. 
Because of the differing energy resolutions of the respective instruments, IN16 (Figure 
2) probes motions on a nano-second timescale while IN5 (Figure 2) probes motion on a 
pico-second timescale. With IN16, a steeper change in slope with hydration is observed 
than for IN5 29, indicative of  the effect of the energy resolution on the mean-square 
displacement (MSD): IN16 has a finer resolution and thus incorporates additional, 
slower motions into the MSD.  
The present work shows clear evidence that enzyme activity does not necessarily 
require that the enzyme be significantly hydrated: within the limits of the water 
detection method used, activity at very near zero hydration has been observed. These 
results raise questions concerning the role of hydration in enzyme activity. Clearly 
hydrolysis reactions require the participation in the chemical steps of at least one water 
molecule, and some proteins contain strongly-bound structurally-important water 
molecules that may be difficult to remove by drying. However, the present results raise 
5 
questions concerning the role of surface hydration water in protein function. The results 
show that, although hydration facilitates function, due possibly in part to the dynamical 
effects manifested above the “glass transition” in the neutron spectra in Figure 2, 
solvation is not an absolute requirement. The role of water as a reactant or as a diffusion 
medium for the products and substrates of the reaction is precluded here by the use of a 
gas phase transesterification catalytic system. Given that water is the only readily-
available terrestrial liquid solvent, it is unsurprising to find its incorporation in proteins, 
and dependence upon it as diffusion medium. However, the present results are 
consistent with the main role of water in enzymology being as a (non-specific) solvent 
and diffusion medium rather than as a chemically-unique essential component.  
Figure 2:u2 of PLE as a function of the temperature for the three hydrations 
measured and obtained 29 from data collected with IN16 and IN5.  
Methods summary 
Protein preparation 
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. 
Water quantification 
The protein powder was equilibrated against 1 g of pure 18O-labeled (18O atom 
95%), Cambridge Isotope Laboratories (CIL, USA) water for 2 to 3 days in a confined 
6 
environment until a hydration level of 30-40 % (w/w) was reached. After drying, the 
enrichment in 18O of the protein sample was determined by mass spectrometry.  
Gas phase activity measurements 
The enzyme catalysed rate of butyl transfer between the methyl butyrate and 
propanol was measured using gas phase chromatography in a dual-mininert® system 30, 
allowing the drying of 5 mg of enzyme powder and isolation of the drying agent before 
measurement of the enzyme activity. The gas phase chromatograph (Varian 3000) was 
equipped with a flame ionization detector and a slightly polar packed column 
(Chromosorb 101, Supelco). The column was maintained at 170 °C. The flow rates 
were 30 mL/min for the dry N2 and H2, and 300 mL/min for the dry air.  
 
Neutron Scattering 
The samples were prepared and analysed in the manner described in 29. The 
incident neutron wavelengths were 5.1 Å and 6.27 Å on IN5 and IN16, respectively. All 
data were collected with the sample holder oriented at 135º relative to the incident 
beam. The samples were contained in aluminium flat-plate cells, of 0.3 mm thickness. 
Spectra were measured with a temperature ramp starting at 80 K and increasing to 300 
K in steps of 10 K every half an hour. The measured transmission for all the samples 
was 0.96 indicating that multiple scattering was negligible. 
 
 
 
 
7 
 
 
 
 
Acknowledgements:  
We thank Steve Cooke, Earth Science Department, Waikato University for processing the protein samples 
in the mass spectrometer; Colin Monk for general technical assistance, and the ILL for neutron beam time 
and associated support. 
 
Author contributions: 
All the authors equally participated in the discussions of the data interpretation. M. L. performed GC 
analysis, 18O-labelled water quantification experiments, neutron scattering sample preparation and wrote 
the paper. V K-S. Performed the neutron scattering data analysis. M. T. Supervised the neutron scattering 
experimental work. RV D. Implemented and developed the 18O-labelled experiments. JL F. participated in 
the experimental work, brought his expertise in protein hydration to the project and contributed to the 
writing of the paper. JC S. brought his expertise in protein dynamics to the supervision of parts of the 
research,participated in the analysis of the results and wrote the paper. RM D brought his expertise in 
enzymology under extreme conditions for research supervision, analysis of the results and wrote the 
paper.  
 
 
 Author information: 
8 
Correspondence and requests for materials should be addressed to Lopez M. (e-mail: lopez-
murielle@orange.fr). 
 
 
9 
 
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Protein Hydration (h)
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Biochemistry - How soft is a protein? A protein dynamics force constant measured by neutron scattering.

Coincidence of dynamical transitions in a soluble protein and its hydration water: direct measurements by neutron scattering and MD simulations.

Correlation of IR spectroscopic, heat capacity, diamagnetic susceptibility and enzymatic measurements on lysozyme powder.

Enzymatic catalysis and dynamics in low-water environments.

Enzymatic catalysis in anhydrous organic solvents.

Enzymatic catalysis in nonaqueous solvents.

Enzyme activity and dynamics: xylanase activity in the absence of fast anharmonic dynamics.

Enzyme activity and flexibility at very low hydration.

Enzyme activity down to -100 degrees C. Biochim Biophys Acta.

Esterase catalysis of substrate vapour: enzyme activity occurs at very low hydration.

How dry are anhydrous enzymes? Measurement of residual and buried

Hydration-coupled dynamics in proteins studied by neutron scattering and NMR: The case of the typical EFhand calcium-binding parvalbumin.

Hydration-induced conformational and flexibility changes in lysozyme at low water content.

Molecular Dynamics of Solid-State Lysozyme as Affected by Glycerol and Water: A Neutron Scattering Study.

Neutron frequency windows and the protein dynamical transition.

O-labeled water molecules using mass spectrometry.

Overview lecture. Hydration processes in biological and macromolecular systems.

Propyl butyrate production Methanol production Protein Hydration (h) P r o d u c t r e l e a s e d ( p m o l / m i n / m g )

Radially softening diffusive motions in a globular protein.

Relationship between Water Activity and Catalytic Activity of Lipases in Organic Media -Effects of Supports, Loading and Enzyme Preparation.

Rhizomucor miehei lipase remains highly active at water activity below 0.0001. Febs Letters 301,

Role of protein-water hydrogen bond dynamics in the protein dynamical transition.

Solid/gas biocatalysis: an appropriate tool to study the influence of organic components on kinetics of lipase-catalyzed alcoholysis.

The dynamic transition in proteins may have a simple explanation. Faraday Discuss.

The effect of low temperatures on enzyme activity.

The Role of Dynamics in Enzyme Activity.

The role of water in recognition and catalysis by enzyme. Biochemist,

The use of gas-phase substrates to study enzyme catalysis at low hydration.

Translational hydration water dynamics drives the protein glass transition.

Water content, one of the most important properties of food.

Water plays a different role on activation thermodynamic parameters of 10 alcoholysis reaction catalyzed by lipase in gaseous and organic media.

Enzyme activity and dynamics in near-anhydrous conditions

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Enzyme activity and dynamics in near-anhydrous conditions

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