Verma, Pramod Kumar and Rakshit, Surajit and Mitra, Rajib Kumar and Pal, Samir Kumar (2011) Role of hydration on the functionality of a proteolytic enzyme α-chymotrypsin under crowded environment. Biochimie, 93 (9). pp. 1424-1433. ISSN 0300-9084 http://resolver.caltech.edu/CaltechAUTHORS:20110912-094542110
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Enzymes and other bio-macromolecules are not only sensitive to physical parameters such as pH, temperature and solute composition but also to water activity. A universally instructive way to vary water activity is the addition of osmotically active but otherwise inert solvents which also mimic the condition of an intercellular milieu. In the present contribution, the role of hydration on the functionality of a proteolytic enzyme α-chymotrypsin (CHT) is investigated by modulating the water activity with the addition of polyethylene glycols (PEG with an average molecular weight of 400). The addition of PEG increases the affinity of the enzyme to its substrate, however, followed by a decrease in the turnover number (k_(cat)). Energetic calculations show that entrance path for the substrate is favoured, whereas the exit channel is restricted with increasing concentration of the crowding agent. This decrease is attributed to the thinning of the hydration shell of the enzyme due to the loss of critical water residues from the hydration surface of the enzyme as evidenced from volumetric and compressibility measurements. The overall secondary and tertiary structures of CHT determined from far-UV and near-UV circular dichroism (CD) measurements show no considerable change in the studied osmotic stress range. From kinetic and equilibrium data, we calculate 115 ± 30 numbers of water molecules to be altered during the enzymatic catalysis of CHT. Spectroscopic observation of water relaxation and rotational dynamics of ANS–CHT complex at various concentrations of the osmoting agent also support the dehydration of the hydration layer. Such dehydration/hydration processes during turnover imply a significant contribution of solvation to the energetics of the conformational changes.
|Additional Information:||© 2011 Elsevier Masson SAS. Received 21 October 2010; Accepted 20 April 2011. Available online 29 April 2011. P.K.V and S.R. thank CSIR for research fellowships. We thank DST for a financial grant (SR/SO/BB-15/2007).|
|Official Citation:||Pramod Kumar Verma, Surajit Rakshit, Rajib Kumar Mitra, Samir Kumar Pal, Role of hydration on the functionality of a proteolytic enzyme α-chymotrypsin under crowded environment, Biochimie, Volume 93, Issue 9, September 2011, Pages 1424-1433, ISSN 0300-9084, 10.1016/j.biochi.2011.04.017. (http://www.sciencedirect.com/science/article/pii/S0300908411001362)|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Ruth Sustaita|
|Deposited On:||12 Sep 2011 17:35|
|Last Modified:||12 Sep 2011 17:35|
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