Theoretical Investigation into the Change in the Number of Water Molecules in Solvent Inaccessible Region of an Enzyme and Enzyme-substrate Complex

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Ikechukwu Iloh Udema
Abraham Olalere Onigbinde


Background: There may be dry enzymes, but water remains indispensable for the catalytic action of enzymes. There is not as much interest in how the presence of a drug such as aspirin and a psychoactive compound such as ethanol may affect the water-mediated role of the enzyme.

Objectives: The objectives of this research are: 1) To assess the changes in the number of water molecules interacting with the enzyme-substrate complex and the solvent inaccessible region of a protein, 2) to determine the free energy difference due to preferential solvation and hydration, and 3) to re-examine theoretical issues in literature and relate them to the interpretation of the results.

Methods: A major theoretical research and minor experimentation using Bernfeld method.

Results and Discussion: The presence of ethanol/aspirin alone yielded only dehydration of the osmolyte inaccessible region and the enzyme substrate complex (ES). There was positive free energy difference (DDG) if the equilibrium constant for hydration change (Keq(1))> the equilibrium constant for folding-unfolding transition (Keq(3)); it is negative where Keq(3)> Keq(1). Analysis of various models made them valuable for the interpretation of result for feature application.

Conclusion: The change in the number of water molecules in an osmolyte inaccessible region of the enzyme and those interacting with the ES may be either positive or negative due respectively to sucrose and ethanol/aspirin. The spontaneity of two processes, hydration and folding-unfolding transition, the free energy difference, differs. The model for water stripping, preferential interaction concept, and the KBI for osmolation and hydration can guide the interpretation of the effects of any cosolute.

Porcine pancreatic alpha amylase, change in gibbs free energy, change in the number of water molecules, enzyme-substrate complex, osmolyte-inaccessible region of enzyme, cosolutes, Kirkwood-Buff Integrals(KBI).

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Udema, I., & Onigbinde, A. (2019). Theoretical Investigation into the Change in the Number of Water Molecules in Solvent Inaccessible Region of an Enzyme and Enzyme-substrate Complex. Asian Journal of Research in Biochemistry, 5(2), 1-17.
Original Research Article


Nakagawa H, Kitao JA, Kataoka M. Hydration affects both harmonic and anharmonic nature of protein dynamics. Biophys. J. 2008;95(6):2916–2923.

Bernazzani P. Structural changes associated with interactions between starch and particles of TiO2 ZnSe. J. Chem. Biochem. Mol. Biol. 2008;2(1):1–13.

Gangadharan D, Nampoothiri KM, Sivaramakrishnan S, Pandey A. Immobilized bacterial a-amylase for effective hydrolysis of raw and soluble starch. Food Res. Int. 2009;42:436-442.

Knight J, Hamelberg D, McCammon AJ, Kothary R. The role of conserved water molecules in the catalytic domain of protein kinases. 2009;76(3):527-535.

Affleck R, Xu Z-F, Suzawa V, Focht K, Clark DS. Enzymatic catalysis and dynamics in low-water environments. Biochemistry. 1992;89:1100-1104.

Kurkal V, Daniel RM, Finney JL, Tehei M, Dunn RV, Smith JC. Enzyme activity and flexibility at very low hydration. Biophys. J. 2005;89:1282–1287.

Laage D, Elsaesser T, Hynes JT. Water dynamics in the hydration shells of biomolecules. Chem. Rev. 2017;117: 10694-10725.

Maharolkar AP, Murugkar AG, Khirade PW, Mehrotra SC. Microwave dielectric relaxation and polarization study of binary mixture of methylethylketone with nitrobenzene. Bull. Chem. Soc. Ethiop. 2019;33(2):349-358.

Buurma, NJ, Pas Torello L, Blandermer JM, Engberts JBFN. Kinetic evidence for hydrophobically stabilized encounter complexes formed by hydrophobic esters in aqueous solutions containing monohydric alcohols. J. Am. chem. Soc. 2001;123:11848–11853.

Udema II, Onigbinde AO. Effect of interacting organic co-solutes with enzyme substrate complex on the hydrolysis of raw soluble starch with alpha-amylase: Theory and experimentation. Adv. Res. 2016;7(1): 1-19.

Schnell S, Maini PK. Enzyme kinetics at high enzyme concentration. Bull. Math. Biol. 2000;62:483–499.

Udema II. Substrate mass conservation in enzyme catalyzed amylolytic activity. Int. J. Biochem. Res. Rev. 2017;18(1):1-10.

Mitchell DC, Litman DT. Effect of ethanol and osmotic stress on receptor conformation reduced water activity amplifies the effect of ethanol on Metarhodopsin II formation. J. Biol. Chem. 2000;275(8):5355-5360.

Sirotkin VA, Kuchierskaya AA. Alpha-Chymotrypsin in water-ethanol mixtures: Effect of preferential interactions. Chem. Phys. Lett. 2017;689:156-161.

Udema II, Onigbinde AO. Basic Kirkwood –Buff theory of solution structure and appropriate application of Wyman linkage equation to biochemical phenomena. Asian J. Phys. Chem. Sci. 2019;7(1):1-14.

Sheng Y, Capri J, Waring A, Valentine JS, Whitelegge J. Exposure of solvent-inaccessible regions in the amyloidogenic protein human SOD1 determined by hydroxyl radical foot printing. J. Am. Soc. Mass Spectrum. 2019;30(2):218-226.

Blundell TL, Gong S. Structural and functional restraints on the occurrence of single amino acid variations in human proteins. PLoS one. 2010;5(2):e9186,1-12.

Gains JC, Acebes S, Virueta A, Butter M, Regan L, O’ Hern CS. Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins. Proteins. 2018;85(5):581-591.

Bolen DW, Baskakov IV. The osmophobic effect: Natural selection of a thermodynamic force in protein folding. J. Mol. Biol. 2001;310(5):955-963.

Pace CN. Measuring and increasing protein stability. Trends Biotechnol. 1990;8:93–98.

Udema II, Onigbinde AO. Activity coefficient of solution components and salts as special osmolyte from Kirkwood-Buff theoretical perspective. Asian Res. Biochem. 2019;4(3):1-20.

Lineweaver H, Burk D. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 1934;56:658–666.

Kramer RM, Shende VR, Motl N, Pace N, Scholtz JM. Toward a molecular understanding of protein solubility: Increased negative surface charge correlates with increased solubility. Biophys. J. 2012;102:1907–1915.

Bernfeld P. Amylases, alpha and beta. Methods. Enzymol. 1955;1:149–152.

Udema II. The effect of additives and temperature on the velocity of hydrolysis of raw starch with human salivary a - amylase. J. Biochem. Res. Rev. 2016; 10(2):1-17.

Sanyal NS, Kaushal N. Effect of two non-steroidal anti-inflammatory drugs, aspirin and nimesulide on the G-glucose transport and disaccharide hydrolases in the intestinal brush border membrane. Pharmacol Rep. 2005;57:833-838.

de Piña MZ, Saldaña-Balmori Y, Hermández-Tobias A, Piña E. Nonsteroidal anti-inflammatory drugs lower ethanol-mediated liver increase in lipids and thiobarbituric acid reactive substances. Alcohol Clin Exp Res. 1994;17(6):1228-12232.

Levitt M, Sharon R. Accurate simulation of protein dynamics in solution. Proc. Nat. Acad. Sci. U.S.A. 1988;85:7557-7561.

Petukhov M, Rychkov G, Firsov L, Serrano L. H-bonding in protein hydration revisited. Protein Sci. 2004;13(8):22120-2129.

Ooi T, Oobatake M. Prediction of the thermodynamics of protein unfolding: The helix-coil transition of polyl (L-alannine). Biochemistry. 1991;88:974-975.

Yang L, Dordick JS, Garde S. Hydration in nonaqueous media is consistent with solvent dependence of its activity. Biophys. J. 2004;87:812-821.

Lynch TW, Sligar SG. Macromolecular hydration changes associated with BamHI binding and catalysis. J. Biol. Chem. 2000;275(39):30561-30565.

Dai L, Klibanov AM. Striking activation of oxidative enzymes suspended in nonaqueous media. Proc. Nat. Acad. Sci. U.S.A. 1999;96:9475-9478.

Zaks A, Klibanov AM. The effect of water on enzyme action in organic media. J. Biol. Chem. 1988;263(17):8017-8021.

Pal SKJ, Zewail AH. Biological water at the protein surface, dynamical solvation probed directly with femtosecond resolution. Proc. Nat. Acad. Sci. U.S.A. 2001;94(4):1763-1768.

Csermely P. Water and cellular folding process. Cell. Mol. Biol. 2001;47(5):1-9.

Poole P. Hydration and enzymatic activity. J. Phys. Colloq. 1984;45(c7):249-253.

Timasheff SN. Protein solvent preferential interaction, protein hydration, and the modulation of biochemical reactions by solvent components. Biochemistry. 2002; 99(15):9721-9726.

Harano Y, Kinoshita M. Translational-entropy gain of the solvent upon protein folding. Biophys. J. 2005;89:2701-2710.

Robinson CR, Sligar SG. Molecular recognition mediated by bound water: A mechanism for star activity of the restriction endonuclease EcoRI. J. Mol. Biol. 1993;234(2):302-306.

Rösgen J, Pettit MB, Bolen DW. Protein folding, stability, and solvation structure in osmolyte solution. Biophys. J. 2005;89: 2988–2997.

Bolen DW, Baskakov IV. The osmophobic effect: Natural selection of a thermo-dynamic force in protein folding. J. Mol. Biol. 2001;310(5):955-963.

Baskakov I, Bolen DW. Forcing thermodynamically unfolded proteins to fold (communication). J. Biol. Chem. 1998;273(9):1-5.

Schellman JA. Protein stability in mixed solvents: A balance of contact interaction and excluded volume. Biophys J. 2003;85: 108-125.