TAILIEUCHUNG - Báo cáo khoa học: Relationship between functional activity and protein stability in the presence of all classes of stabilizing osmolytes

We report the effects of stabilizing osmolytes (low molecular mass organic compounds that raise the midpoint of thermal denaturation) on the stabil-ity and function of RNase-A under physiological conditions (pH and 25 C). Measurements of Gibbs free energy change at 25 C(DGD ) and kinetic parameters, Michaelis constant (Km) and catalytic constant (kcat )of the enzyme mediated hydrolysis of cytidine monophosphate | ễFEBS Journal Relationship between functional activity and protein stability in the presence of all classes of stabilizing osmolytes Shazia Jamal Nitesh K. Poddar Laishram R. Singh f Tanveer A. Dar 4 Vikas Rishi and Faizan Ahmad Centre for Interdisciplinary Research in Basic Sciences Jamia Millia Islamia New Delhi India Keywords catalytic efficiency denaturation equilibrium enzyme activity osmolytes protein stability Correspondence F. Ahmad Centre for Interdisciplinary Research in Basic Sciences Jamia Millia Islamia New Delhi India 110025 Fax 91 11 2698 3409 Tel 91 11 2698 1733 E-mail faizan_ahmad@ These authors contributed equally to this work Present addresses fDivision of Population Science Fox Chase Cancer Center Philadelphia PA USA ỊDepartment of Chemistry Biochemistry University of Montana Missoula MT USA NationalCancer Institute NIH Bethesda MD USA Received 29 May 2009 revised 10 August 2009 accepted 19 August 2009 doi We report the effects of stabilizing osmolytes low molecular mass organic compounds that raise the midpoint of thermal denaturation on the stability and function of RNase-A under physiological conditions pH and 25 C . Measurements of Gibbs free energy change at 25 C AGD and kinetic parameters Michaelis constant Km and catalytic constant kcat of the enzyme mediated hydrolysis of cytidine monophosphate enabled us to classify stabilizing osmolytes into three different classes based on their effects on kinetic parameters and protein stability. a Polyhydric alcohols and amino acids and their derivatives do not have significant effects on AGD and functional activity Km and kcat . b Methylamines increase AGD and kcat but decrease Km. c Sugars increase AGD but decrease both Km and kcat. These findings suggest that among the stabilizing osmo-lytes a polyols amino acids and amino acid derivatives are compatible solutes in terms of both stability and function b methylamines are the best refolders stabilizers

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