Kinetic manifestation of conformational dynamics, accompanying enzymatic hydrolysis of benzoylglycylphenyllactate by carboxypeptidase A
D. E. Khoshtariya, N. G. Goguadze, J. Ulstrup
Institute of Inorganic Chemistry and Electrochemistry, Georgian Academy of Sciences, Tbilisi; Chemistry Department A, The Technical University of Denmark, Lyngby
Abstract: The influence of temperature, viscosity and H–D isotope substitution on kinetic parameters of the carboxypeptidase-catalysed hydrolysis of O-(benzoylglycyl)-L-β-phenyl-lactate has been studied. In a wide temperature range (15–50°C) the catalytic constant, κcat displays a linear Arrhenius dependence yielding the activation energy value Eα=45.5 kJ/mol. In a wide range of viscosities (0.9–43.9 mPa·s) in water-sucrose solutions (0–60% w/w), κcat displays a negative power-low dependence (linear in logarithmic coordinates) with an index of -0.65. In water-glycerol solutions we observed a significant deviation from linearity due to a specific interaction of glycerol molecules with the catalytic groups. Substitution of H2O by D2O as a solvent yields a kinetic isotope effect, kHcat/kDcat ≈1.20, caused by a greater viscosity of D2O.
The obtained data analysed in terms of the modern elementary chemical act theory led to the conclusion that the rate-limiting step, probably the tetrahedral adduct formation via the substrate carbonyl nucleophilic attack by (Glu-270)—C02-, has a conformational-nonadiabatic nature.
Russian Journal of Bioorganic Chemistry 1991, 17 (5):618-625