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, κ_cat displays a linear Arrhenius dependence yielding the activation energy value E_α=45.5 kJ/mol. In a wide range of viscosities (0.9–43.9 mPa·s) in water-sucrose solutions (0–60% w/w), κ_cat 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, kH_cat/kD_cat ≈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

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