A novel approach for estimating the relationship between the kinetics and thermodynamics of glycoside hydrolases

摘要

A series of experiments were performed, in which p-nitrophenyl-β-d-cellobioside (PNPC) was hydrolyzed by 1, 4-β-d-glucan-cellobiohydrolase (CBHI: EC 3.2.1.91), and O-nitrophenyl-β-d-galactoside (ONPG) was hydrolyzed by β-galactosidase (EC 3.2.1.23) under different combinations of temperature and time period. The combined effects of temperature and time on p-nitrophenyl and O-nitrophenyl formation were characterized as the change of the instantaneous reaction velocity occurrence per temperature range termed as v_inst· T−1. This parameter was used as a stable index to evaluate the apparent activation energy (E_a) based on the Arrhenius approach, instead of the reaction velocity constant, k. It was found that E_a for PNPC hydrolysis by CBHI first decreased with temperature increase and then slightly increased at higher temperature, and its minimum value was obtained just at the maximum point of v_inst. In addition, E_a for PNPC hydrolysis by dilute sulfuric acid was not a constant, but was continuously increased with temperature. The present studies demonstrated that E_a obtained by Arrhenius approach for the hydrolysis reaction of β-hydrolases appears to be only an empirical kinetic parameter for the dependence of the reaction velocity on temperature and time, and has no meaning in the sense of thermodynamic energy.