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

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.