The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (K m =21 mM, K ic =0.035 mM; K icl =1.5×1015mM; kcat=12 h-1) or assuming variable substrate (K m =16 mM, K ic =0.037 mM; K icl =5.8×1014 mM; kcat=9 h-1), showed a good similarity between these two alternative methodologies and pointed out that bothethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant K icl . In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration.
Keywords: Cellulase kinetics; ethanol inhibition; exoglucanase Cel7A; integrated Michaelis-Menten equations.