Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding.

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K(m) values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 +/- 0.2 x 10(-8) mol L(-1), 1.4 +/- 0.2 x 10(-8) mol L(-1), and 1.6 +/- 0.2 x 10(-8) mol L(-1), respectively. The dissociation constant (Kd) showed changes in the affinity of the enzyme for the substrate with increases in the Kcat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.