Recovery of catalytic activity from an inactive aggregated mutant of L-aspartase

Two highly conserved lysyl residues have been replaced with an arginine to examine their role in the mechanism of L-aspartase from Escherichia coli. R eplacement of an active-site lysine results in a significant loss of cataly tic efficiency [A. S. Saribas, J. F. Schindler, and R. E. Viola (1994) J. B iol. Chern. 269, 6313-6319], while replacement of the second lysine leads t o a completely inactive and insoluble protein. Fluorescence spectral eviden ce has suggested that the loss of activity is due to the misfolding of this aspartase mutant. Some catalytic activity is recovered when the mutant is treated with varying levels of denaturants, and extended treatment with hig h levels of guanidine.HCl results in the recovery of a substantial fraction of the wild-type activity from this inactive mutant. However, upon removal of the denaturant this mutant enzyme slowly reverts to its inactive and in soluble form, Treatment with an artificial chaperone system in which solubi lization by detergent is followed by its removal with beta-cyclodextrin lea ds to a stable enzyme under nondenaturing conditions with about half-the ca talytic activity of the wild-type enzyme. These results confirm a structura l role for lysine-55 in L-aspartase and demonstrate that additional charact erization is required before conclusions can be drawn from the production o f an inactive mutant. (C) 1999 Academic Press.