title: Recovery of catalytic activity from an inactive aggregated mutant of l-aspartase.
abstract: Two highly conserved lysyl residues have been replaced with an arginine to examine their role in the mechanism of l-aspartase from Escherichia coli. Replacement of an active-site lysine results in a significant loss of catalytic efficiency [A. S. Saribas, J. F. Schindler, and R. E. Viola (1994) J. Biol. Chem. 269, 6313-6319], while replacement of the second lysine leads to a completely inactive and insoluble protein. Fluorescence spectral evidence 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 high 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 insoluble form. Treatment with an artificial chaperone system in which solubilization by detergent is followed by its removal with beta-cyclodextrin leads to a stable enzyme under nondenaturing conditions with about half the catalytic activity of the wild-type enzyme. These results confirm a structural role for lysine-55 in l-aspartase and demonstrate that additional characterization is required before conclusions can be drawn from the production of an inactive mutant.
authors.affiliation: Department of Chemistry, University of Akron, Akron, Ohio, 44325-3601, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Jayasekera
authors.forename: R E
authors.initials: RE
authors.lastname: Viola
mesh_terms: D001220:Aspartate Ammonia-Lyase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D008239:Lysine / Q000737:chemistry; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D001220:Aspartate Ammonia-Lyase; D008239:Lysine