MUTAGENIC INVESTIGATION OF CONSERVED FUNCTIONAL AMINO-ACIDS IN ESCHERICHIA-COLI L-ASPARTASE

The potential importance of several functional amino acids in the acti vity of L-aspartase from Escherichia coli has been examined by site-di rected mutagenesis. Amino acids whose importance in enzyme activity wa s suggested by chemical modification and pH dependence studies were ch osen as candidates for investigation. The selection of the particular amino acid targets was guided by homology comparisons among the other sequenced bacterial L-aspartases and by the broader comparison among t he fumarase-aspartase enzyme family, Substitution of the most highly c onserved cysteine with either serine or alanine, or the most highly co nserved histidine with leucine, had no significant effect on the activ ity of L-aspartase or on the sensitivity of these mutated L-aspartases to cysteine and histidine specific modifying reagents. However, alter ation of each of the two conserved lysines to arginine did cause drama tic changes in the catalytic properties of the enzyme. Modification of lysine 54 results in the complete loss of enzyme activity, However, t his activity loss appears to be related to changes in the subunit asso ciation properties of the arginine 54 mutant. Lysine 326 appears to be involved in substrate binding. Modification of this residue causes a 5-fold increase in the K-m for aspartic acid, a drastic decrease in K- cat/K-m, and a change in the divalent metal ion requirements of the en zyme.