Mutagenic investigation of conserved functional amino acids in Escherichia coli L-aspartase.
The potential importance of several functional amino acids in the activity of L-aspartase from Escherichia coli has been examined by site-directed mutagenesis. Amino acids whose importance in enzyme activity was suggested by chemical modification and pH dependence studies were chosen 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 the fumarase-aspartase enzyme family. Substitution of the most highly conserved cysteine with either serine or alanine, or the most highly conserved histidine with leucine, had no significant effect on the activity of L-aspartase or on the sensitivity of these mutated L-aspartases to cysteine and histidine specific modifying reagents. However, alteration of each of the two conserved lysines to arginine did cause dramatic changes in the catalytic properties of the enzyme. Modification of lysine 54 results in the complete loss of enzyme activity. However, this activity loss appears to be related to changes in the subunit association 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 Km for aspartic acid, a drastic decrease in kcat/Km, and a change in the divalent metal ion requirements of the enzyme.
Saribaş
A S
AS
Department of Chemistry, University of Akron, Ohio 44325-3601.
Schindler
J F
JF
Viola
R E
RE
D000595:Amino Acid Sequence; D001220:Aspartate Ammonia-Lyase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001220:Aspartate Ammonia-Lyase