Activation of aspartase by site-directed mutagenesis.
To elucidate the role of sulfhydryl groups in the enzymatic reaction of the aspartase from Escherichia coli, we used site-directed mutagenesis which showed that the enzyme was activated by replacement of Cys-430 with a tryptophan. This mutation produced functional alterations without appreciable structural change: The kcat values became 3-fold at pH 6.0; the Hill coefficient values became higher under both pH conditions; the dependence of enzyme activity on divalent metal ions increased; and hydroxylamine, a good substrate for the wild-type enzyme, proved a poor substrate for the mutant.
Murase
S
S
Department of Chemistry, Faculty of Science, Kyoto University, Japan.
Takagi
J S
JS
Higashi
Y
Y
Imaishi
H
H
Yumoto
N
N
Tokushige
M
M
D000595:Amino Acid Sequence; D001220:Aspartate Ammonia-Lyase / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003545:Cysteine; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D014364:Tryptophan
D010446:Peptide Fragments; D014364:Tryptophan; D001220:Aspartate Ammonia-Lyase; D003545:Cysteine