title: Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis.
abstract: Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for L-aspartic acid beta-hydroxamate or impaired aspartate binding. The relative activities towards L-Asn, L-Gln, and l-aspartic acid beta-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis. Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H-NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. 1H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.
authors.affiliation: Institut für Physiologische Chemie, Philipps-Universität, Marburg, Federal Republic of Germany.
authors.forename: A
authors.initials: A
authors.lastname: Wehner
authors.forename: E
authors.initials: E
authors.lastname: Harms
authors.forename: M P
authors.initials: MP
authors.lastname: Jennings
authors.forename: I R
authors.initials: IR
authors.lastname: Beacham
authors.forename: C
authors.initials: C
authors.lastname: Derst
authors.forename: P
authors.initials: P
authors.lastname: Bast
authors.forename: K H
authors.initials: KH
authors.lastname: Röhm
mesh_terms: D000595:Amino Acid Sequence; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics*; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014508:Urea / Q000494:pharmacology
chemical_list: D046911:Macromolecular Substances; D001224:Aspartic Acid; D006639:Histidine; D014508:Urea; D001215:Asparaginase