PMID:1521538
| Citation |
Wehner, A, Harms, E, Jennings, MP, Beacham, IR, Derst, C, Bast, P and Röhm, KH (1992) Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis. Eur. J. Biochem. 208:475-80 |
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| 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. |
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| Keywords |
Amino Acid Sequence; Asparaginase/chemistry; Asparaginase/genetics; Aspartic Acid/metabolism; Base Sequence; Catalysis; Enzyme Stability; Escherichia coli/enzymology; Escherichia coli/genetics; Histidine/chemistry; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Spectrometry, Fluorescence; Structure-Activity Relationship; Urea/pharmacology |
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