Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis.
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.[1]References
- Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis. Wehner, A., Harms, E., Jennings, M.P., Beacham, I.R., Derst, C., Bast, P., Röhm, K.H. Eur. J. Biochem. (1992) [Pubmed]
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