Site-specific mutagenesis of Escherichia coli asparaginase II : none of the three histidine residues is required for catalysis

• Published in: European journal of biochemistry Vol. 208; no. 2; pp. 475 - 480
• Main Authors: WEHNER, A, HARMS, E, JENNINGS, M. P, BEACHAM, I. R, DERST, C, BAST, P, RÖHM, K. H
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 01.09.1992
• Subjects: Amino Acid Sequence; asparaginase; Asparaginase - chemistry; Asparaginase - genetics; Aspartic Acid - metabolism; Bacteriology; Base Sequence; Biological and medical sciences; Catalysis; Enzyme Stability; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; histidine; Histidine - chemistry; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Magnetic Resonance Spectroscopy; Metabolism. Enzymes; Microbiology; Molecular Sequence Data; Mutagenesis, Site-Directed; N.M.R; Protein Conformation; site-specific mutagenesis; Spectrometry, Fluorescence; Structure-Activity Relationship; Urea - pharmacology; Enzymatic activity; Investigation method; Structure activity relation; Enzyme; Escherichia coli; Site directed mutagenesis; Bacteria; Asparaginase; Enterobacteriaceae
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1992.tb17210.x

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.