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Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis. 1992

A Wehner, and E Harms, and M P Jennings, and I R Beacham, and C Derst, and P Bast, and K H Röhm
Institut für Physiologische Chemie, Philipps-Universität, Marburg, Federal Republic of Germany.

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009682 Magnetic Resonance Spectroscopy Spectroscopic method of measuring the magnetic moment of elementary particles such as atomic nuclei, protons or electrons. It is employed in clinical applications such as NMR Tomography (MAGNETIC RESONANCE IMAGING). In Vivo NMR Spectroscopy,MR Spectroscopy,Magnetic Resonance,NMR Spectroscopy,NMR Spectroscopy, In Vivo,Nuclear Magnetic Resonance,Spectroscopy, Magnetic Resonance,Spectroscopy, NMR,Spectroscopy, Nuclear Magnetic Resonance,Magnetic Resonance Spectroscopies,Magnetic Resonance, Nuclear,NMR Spectroscopies,Resonance Spectroscopy, Magnetic,Resonance, Magnetic,Resonance, Nuclear Magnetic,Spectroscopies, NMR,Spectroscopy, MR
D011487 Protein Conformation The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain). Conformation, Protein,Conformations, Protein,Protein Conformations
D002384 Catalysis The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction. Catalyses
D004795 Enzyme Stability The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat. Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006639 Histidine An essential amino acid that is required for the production of HISTAMINE. Histidine, L-isomer,L-Histidine,Histidine, L isomer,L-isomer Histidine
D006863 Hydrogen-Ion Concentration The normality of a solution with respect to HYDROGEN ions; H+. It is related to acidity measurements in most cases by pH pH,Concentration, Hydrogen-Ion,Concentrations, Hydrogen-Ion,Hydrogen Ion Concentration,Hydrogen-Ion Concentrations
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein

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