States and functions of tyrosine residues in Escherichia coli asparaginase II.
The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site-directed mutagenesis, chemical modification of wild-type and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer. pH titration curves showed that the active-site residue Y25 has a normal pKa while the C-terminal Y326 is unusually acidic. 1H-NMR signals of a peculiar ligand-sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14-27) which shields the active site during catalysis is highly flexible in the free enzyme.
Derst
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Institut für Physiologische Chemie, Philipps-Universität, Marburg, Germany.
Wehner
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Specht
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Röhm
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D001215:Asparaginase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D001974:Bromosuccinimide; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D014443:Tyrosine
D017931:DNA Primers; D006146:Guanidines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014443:Tyrosine; D001215:Asparaginase; D019791:Guanidine; D001974:Bromosuccinimide