Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248.
Journal - Protein science : a publication of the Protein Society (UNITED STATES )
The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.
|ISSN : ||0961-8368|
|Mesh Heading : ||Amino Acid Sequence Amino Acid Substitution Asparaginase Binding Sites Escherichia coli Glutaminase Kinetics Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Protein Conformation Protein Engineering Recombinant Proteins Sequence Alignment Sequence Homology, Amino Acid Thermodynamics chemistry chemistry metabolism|
|Mesh Heading Relevant : ||chemistry metabolism enzymology metabolism|
States and functions of tyrosine residues in Escherichia coli asparaginase II.
Journal - European journal of biochemistry / FEBS (GERMANY )
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.
|ISSN : ||0014-2956|
|Mesh Heading : ||Asparaginase Base Sequence Bromosuccinimide DNA Primers Escherichia coli Guanidine Guanidines Hydrogen-Ion Concentration Macromolecular Substances Magnetic Resonance Spectroscopy Models, Structural Molecular Sequence Data Mutagenesis, Site-Directed Protein Denaturation Recombinant Proteins Thermodynamics biosynthesis biosynthesis chemistry metabolism|
|Mesh Heading Relevant : ||Protein Structure, Secondary Tyrosine chemistry metabolism enzymology|