PMID:9139684
| Citation |
Boehlein, SK, Walworth, ES, Richards, NG and Schuster, SM (1997) Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by Escherichia coli asparagine synthetase B. J. Biol. Chem. 272:12384-92 |
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| Abstract |
Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis. |
| Links | |
| Keywords |
Adenosine Monophosphate/analogs & derivatives; Adenosine Monophosphate/metabolism; Amino Acid Sequence; Arginine; Asparagine/biosynthesis; Aspartate-Ammonia Ligase/chemistry; Aspartate-Ammonia Ligase/genetics; Aspartic Acid/analogs & derivatives; Aspartic Acid/metabolism; Escherichia coli; Glutamine/metabolism; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Sequence Alignment; Software; Structure-Activity Relationship; Threonine |
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