MUTAGENESIS AND CHEMICAL RESCUE INDICATE RESIDUES INVOLVED IN BETA-ASPARTYL-AMP FORMATION BY ESCHERICHIA-COLI ASPARAGINE SYNTHETASE-B

Site-directed mutagenesis and kinetic studies have been employed to id entify amino acid residues involved in aspartate binding and transitio n state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Eschen'chia coli asparasne synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-33 0 appear particularly crucial for enzymatic activity. For example, whe n Arg-325 is replaced by alanine or lysine, the resulting mutant enzym es possess no detectable asparasne 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 (Gdm-H Cl). Detailed kinetic analysis of the rescued activity suggests that A rg-325 is involved in stabilization of a pentacovalent intermediate le ading to the formation beta-aspartyl-AMP. This rescue experiment is th e second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutati on of Thr-322 and Thr-323 also produces enzymes with altered kinetic p roperties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-asparty l-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any function al role in asparagine synthesis.