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 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.
Boehlein
S K
SK
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
Walworth
E S
ES
Richards
N G
NG
Schuster
S M
SM
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine; D001216:Asparagine / Q000096:biosynthesis*; D001217:Aspartate-Ammonia Ligase / Q000737:chemistry* / Q000235:genetics; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D004926:Escherichia coli; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D012984:Software; D013329:Structure-Activity Relationship; D013912:Threonine
C106094:beta-aspartyl-adenosine monophosphate; D005973:Glutamine; D013912:Threonine; D001224:Aspartic Acid; D000249:Adenosine Monophosphate; D001216:Asparagine; D001120:Arginine; D001217:Aspartate-Ammonia Ligase