Identification of cysteine-523 in the aspartate binding site of Escherichia coli asparagine synthetase B.
The site-directed chemical modifier [p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA) inactivates Escherichia coli asparagine synthetase B activity following pseudo-first-order kinetics, with ATP providing specific protection, with a Kd of 12 microM. The 5'-FSBA modification appears to be covalent, even though a nonstoichiometric amount (less than 10%) of radiolabeled 5'-FSBA was associated with a totally inactivated enzyme. However, the inactivation by 5'-FSBA could be reversed upon the addition of dithiothreitol. These results are indicative of 5'-FSBA-induced disulfide bond formation, which requires the presence of at least two cysteine residues in the proximity of the ATP binding site. Identification of the critical cysteine residue was accomplished by sequential replacement of each cysteine in the protein by site-directed mutagenesis. Cys 523 was identified as the key residue involved in the formation of the 5'-FSBA-induced disulfide bond. Detailed kinetic analyses and comparison with similar enzymes, suggest that this cysteine residue, while in close proximity to the ATP binding site, is actually involved in aspartate binding in asparagine synthetase B.
Boehlein
S K
SK
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
Walworth
E S
ES
Schuster
S M
SM
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D001217:Aspartate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed
D004220:Disulfides; D004791:Enzyme Inhibitors; D001224:Aspartic Acid; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D001217:Aspartate-Ammonia Ligase; D000241:Adenosine; D003545:Cysteine