IDENTIFICATION OF CYSTEINE-523 IN THE ASPARTATE BINDING-SITE OF ESCHERICHIA-COLI ASPARAGINE SYNTHETASE-B

The site-directed chemical modifier [p-(fluorosulfonyl)benzoyl] adenos ine (5'-FSBA) inactivates Escherichia coli asparagine synthetase B act ivity following pseudo-first-order kinetics, with ATP providing specif ic protection, with a K-d of 12 mu M. 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 e nzyme, However, the inactivation by 5'-FSBA could be reversed upon the addition of dithiothreitol. These results are indicative of 5'-FSBA-i nduced disulfide bond formation, which requires the presence of at lea st two cysteine residues in the proximity of the ATP binding site. ide ntification of the critical cysteine residue was accomplished by seque ntial replacement of each cysteine in the protein by site-directed mut agenesis. Cys 523 was identified as the key residue involved in the fo rmation of the 5'-FSBA-induced disulfide bond, Detailed kinetic analys es and comparison with similar enzymes, suggest that this cysteine res idue, while in close proximity to the ATP binding site, is actually in volved in aspartate binding in asparagine synthetase B.