Boehlein S K, Walworth E S, Schuster S M
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
Biochemistry. 1997 Aug 19;36(33):10168-77. doi: 10.1021/bi970494l.
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.
位点特异性化学修饰剂[对（氟磺酰基）苯甲酰基]腺苷（5'-FSBA）使大肠杆菌天冬酰胺合成酶B的活性按照假一级动力学失活，ATP提供特异性保护作用，解离常数为12微摩尔。5'-FSBA的修饰似乎是共价性的，尽管非化学计量的放射性标记5'-FSBA（少于10%）与完全失活的酶相关联。然而，加入二硫苏糖醇后，5'-FSBA引起的失活作用可以逆转。这些结果表明5'-FSBA诱导了二硫键的形成，这需要在ATP结合位点附近至少存在两个半胱氨酸残基。通过定点诱变依次替换蛋白质中的每个半胱氨酸来鉴定关键的半胱氨酸残基。Cys 523被鉴定为参与5'-FSBA诱导的二硫键形成的关键残基。详细的动力学分析以及与相似酶的比较表明，这个半胱氨酸残基虽然紧邻ATP结合位点，但实际上参与了天冬酰胺合成酶B中天冬氨酸的结合。
