title: 5'-p-Fluorosulfonylbenzoyladenosine. Inactivation of myosin subfragment 1 and a model reaction with cysteine.
abstract: Myosin subfragment 1 ((S-1) is inactivated upon incubation with the nucleotide analogue 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo). The rate of inactivation is increased in the presence of MgADP and MgPPi and decreased in the presence of MgATP. Complete loss of ATPase activity correlates with the loss of two sulfhydryl groups on S-1, and for reactions carried out in the presence of MgADP, this is accompanied by noncovalent trapping of the nucleotide (0.75 ADP/S-1). Treatment of the inactivated S-1 with dithiothreitol leads to the recovery of the lost sulfhydryl groups, release of the trapped MgADP, and recovery of the protein's activity. Importantly, all of these changes are achieved without any significant release of the incorporated radioactive nucleotide analogue. Remarkably similar results were obtained for the reaction of 5,5'-dithiobis-(2-nitrobenzoic acid) with S-1 (Wells, J. A., and Yount, R. G. (1980) Biochemistry 19, 1711-1717) and were interpreted in terms of disulfide bond formation between the reactive SH1 and SH2 thiols. It is proposed that the inactivation of S-1 by 5'-FSO2BzAdo is caused by a two-step reaction involving the initial formation of a thiosulfonate, with the SH1 group, followed by a displacement of the analogue by the SH2 residue to yield a disulfide. The stable and measurable incorporation of the analogue into S-1 proceeds in a parallel reaction at site(s) which did not affect the ATPase activity. The general feasibility of the proposed role of 5'-FSO2BzAdo in forming a disulfide bond in proteins is demonstrated by monitoring its reaction with free cysteine. As detected by pH stat titrations, 5,5'-dithiobis-(2-nitrobenzoic acid) titrations, and amino acid analysis, 5'-FSO2BzAdo rapidly converts cysteine into cystine.
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D003545:Cysteine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D015879:Myosin Subfragments; D009218:Myosins / Q000037:antagonists & inhibitors*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors*; D011817:Rabbits
chemical_list: D015879:Myosin Subfragments; D010446:Peptide Fragments; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000251:Adenosine Triphosphatases; C026049:EDTA-ATPase; D009218:Myosins; D000252:Calcium-Transporting ATPases; D000241:Adenosine; D003545:Cysteine