Inactivation of isocitrate dehydrogenase kinase/phosphatase by 5 '-[p-(fluorosulfonyl)benzoyl] adenosine is not due to the labeling of the invariant lysine residue found in the protein kinase family

The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/pho sphatase was rapidly lost after prior incubation with the ATP analogue 5'-[ p-(fluorosulfonyl)benzoyl]adenosine (FSBA). This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could b e fully reversed by subsequent addition of dithiothreitol, thereby indicati ng the involvement of cysteine residue(s) in this process. About 2 mol [H-3 ]FSBA/mol IDHK/P were bound during the time course of the inactivation. How ever, this binding was not significantly modified by either prior incubatio n with ATP or subsequent addition of dithiothreitol. This suggested that FS BA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occ urred via the formation of a disulfide bond. Accordingly, mass spectral ana lysis revealed that on addition of FSBA, a disulfide bond was formed betwee n residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme ins ensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue. However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480.