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/phosphatase 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 be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process. About 2 mol [3H]FSBA/mol IDHK/P were bound during the time course of the inactivation. However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol. This suggested that FSBA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond. Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme insensitive 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.
Oudot
C
C
Institut de Biologie et Chimie des Protéines, CNRS, Lyon, France.
Jault
J M
JM
Jaquinod
M
M
Negre
D
D
Prost
J F
JF
Cozzone
A J
AJ
Cortay
J C
JC
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics; D011494:Protein Kinases / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D004220:Disulfides; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C042301:isocitrate dehydrogenase kinase; C042300:isocitrate dehydrogenase phosphatase; D010749:Phosphoprotein Phosphatases; D014357:Trypsin; D000251:Adenosine Triphosphatases; D000241:Adenosine