• Citer

Active site labelling of inositol 1,4,5-trisphosphate 3-kinase by phenylglyoxal

par Communi, David ;Lecocq, Raymond ;Vanweyenberg, Valérie ;Erneux, Christophe
Référence Biochemical Journal, 310, 1, page (109-115)
Publication Publié, 1995

Article révisé par les pairs

• Accès en ligne
• Détails
• Contenu
• Statistiques

Résumé :

Chemical modification by phenylglyoxal, an arginine-specific reagent, of both native and recombinant rat brain inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase A was accompanied by irreversible inhibition of enzyme activity. This effect was prevented in the presence of the substrate ATP but not Ins(1,4,5)P3. The modification reaction obeyed pseudo-first-order rate kinetics. Complete inhibition of activity corresponded to incorporation of 1.2 mol of phenylglyoxal per mol of protein. A single [14C]phenylglyoxal-modified peptide was isolated following alpha-chymotrypsin digestion of the radiolabelled Ins(1,4,5)P3 3-kinase and reverse-phase HPLC. ATP prevented the incorporation of radioactivity to this peptide. The peptide sequence (i.e. QWREGISSSTTL) corresponded to amino acids 315 to 326 of rat brain Ins(1,4,5)P3 3-kinase A. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivative showed the modified amino acid to be Arg-317. The data directly identify a reactive arginine residue as part of the ATP-binding site. Arg-317 is located within a sequence segment which is conserved among the catalytic domain of Ins(1,4,5)P3 3-kinase isoenzymes A and B in human and rat species.