Active site labelling of inositol 1,4,5-trisphosphate 3-kinase A by phenylglyoxal.
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.
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Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire (IRIBHN), Université Libre de Bruxelles, Belgium.
Lecocq
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Vanweyenberg
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Erneux
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008969:Molecular Sequence Data; D010658:Phenylglyoxal / Q000494:pharmacology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D010658:Phenylglyoxal