ACTIVE-SITE LABELING 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)P-3] 3-kinase A was accompanied by irreversible inhibition of enzyme activity. This effect was prevented in the presence of the s ubstrate ATP but not Ins(1,4,5)P-3. The modification reaction obeyed p seudo-first-order rate kinetics. Complete inhibition of activity corre sponded to incorporation of 1.2 mol of phenylglyoxal per mol of protei n. A single [C-14]phenylglyoxal-modified peptide was isolated followin g alpha-chymotrypsin digestion of the radiolabelled Ins(1,4,5)P-3 3-ki nase and reverse-phase HPLC. ATP prevented the incorporation of radioa ctivity to this peptide. The peptide sequence (i.e. QWREGISSSTTL) corr esponded to amino acids 315 to 326 of rat brain Ins(1,4,5)P-3 3-kinase A. An estimate of the radioactivity of the different phenylthiohydant oin amino acid derivatives showed the modified amino acid to be Arg-31 7. The data directly identify a reactive arginine residue as part of t he ATP-binding site. Arg-317 is located within a sequence segment whic h is conserved among the catalytic domain of Ins(1,4,5)P-3 3-kinase is oenzymes A and B in human and rat species.