The ATP-binding site of brain phosphatidylinositol 4-kinase PI4K230 as revealed by 5 '-p-fluorosulfonylbenzoyladenosine

The ATP-binding site of purified bovine brain phosphatidylinositol 4-kinase 230 (PI4K230) was studied by its reaction with 5 ' -p-fluorosulfonylbenzoy ladenosine (FSBA), an ATP-like alkylating reagent. Four hundred to eight hu ndred micromolar FSBA inactivated PI4K230 specifically with apparently firs t-order kinetics and resulted in 50% loss of enzyme activity in 36-130 min. The specificity of the reaction with FSBA Was demonstrated by the lack of inactivation with 5 ' -p-fluorosulfonylbenzoyl chloride and by protection w ith ATP and ATP analogues against inactivation. Most ATP analogues competed with FSBA inactivation in order of their increasing hydrophobicity, parall el to their inhibitory potency in activity measurements. The specific bindi ng of FSBA to PI4K230 was demonstrated also by Western-blot experiments. Th ese results suggest that FSBA-reactive group(s) involved in the enzyme acti vity are located near to the ATP-binding site in a hydrophobic region of na tive PI4K230. Experiments with site-directed mutagenesis indicate that the conserved Lys-1792 plays essential role ill the enzyme activity and serves as one target of affinity labelling by FSBA. Prevention of both Lys-1792-di rected and Lys-1792-independent binding of FSBA by Cibacron Blue 3GA sugges t that these sites are located spatially close to each other. (C) 2001 Else vier Science Ltd. All rights reserved.