Affinity labelling of the active site of brain phosphatidylinositol 4‐kinase with 5′‐fluorosulphonylbenzoyl‐adenosine

5′‐p‐Fluorosulphonylbenzoyl‐adenosine (FSO₂BzAdo), an affinity labelling analogue of ATP, was used to label the active site of sheep brain phosphatidylinositol 4‐kinase (PtdIns 4‐kinase). The incubation of PtdIns 4‐kinase with concentrations of FSO₂BzAdo as low as 50μ resulted in considerate inactivation of the enzyme. (e.g. 55% less after 60 min with 50μ FSO₂BzAdo). The kinetics of inactivation of PtdIns 4‐kinase by FSO₂BzAdo suggest a two‐step mechanism, in which a rapid reversible binding of FSO₂BzAdo to the enzyme is followed by a covalent sulphonation step. The first‐order rate constant (k₂) for the inactivation of PtdIns 4‐kinase was calculated to be 0.063 min⁻¹, and the steady‐state constant of inactivation (k_i) to be 200μ. Preincubation of the enzyme with either ATP plus Mg²⁺, or PtdIns alone, prior to addition of FSO₂BzAdo reduced the degree of inactivation of the enzyme; suggesting that FSO₂BzAdo binds within the active site PtdIns 4‐kinase. Moreover, since ATP plus Mg²⁺ provided the greatest protection against inactivation, it is concluded that the main site of labelling of PtdIns 4‐kinase by FSO₂BzAdo is within the ATP‐binding site of the enzyme. Results obtained from chemical modification experiments, which employed pyridoxal 5′‐phosphate and tetranitromethane, are consistent with a catalytically‐essential lysine being present within the ATP‐binding site of PtdIns 4‐kinase. Therefore, it is hypothesised that the inactivation of PtdIns 4‐kinase by FSO₂BzAdo may be due to the labelling of this lysine residue.