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 (ki) 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.
|Number of pages||6|
|Journal||European Journal of Biochemistry|
|Publication status||Published - Dec 1992|