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 50 microM resulted in considerate inactivation of the enzyme. (e.g. 55% less after 60 min with 50 microM 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 200 microM. 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.
Scholz
G
G
Department of Medical Biochemistry, School of Medicine, Flinders University of South Australia.
Barritt
G J
GJ
Kwok
F
F
D019870:1-Phosphatidylinositol 4-Kinase; D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D010770:Phosphotransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D012756:Sheep; D013774:Tetranitromethane / Q000494:pharmacology
D000345:Affinity Labels; D011732:Pyridoxal Phosphate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D013774:Tetranitromethane; D000241:Adenosine