Loss of unisite and multisite catalyses by Escherichia coli F1 through modification with adenosine tri- or tetraphosphopyridoxal.
Pyridoxal phosphate (PLP) and adenosine diphospho (AP2-PL)-, triphospho (AP3-PL)-, and tetraphospho (AP4-PL)-pyridoxals (Tagaya, M., and Fukui, T. (1986) Biochemistry 25, 2958-2964) were tested as potential affinity probes for F1 ATPase of Escherichia coli. Both AP3-PL and AP4-PL bound and inhibited F1 ATPase, whereas PLP and AP2-PL were weak inhibitors. The concentrations of AP3-PL and AP4-PL for half-maximal inactivations of the multisite (steady state) ATPase activity were both 18 microM. The binding of these reagents to a reactive lysyl residue(s) was confirmed from the difference absorption spectra, and the stoichiometry of binding of [3H]AP3-PL to F1 at the saturating level was about 1 mol/mol F1. The analogue bound to both the alpha subunit (about two-thirds of the radioactivity) and the beta subunit (about one-third of the radioactivity). No inactivation of multisite ATPase activity or binding of AP3-PL was observed in the presence of ATP. F1 modified with about one mol of AP3-PL had essentially no uni- and multisite hydrolysis of ATP. The rate of binding of ATP decreased to 10(-2) of that of unmodified F1, and the rate of release of ATP was about two times faster. The equilibrium F1 X ATP in equilibrium F1 X ADP X Pi was shifted toward F1 X ATP, and no promotion of ATP hydrolysis at unisite was observed with excess ATP. These results suggest that the AP3-PL or AP4-PL bound to an active site, and catalysis by the two remaining sites was completely abolished.
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011730:Pyridoxal / Q000031:analogs & derivatives*; D013053:Spectrophotometry
D000227:Adenine Nucleotides; D046911:Macromolecular Substances; C049517:adenosine triphosphopyridoxal; C049518:adenosine tetraphosphopyridoxal; D011730:Pyridoxal; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases