Promotion and inhibition of catalytic cooperativity of the Ca2+-dependent ATPase activity of spinach chloroplast coupling factor 1 (CF1).
ATP- and ITP-stimulation of the Ca2+-dependent hydrolysis of low concentrations of [gamma-32P]ATP was used as a direct demonstration of catalytic cooperativity in CF1. CF1 activated by epsilon-subunit removal or dithiothreitol, or by the presence of ethanol in the ATPase assay medium, shows pronounced catalytic cooperativity, with maximal stimulation of [gamma-32P]ATP hydrolysis at about 20 microM CaATP. Catalytic cooperativity is diminished by the presence of the epsilon-subunit or by pretreatment of either untreated or epsilon-depleted CF1 with azide (C1/2=30 microM). Both activated and untreated forms of CF1 also exhibit hydrolysis of CaATP by a high-affinity, low-capacity mode of turnover, which is unaffected by any of the preceding treatments and shows normal Michaelis-Menten behaviour. We propose that this high-affinity mode represents unisite catalysis, and that the endogenous inhibitor, epsilon, and the exogenous inhibitor, azide, both act exclusively on cooperative interactions between the catalytic sites.
Andralojc
P J
PJ
Department of Biochemistry, University of Oxford (U.K.).
Harris
D A
DA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D000431:Ethanol / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D010944:Plants / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019810:Sodium Azide
D001386:Azides; D010761:Phosphorus Radioisotopes; D000431:Ethanol; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases