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.