Structural alterations and inhibition of unisite and multisite ATP hydrolysis in soluble mitochondrial F1 by...

The effect of guanidinium chloride (GdnHCl) on the ATPase activity and structure of soluble mitochondrial F1 was studied. At high ATP concentrations, hydrolysis is carried by the three catalytic sites of F1; this reaction was strongly inhibited by GdnHCl concentrations of <50 mM. With substoichiometric ATP concentrations, hydrolysis is catalyzed exclusively by the site with the highest affinity. Under these conditions, ATP binding and hydrolysis took place with GdnHCl concentrations of >100 mM; albeit at the latter concentration, the rate of hydrolysis of bound ATP was lower. Similar results were obtained with urea, although nearly 10-fold higher concentrations were required to inhibit multisite hydrolysis. GdnHCl inhibited multisite ATPase activity by diminishing the V(max) of the reaction without significant alterations of the Km for MgATP. GdnHCl prevented the effect of excess ATP on hydrolysis of ATP that was already bound to the high-affinity catalytic site. With and without 100 mM GdnHCl and 100 microM [3H]ATP in the medium, F1 bound 1.6 and 2 adenine nucleotides per F1, respectively. The effect of GdnHCl on some structural features of F1 was also examined. GdnHCl at concentrations that inhibit multisite ATP hydrolysis did not affect the exposure of the cysteines of F1, nor its intrinsic fluorescence. With 100 mM GdnHCl, a concentration at which unisite ATP hydrolysis was still observed, 0.7 cysteine per F1 became solvent-exposed and small changes in its intrinsic fluorescence of F1 were detected. GdnHCl concentrations on the order of 500 mM were required to induce important decreases in intrinsic fluorescence. These changes accompanied inhibition of unisite ATP hydrolysis. The overall data indicate that increasing concentrations of GdnHCl bring about distinct and sequential alterations in the function and structure of F1. With respect to the function of F1, the results show that at low GdnHCl concentrations, only the high-affinity site expresses catalytic activity, and that inhibition of multisite catalysis is due to alterations in the transmission of events between catalytic sites.