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

The effect of guanidinium chloride (GdnHCl) on the ATPase activity and stru cture of soluble mitochondrial Fl was studied. At high ATP concentrations, hydrolysis is carried by the three catalytic sites of Fl; this reaction was strongly inhibited by GdnHCl concentrations of <50 mM. With substoichiomet ric ATP concentrations, hydrolysis is catalyzed exclusively by the site wit h the highest affinity. Under these conditions, ATP binding and hydrolysis took place with GdnHCl concentrations of > 100 mM; albeit at the latter con centration, 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 ATPas e activity by diminishing the V-max of the reaction without significant alt erations of the K-m for MgATP. GdnHCl prevented the effect of excess ATP on hydrolysis of ATP that was already bound to the high-affinity catalytic si te. With and without 100 mM GdnHCl and 100 muM [H-3]ATP in the medium, Fl b ound 1.6 and 2 adenine nucleotides per Fl, respectively. The effect of GdnH Cl on some structural features of Fl was also examined. GdnHCl at concentra tions that inhibit multisite ATP hydrolysis did not affect the exposure of the cysteines of Fl, nor its intrinsic fluorescence. With 100 mM GdnHCl, a concentration at which unisite ATP hydrolysis was still observed, 0.7 cyste ine per Fl became solvent-exposed and small changes in its intrinsic fluore scence of Fl were detected. GdnHCl concentrations on the order of 500 mM we re 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 Fl. With respect to the function of F1, the results show that at low GdnHCl concentrations, on ly 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.