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De Gomez-Puyou, MT; GomezPuyou A; Dominguez-Ramirez, L; Reyes-Vivas, H (2001)

STRUCTURAL ALTERATIONS AND INHIBITION OF UNISITE AND MULTISITE ATP HYDROLYSIS IN SOLUBLE MITOCHONDRIAL F1 BY GUANIDINIUM CHLORIDE

BIOCHEMISTRY-US 40(11):3396-3402
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The effect of guanidinium chloride (GdnHCl) on the ATPase activity and structure 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 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 K-m 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 muM [H-3]ATP in the medium, Fl bound 1.6 and 2 adenine nucleotides per Fl, respectively. The effect of GdnHCl on some structural features of Fl was also examined. GdnHCl at concentrations 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 cysteine per Fl became solvent-exposed and small changes in its intrinsic fluorescence of Fl 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 Fl. 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.