Escherichia coli F1 ATPase is reversibly inhibited by intra- and intersubunit crosslinking: an approach to assess rotational catalysis.
Reaction of the multisubunit F1 ATPase from Escherichia coli (EF1) with a bifunctional cleavable crosslinker, 3,3'-dithiobis(succinimidylpropionate) (DSP), has been used to explore the possibility that during catalysis a rotational movement of catalytic subunits relative to noncatalytic subunits occurs. The premise is that such rotational catalysis is tenable if intersubunit crosslinking of a major subunit with one of the minor subunits inhibits the enzyme activity and if upon cleavage of the crosslinks, the enzyme regains activity. The results presented in this paper show that crosslinking of about 5-6 reactive groups on EF1 with DSP is accompanied by a loss of 2/3 of the enzyme activity. Both intra- and intersubunit crosslinks are formed. The most prominent intersubunit crosslinks are those of gamma and delta subunits with the alpha subunit. Nearly complete recovery of activity can be attained by cleaving the disulfide bond in the crosslinker with dithiothreitol. Because the chemical modification of enzyme groups remains after the crosslinker is cleaved, the loss in activity before cleavage can be ascribed to conformational restraints. The results show that catalysis by the EF1 ATPase is highly sensitive to the restrictions of crosslinking, and are consistent with the view that catalysis is accompanied by appreciable movements of the major subunits with respect to the minor subunits, as suggested for rotational catalysis.
D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D013388:Succinimides / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D013388:Succinimides; D006180:Proton-Translocating ATPases; C011240:dithiobis(succinimidylpropionate); D004229:Dithiothreitol