Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion.
Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme. A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP. In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit. These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin. The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed. ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2. In a similar manner to native E. coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide. Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001313:Aurovertins / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009154:Mutation; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000032:analysis*; D014357:Trypsin / Q000494:pharmacology*
D001313:Aurovertins; D008024:Ligands; D015636:Magnesium Chloride; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D008274:Magnesium