Amino acid substitutions in the a subunit affect the epsilon subunit of F1F0 ATP synthase from Escherichia coli

Amino acid substitutions at many positions in the a subunit of F1F0 ATP syn thase result in impaired proton translocation and altered catalytic activit y. In this work, we demonstrate that amino acid substitutions in the a subu nit affect the epsilon subunit. In mutant F1F0 ATP synthases, the epsilon s ubunit was studied by determining its sensitivity to proteolysis and by che mical crosslinking under conditions of active turnover and in quiescent enz yme. Like native F1F0 ATP synthase, the epsilon subunit in enzymes carrying either the a(arg-210-->ile) or a(gly-218-->asp) substitutions proved resis tant to trypsin digestion during ATP hydrolysis. In each case, the epsilon subunit was rapidly digested in the presence of a nonhydrolyzable ligand, b ut this did not result in the activation of hydrolytic activity typically s een in wild-type enzyme. In enzyme carrying the a(ala-217-->arg) substituti on, the trypsin digestion of the epsilon subunit occurred regardless of lig and and was accompanied by a limited hydrolytic activation. Relative to the native F1F0 ATP synthase, the a(ala-217-->arg) substitution resulted in re duced efficiency of crosslinking between the epsilon and beta subunits usin g 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide. These observations indi cate that the structural changes resulting from amino acid substitutions in the a subunit are propagated to the epsilon subunit and are specific to th e individual substitutions. (C) 1999 Academic Press.