Structure of the γ subunit of Escherichia coli F₁ ATPase probed in trypsin digestion and biotin-avidin binding studies

The arrangement and functional role of the γ subunit of the Escherichia coli F₁ATPase (ECF₁) has been probed by protease digestion and avidin- biotin labeling experiments using wild-type enzyme and four mutants, γS8C, γT106C, γS179C, and γV286C, respectively. Trypsin was found to cleave the γ subunit at four sites, Arg⁷⁰, Lys¹⁹⁹, Lys²⁰¹, and Lys²¹². Cleavage at these four sites did not greatly reduce the high ATPase activity of the enzyme that is obtained when the ε subunit is removed by the protease treatment. However, prolonged trypsin cleavage led to loss of inhibition by ε subunit added back to the trypsin-treated enzyme. Endoproteinase-Lys-C cleaves the γ subunit of ECF₁ at three of the four sites, i.e. Lys¹⁹⁹, Lys²⁰¹, and Lys²¹², but not at Arg⁷⁰. The enzyme was activated by treatment with this protease because of degradation and release of the ε subunit, but added pure ε subunit still caused inhibition of ATPase activity. Therefore, cleavage at Arg⁷⁰ by trypsin is responsible for the loss of response to the ε subunit inhibition. Biotin was reacted with Cys residues at positions 8, 106, 179, and 286 in different γ subunit mutants and the accessibility of the biotin to avidin monitored in the intact ECF₁ from the different mutants. Avidin was able to react with biotin when incorporated at position 106, not at 8, 179, or 286. The four trypsin cleavage sites, Arg⁷⁰, Lys¹⁹⁹, Lys²⁰¹, and Lys²¹², as well as Thr¹⁰⁶ are in regions of the γ subunit predicted to be mainly β-sheet and β-turn structures.