Spatial precision of a catalytic carboxylate of F1‐ATPase β subunit probed by introducing different carboxylate‐containing side chains

Abstract

Combining mutation and chemical modification, we have introduced Asp, Gln, Cys, S‐carboxymethylcysteine (Cax) and S‐carbamoylmethylcysteine (Cam) into the positions of Glu¹⁹⁰ and Glu²⁰¹ of the β subunit of F₁‐ATPase from the thermophilic Bacillus PS3. The steady‐state ATPase activities of α₃β₃γ complexes containing these changed β subunits were 12% (E190Cax), 7% (E190D), 3% (E190Cam), 3β₃γ complex. For the complexes containing E190C or E190Q, even the ability of single‐site catalysis was lost. Thus, the presence of a carboxylate at 190 (but not at 201) is absolutely required for catalysis and its spatial precision is very strict. Analysis of inactivation of the complexes by dicyclohexylcarbodiimide suggests that Glu¹⁹⁰ and Glu²⁰¹ are interacting in the F₁‐ATPase.