N‐Ethylmaleimide‐sensitive mutant (βVal‐153→Cys) Escherichia coli F1‐ATPase: Cross‐linking of the mutant β subunit with the α subunit

摘要

>A β subunit mutation, βVal-153→Cys, in the glycine-rich sequence (phosphate-binding loop) of Escherichia coli F₁ was constructed. Like vacuolar-type ATPase, the mutant enzyme was inhibited by N-ethylmaleimide (NEM) and labeled with [14C]NEM. The inhibition and labeling were prevented by ATP. m-Maleimidobenzoyl-N-hydroxysuccinimide (MBS) (3 μM) almost completely inhibited the mutant enzyme, and cross-linked one pair of α and β subunits. These results suggest that the interaction of the domain near βVal-153 with the α subunit is essential for catalytic cooperativity of the enzyme and that βVal-153 is within 10 Å of the α subunit.