Product-activation of Escherichia coli membrane-bound H+-ATPase (F1F0-ATPase) connected with ϵ-subunit at alkaline pH

Abstract Mutant strain AN1518 or AN2387 (Gly48 → Asp in ϵ-subunit) and partial revertant strain AN2540 (Gly48 → Asp, Pro47 → Ser in ϵ-subunit) of E. coli were used in a kinetic study of membrane-bound H + -ATPase. It was found that at pH 9.0 mutant strain AN1518 or AN2387 and partial revertant strain AN2540 gave a low initial rate, which increased with time until linearity was reached after 1–2 min. This phenomenon was prominent in mutant strains, but was not so obvious in wild-type AN346 of E. coli ; this property is similar to F 1 -ATPase reported by Cox [1]. The mechanism of the slow activation of membrane-bound H + -ATPase was further investigated in this paper. The experimental results indicated that the hydrolytic rate of E. coli F 1 F 0 -ATPase that increased with time was membrane protein concentration- and pH-dependent, and that the product ADP produced during ATP hydrolysis is the factor causing the slow activation. Preincubation of the hydrolytic product ADP with a concentration comparable to that produced in the assay (20 μM) caused initial activation of ATP hydrolysis and abolished the slow activation. On the other hand, with the removal of ADP during the progress of the hydrolytic reaction it could be seen that the slow activation was abolished as well. In order to test the relationship between the ϵ-subunit and ADP involved in the slow activation, trypsin treatment was carried out on the membrane-bound H + -ATPase of various strains. The activation observed after trypsin treatment was on the order of AN1518 > AN2540 > AN346. The activation effects of ADP and trypsin were not found to be additive. This implies that ADP acted in a similar way to trypsin, i.e., to cause removal of the ϵ-subunit. A tentative mechanism of the slow activation was proposed that ADP, a product of ATP hydrolysis, could induce conformational changes of F 1 F 0 at alkaline pH 9.0, thus weakening the binding strength between the ϵ-subunit and other subunits of F 1 F 0 , and resulting in removal or partial removal of the ϵ-subunit. This further impaired the coupling of F 1 and F 0 in the mutant strains; as a consequence the rate of ATP hydrolysis was increased.