Product-activation of Escherichia coli membrane-bound H(+)-ATPase (F1F0-ATPase) connected with epsilon-subunit at alkaline pH.
Mutant strain AN1518 or AN2387 (Gly48-->Asp in epsilon-subunit) and partial revertant strain AN2540 (Gly48-->Asp, Pro47-->Ser in epsilon-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 F1-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 F1F0-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 microM) 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 epsilon-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 epsilon-subunit. A tentative mechanism of the slow activation was proposed that ADP, a product of ATP hydrolysis, could induce conformational changes of F1F0 at alkaline pH 9.0, thus weakening the binding strength between the epsilon-subunit and other subunits of F1F0, and resulting in removal or partial removal of the epsilon-subunit. This further impaired the coupling of F1 and F0 in the mutant strains; as a consequence the rate of ATP hydrolysis was increased.
Wu
J H
JH
Institute of Biophysics, Academia Sinica, Beijing, People's Republic of China.
Li
S G
SG
Lin
Z H
ZH
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008565:Membrane Proteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D008565:Membrane Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases