Steady-state kinetic analysis of an electroenzyme.
Primary active transport of ions through the plasma membranes of plants and fungi is driven by a proton-dependent ATPase, which consists of a membrane-embedded (Mr 104,000) polypeptide, forms a beta-aspartylphosphate intermediate and is blocked by orthovanadate. It can be extracted from cell membranes and reactivated in native lipid micelles or in exogenous phospholipid vesicles. For the fungus Neurospora, vesicle preparations directly display proton-pumping, and can develop membrane potentials (delta psi) of 120 mV or pH differences (delta pH) of 2 units, with a stoichiometry of 1 H+ transported per ATP molecule split. In vivo, the proton pump sustains delta psi values of 150-350 mV (cytoplasm negative) and delta pH values up to 3.5 units (pHi congruent to 7, with pHo = 3.5). Since the total proton-motive force thus can exceed 400 mV, compared with a delta GATP of 500 mV, the stoichiometry must be 1 H+/ATP, with little leeway for neutralizing ions. Kinetic analysis of pump-currents measured during forcing of [ATP]i, pHo, pHi, and delta psi yields three main conclusions: again, the stoichiometry is 1 H+/ATP; energy conversion occurs during transmembrane charge transfer, which therefore is probably the E1 approximately P--E2 X P transition (see Na+,K+-ATPase); protons are strongly dissociated at both membrane surfaces, with pKi congruent to 5.4 versus pHi = 7.2, and pKo congruent to 2.9 versus pHo = 5.8. Considerations of structure and partial-reaction chemistry (by analogy with the Na+,K+-ATPase) suggest a kinetically testable model for the transport mechanism: a sequential, double-gated channel, through which the membrane field is transported across the ion, rather than vice versa.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000201:enzymology; D004560:Electricity; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels; D007700:Kinetics; D008564:Membrane Potentials; D009491:Neurospora / Q000201:enzymology; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism
D007473:Ion Channels; D006859:Hydrogen; D012964:Sodium; D000251:Adenosine Triphosphatases; D011188:Potassium