Steady-state kinetic analysis of an electroenzyme

Area of Science:

• Biochemistry
• Cell Biology
• Plant Physiology

Background:

• Primary active transport of ions across plant and fungal plasma membranes is crucial for cellular function.
• This transport is primarily driven by a proton-dependent ATPase, a complex protein embedded in the membrane.

Purpose of the Study:

• To investigate the mechanism and stoichiometry of the proton-dependent ATPase in fungal plasma membranes.
• To elucidate the energy conversion process during ion transport and characterize the proton pump's function in vivo and in vitro.

Main Methods:

• Extraction and reactivation of the proton-dependent ATPase in lipid micelles and phospholipid vesicles.
• Measurement of proton pumping, membrane potential (delta psi), and pH differences (delta pH) in vesicle preparations.
• Kinetic analysis of pump-currents under varying conditions ([ATP]i, pHo, pHi, delta psi).

Main Results:

• The proton pump exhibits a stoichiometry of 1 H+ transported per ATP molecule split.
• In vitro, vesicle preparations generated membrane potentials up to 120 mV and pH differences of 2 units.
• In vivo, the proton pump sustains significant membrane potentials (150-350 mV) and pH gradients (up to 3.5 units).
• Kinetic analysis indicated energy conversion occurs during transmembrane charge transfer, likely the E1~P--E2 X P transition.
• Protons are highly dissociated at both membrane surfaces, with distinct pKi and pKo values.

Conclusions:

• The proton-dependent ATPase functions with a strict 1 H+/ATP stoichiometry, essential for generating substantial proton-motive force.
• Energy conversion is tightly linked to transmembrane charge transfer, suggesting a specific conformational change.
• A sequential, double-gated channel model is proposed for the transport mechanism, where the membrane field moves across the ion.