Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants

Roberts, G.; Berberian, G.; Beauge, L.

We investigated the nature of the complex ATP activation kinetics of plant H+-ATPases. To this aim we analyzed that activation in three isolated isoforms (AHA1, AHA2, and AHA3) of H+-ATPase from Arabidopsis thaliana. The isoforms were obtained by heterologous expression in endoplasmic reticulum of yeast. ATP stimulation was always with low affinity (K0.5) between 500 and 1800 micromolar). In addition, the curves were not Michaelian and displayed positive cooperativity. Detailed studies with AHA2 showed that (a) enzyme solubilized with lysophosphatidylcholine exhibited Michaelian behavior even in the presence of soybean lecithin liposomes free of enzyme, (b) solubilized enzyme incorporated into the same liposomes displayed two-site kindles with negative cooperativity, and (c) enzyme partially digested with trypsin lost the C-terminal portion of the molecule. Under this condition the ATP activation kinetics was Michaelian or had a slight negative cooperativity and the K0.5ATP was reduced 3-fold. These data suggest that the functional unit of the H+-ATPase has two catalytic ATP sites with variable cooperativity and kindles competence of the E(ATP) and E(ATP)2 complexes. Such variability is likely modulated by the association of the enzyme with membrane structures and by a regulatory domain in the C terminus of the enzyme molecule.

Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants

Roberts, G.; Berberian, G.; Beauge, L., 1995: Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants. Plant Physiology 108(2): 813-819