PLANT PHYSIOLOGY , Vol 108, Issue 2 813-819, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants
G. Roberts, G. Berberian and L. Beauge
Division de Biofisica, Instituto de Investigacion Medica "Mercedes y Martin Ferreyra," Casilla de Correo 389, 5000 Cordoba, Argentina
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 [mu]M). 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
kinetics 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 kinetics 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.
