Abstract
The oxygen exchange that occurs between water and the gamma-PO3 of ATP in light-activated chloroplast lamellae was found to proceed with close to full equilibration of the oxygens before ATP returned to the medium. This is in contrast to the entry of approximately one water oxygen when ATP is synthesized from ADP and P1 in the same system. In the latter case, the limitation is kinetic, however, not steric, as shown by the presence of some molecules containing more than one water-derived oxygen in the gamma-PO3. The different extents of exchange can be explained by a relatively faster rate of dissociation of ATP from the chloroplast coupling factor during synthesis from ADP and P1 relative to its dissociation in the absence of net phosphorylation. To determine the mechanism of gamma-PO3:H2O exchange, its rate was compared with the rate of reversible cleavage of ATP as detected by betagamma bridge to beta nonbridge 18O scrambling in [Pbeta-18O-Pgamma]ATP (Midelfort, C. F., and Rose, I. A. (1976) J. Biol. Chem. 251, 5881-5887). The scrambling reaction, which depends on cleavage of the PbetaO--Pgamma bond, was found to occur in nearly the same fraction of ATP molecules that experienced gamma-PO3:H2O exchange in the same incubation, suggesting that the latter is due to multiple cycles of reversible ATP hydrolysis on the chloroplast coupling factor, i.e. [ATP-H2O in equilibrium ADP-Pi].
Highlights
Two mechanisms have been put forth to explain the incorporation of water oxygens into the y-PO, of ATP synthesized during oxidative phosphorylation in mitochondria or photophosphorylation in plant chloroplasts
They suggest that H,180 may attack the y-phosphate of ATP to form a penta coordinated phosphate of trigonal bipyramidal geometry, containing 160H,+ as well as lXOH,+ oxonium ions in the apical positions. (Double protonation of oxygen is required to enable this ligand to effectively compete with ADP for the apical leaving group positions [5] and prevent P,O-P, bond cleavage.) Because either apical group can depart, [yJXO]ATP is formed concomitant with loss of H,‘60
The results indicate that incorporation of water oxygens into the synthesized
Summary
To distinguish between the reversible hydrolysis and the in-line displacement exchange mechanism, a technique is needed which will enable one to detect the transient formation of ADP at the catalytic center of the coupling enzyme. Such a method has recently been introduced by Midelfort and Rose [6] employing ATP containing lx0 in the by bridge position as well as in the y-PO,.
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