Evaluation by steady-state enzyme kinetics of the role of tightly bound nucleotides during photophosphorylation.
The ATP synthetase of chloroplast membranes binds ADP and ATP with high affinity, and the binding becomes quasi-irreversible under certain conditions. One explanation of the function of these nucleotides is that they are transiently tightly bound during ATP synthesis as part of the catalytic process, and that the release of tightly bound ATP from one catalytic site is promoted when ADP and P(i) bind to a second catalytic site on the enzyme. Alternatively, it is possible that the tightly bound nucleotides are not catalytic, but instead have some regulatory function. We developed steady-state rate equations for both these models for photophosphorylation and tested them with experiments where two alternative substrates, ADP and GDP, were phosphorylated simultaneously. It was impossible to fit the results to the equations that assumed a catalytic role for tightly bound nucleotides, whether we assumed that both ADP and GDP, or only ADP, are phosphorylated by a mechanism involving substrate-induced release of product from another catalytic site. On the other hand, the equations derived from the regulatory-site model that we tested were able to fit all the results relatively well and in an internally consistent manner. We therefore conclude that the tightly bound nucleotides most likely do not derive from catalytic intermediates of ATP synthesis, but that substrate (and possibly also product) probably bind both to catalytic sites and to noncatalytic sites. The latter may modulate the transition of the ATP-synthesizing enzyme complex between its active and inactive states.
Tiefert
M A
MA
Department of Biology, Ben-Gurion University of the Negev, 84105 Beer Sheva, Israel.
Shavit
N
N
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D020134:Catalytic Domain; D025222:Chloroplast Proton-Translocating ATPases / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000096:biosynthesis; D007700:Kinetics; D018545:Lactuca / Q000378:metabolism; D008954:Models, Biological; D009711:Nucleotides / Q000378:metabolism*; D010785:Photophosphorylation
D009711:Nucleotides; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D025222:Chloroplast Proton-Translocating ATPases