* To whom correspondence should be addressed.
Received June 18, 2008; Revision received October 3, 2008
The effect of pyrophosphate (PPi) on labeled nucleotide incorporation into noncatalytic sites of chloroplast ATP synthase was studied. In illuminated thylakoid membranes, PPi competed with nucleotides for binding to noncatalytic sites. In the dark, PPi was capable of tight binding to noncatalytic sites previously vacated by endogenous nucleotides, thereby preventing their subsequent interaction with ADP and ATP. The effect of PPi on ATP hydrolysis kinetics was also elucidated. In the dark at micromolar ATP concentrations, PPi inhibited ATPase activity of ATP synthase. Addition of PPi to the reaction mixture at the step of preliminary illumination inhibited high initial activity of the enzyme, but stimulated its activity during prolonged incubation. These results indicate that the stimulating effect of PPi light preincubation with thylakoid membranes on ATPase activity is caused by its binding to ATP synthase noncatalytic sites. The inhibition of ATP synthase results from competition between PPi and ATP for binding to catalytic sites.
KEY WORDS: ATP synthase, CF0CF1, coupling factor CF1, noncatalytic sites, chloroplasts