Assessment of total catalytic sites and the nature of bound nucleotide participation in photophosphorylation.

Application of new hexokinase accessibility method for the measurement of bound (/sup 32/P)ATP during steady state photophosphorylation by chloroplast thylakoids, together with mixing-quenching and filtration techniques has shown the following. (1) Two classes of bound ATP exist: a transitorily tightly bound class that represents a catalytic intermediate and a noncatalytic class of tightly bound ATP that is labeled much more slowly, presumably from medium components. Thylakoids filtered after a few seconds of photophosphorylation have some of both classes present. Thus, ATP may remain bound at a catalytic site on membranes in the absence of continued energization. (2) During steady state photophosphorylation, the sum of catalytically competent bound ATP plus P/sub i/ is considerably greater than 1/chloroplast ATP synthase complex even when substrate concentration limits the synthesis rate. At a given instant, therefore, most or all phosphorylation complexes are functional, with the simultaneous participation of more than one catalytic site/enzyme. (3) During photophosphorlyation, with substrate concentration well below K/sub m/ values, most ATP synthase complexes retain a bound, catalytic ATP. This behavior is not consistent with independent catalytic sites. These results give additional support to the view developed in this laboratory that ATP synthesis is accomplished by a mechanism in whichmore » two catalytic sites are functioning alternately on the ATP synthase. This mechanism proposes that energy-linked conformational changes in the ATP synthase promote binding of P/sub i/ and ADP in mode competent for ATP synthesis at one catalytic site and concomitantly the release of tightly bound ATP from an alternate site.« less