Interaction of ADP and ATP with noncatalytic sites of isolated and membrane-bound chloroplast coupling factor CF1

Abstract

This study of ATP and ADP binding to noncatalytic sites of membrane-bound CF1 (ATP synthase) revealed two noncatalytic sites with different specificities and affinities for nucleotides. One of these is characterized by a high affinity and specificity to ADP (Kd=2.6+/-0.3 microM). However, a certain increase in ADP apparent dissociation constant at high ATP/ADP ratio in the medium allows a possibility that ATP binds to this site as well. The other site displays high specificity to ATP. When the ADP-binding site is vacant, it shows a comparatively low affinity for ATP, which greatly increases with increasing ADP concentration accompanied by filling of the ADP-binding site. The reported specificities of these two sites are independent of thylakoid membrane energization, since both in the dark and in the light the ratios of ATP/ADP tightly bound to the noncatalytic sites were very close. The difference in noncatalytic site affinity for ATP and ADP is shown to depend on the amount of delta subunit in a particular sample. Thylakoid membrane ATP synthase, with stoichiometric content of delta-subunit (one delta-subunit per CF1 molecule), showed the maximal difference in ADP and ATP affinities for the noncatalytic sites. For CF1, with substoichiometric delta subunit values, this difference was less, and after delta subunit removal it decreased still more.

Nucleotide binding to noncatalytic sites is essential for ATP-dependent stimulation and ADP-dependent inactivation of the chloroplast ATP synthase

“…Indeed, the dissociation constant of a site with relatively low affinity for ADP (38 ± 18 lM) corresponds, within the limits of experimental error, to the dissociation constant of the site responsible for inhibition of the ATPase activity induced by light incubation with ADP (45 ± 12 lM). The rate constant of this event (1.0 ± 0.2 min -1 ) agrees with the previously determined time period required to achieve the half-maximum exchange at the noncatalytic sites (about 1 min) (Malyan 2007). It is also the time of light incubation necessary to stimulate the ATPase activity (Shigalova et al 1985).…”

“…The selected preincubation time was based on the nucleotide exchange rate at the noncatalytic sites (Malyan 2007). The initial rate was taken to be the rate measured 20 s after light cessation and addition of 0.5 mM ATP to the reaction mixture.…”

“…These results are in agreement with previous data on the effect of ADP and ATP interactions with the noncatalytic sites of mitochondrial and chloroplast coupling factors F 1 on their ATPase activity (Xue and Boyer 1989;Milgrom et al 1990;Murataliev and Boyer 1992;Jault and Allison 1994). It should be noted that availability of noncatalytic sites widely different in their specificity and affinity for nucleotides is typical of CF o CF 1 and results from differentiation of the noncatalytic sites of CF 1 upon its binding to the membrane part of the complex (Malyan 2006(Malyan , 2007. Under physiological conditions, a site with high specificity and affinity for ADP presumably would not exchange it for ATP and thereby participate in activity regulation.…”

A mechano-chemiosmotic model for the coupling of electron and proton transfer to ATP synthesis in energy-transforming membranes: a personal perspective

“…The head of ATP synthase in E. coli , F 1 , consists of α 3 β 3 γδε -subunits (Kagawa et al 2000 ; Varco-Merth et al 2008 ). Three regulatory nucleotide-binding α -subunits (Malyan 2007 , 2013 ) and three catalytic β -subunits form a symmetrical hexamer, while the δ -subunit is in the form of a lid - cap on the apical part of this hexamer. The b -subunit consists of 156 residues and forms an elongated dimer extending from the periplasmic side of the cytoplasmic membrane to the top of F 1 , where it interacts with a δ -subunit.…”

“…α -subunit protonates additionally two positive residues in the N-terminal part of γ -subunit and transits α -subunit to the open state, from where, an ADP molecule moves to the catalytic center of β 1 -subunit. In “non-catalytic” sites, the rate of nucleotide exchange is about two orders of magnitude lower than the catalysis rate, even with an energized membrane (Malyan 2007 ).…”