Role of short conserved segments of α- and β-subunits that link F<Subscript>1</Subscript>-ATPase catalytic and noncatalytic sites

An analysis of amino acid sequences and 3D structures of chloroplast, mitochondrial, and bacterial F₁-ATPases revealed that in their α- and β-chains there are short highly conserved segments linking in pairs the catalytic and noncatalytic sites. The analysis was based on the reported effect of directed mutagenesis of amino acids forming these segments on catalytic properties of the F₁-ATPases. It is proposed that one of these segments is responsible for transduction of a conformation signal from the noncatalytic to catalytic site upon ADP-for-ATP substitution at the noncatalytic site. At the catalytic site, this signal changes position of the terminal amino acid residue with respect to the adenine part of the molecule and results in a lower tightness of MgADP binding and its dissociation followed by enzyme activation. Mutagenesis of amino acids comprised by the two other segments was shown to produce an effect on the rate of cooperative catalysis, whereas the rate of single-site catalysis remained unaffected. This suggests that these segments are responsible for the cooperative mode of enzyme functioning.     

Light-Dependent Incorporation of Adenine Nucleotide into Noncatalytic Sites of Chloroplast ATP Synthase

The binding of ADP and ATP to noncatalytic sites of dithiothreitol-modified chloroplast ATP synthase was studied. Selective binding of nucleotides to noncatalytic sites was provided by preliminary light incubation of thylakoid membranes with [¹⁴C]ADP followed by its dissociation from catalytic sites during dark ATP hydrolysis stimulated by bisulfite ions (“cold chase”). Incorporation of labeled nucleotides increased with increasing light intensity. Concentration-dependent equilibrium between free and bound nucleotides was achieved within 2–10 min with the following characteristic parameters: the maximal value of nucleotide incorporation was 1.5 nmol/mg of chlorophyll, and the dissociation constant was 1.5 μM. The dependence of nucleotide incorporation on Mg²⁺ concentration was slight and changed insignificantly upon substituting Ca²⁺ for Mg²⁺. Dissociation of nucleotide from noncatalytic sites was illumination dependent. The dissociation kinetics suggested the existence of at least two nucleotide-binding sites with different dissociation rate constants. __________ Translated from Biokhimiya, Vol. 70, No. 11, 2005, pp. 1514–1520. Original Russian Text Copyright ? 2005 by Malyan.     

Covalent Binding of 1,N 6-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP synthase

Catalytic and noncatalytic sites of the chloroplast coupling factor (CF₁) were selectively modified by incubation with the dialdehyde derivative of fluorescent adenosine diphosphate analog 1,N⁶-ethenoadenosine diphosphate. The modified CF₁ was reconstituted with EDTA-treated thylakoid membranes of chloroplasts. The effects of light-induced transmembrane proton gradient and phosphate ions on the fluorescence of 1,N⁶-ethenoadenosine diphosphate, covalently bound to the catalytic sites of ATP synthase, were studied. Quenching of fluorescence of covalently bound 1,N⁶-ethenoadenosine diphosphate was observed under illumination of thylakoid membranes with saturating white light. Addition of inorganic phosphate to the reaction mixture in the dark increased the fluorescence of the label. Quenching reappeared under repeated illumination; however, addition of phosphate ions had no effect on the fluorescence yield in this case. When 1,N⁶-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP synthase, no similar fluorescence changes were observed. The relation between the observed changes of 1,N⁶-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent structural changes in the catalytic site of ATP synthase is discussed.     

Interaction of ADP and ATP with noncatalytic sites of isolated and membrane-bound chloroplast coupling factor CF<Subscript>1</Subscript>

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.     

Catalytic properties of β subunit isolated from chloroplast coupling factor 1

The chloroplast coupling factor (CF₁) was dissociated into subunits by the freezing-thawing procedure in the presence of 0.5 M NaBr and the subunit was purified by ion-exchange chromatography on a DEAE-cellulose column. The subunit did not catalyze ATP hydrolysis either in the presence or in the absence of reagents known to activate Mg²⁺-dependent ATPase activity of CF₁. However, it manifested appreciable adenylate kinase-like and ATP-ADP -phosphate exchange activities. The adenylate kinase-like activity only slightly depended on Mg²⁺ ions. Ethanol, and especially diadenosine pentaphosphate, inhibited the reaction effectively. In contrast, the ATP-ADP exchange activity was Mg²⁺-dependent. Ethanol and diadenosine pentaphosphate were poor inhibitors. Sulfite, the CF₁-ATPase activator, and quercetin, its inhibitor, had a minor effect on catalytic activity of the subunit.Abbreviations CF chloroplast coupling factor 1 - RBP carboxylase-ribulose-1,5-bisphosphate carboxylase - TLC thin layer chromatography - MES morpholinoethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - AP₅A diadenosine pentaphosphate, P¹, P⁵-bis(5-adenosyl)pentaphosphate - DCCD N₁N-dicyclohexylcarbodiimide - SDS sodium dodecylsulfate - PAAG polyacrylamide gel     

Interaction of pyrophosphate with catalytic and noncatalytic sites of chloroplast ATP synthase

The effect of pyrophosphate (PP_i) on labeled nucleotide incorporation into noncatalytic sites of chloroplast ATP synthase was studied. In illuminated thylakoid membranes, PP_i competed with nucleotides for binding to noncatalytic sites. In the dark, PP_i 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 PP_i on ATP hydrolysis kinetics was also elucidated. In the dark at micromolar ATP concentrations, PP_i inhibited ATPase activity of ATP synthase. Addition of PP_i 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 PP_i 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 PP_i and ATP for binding to catalytic sites. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 956–962.     

Interaction of oxyanions with thioredoxin-activated chloroplast coupling factor 1

Interaction between F(1)-ATPase activity stimulating oxyanions and noncatalytic sites of coupling factor CF(1) was studied. Carbonate, borate and sulfite anions were shown to inhibit tight binding of [14C]ATP and [14C]ADP to CF(1) noncatalytic sites. The demonstrated change of their inhibitory efficiency in carbonate-borate-sulfite order coincides with the previously found change in efficiency of these anions as stimulators of CF(1)-ATPase activity [Biochemistry (Mosc.) 43 (1978) 1206-1211]. Inhibition of tight nucleotide binding to noncatalytic sites was accompanied by stimulation of nucleotide binding to catalytic sites. This suggests that stimulation of CF(1)-ATPase activity is caused by interaction between oxyanions and noncatalytic sites. A most efficient stimulator of CF(1)-ATPase activity, sulfite oxyanion, appeared to be a competitive inhibitor with respect to ATP and a partial noncompetitive inhibitor with respect to ADP. The inhibition weakened with increasing time of CF(1) incubation with sulfite and nucleotides. Sulfite is believed to inhibit fast reversible interaction between nucleotides and noncatalytic sites and to produce no effect on subsequent tight binding of nucleotides. A possible mechanism of the oxyanion-stimulating effect is discussed.     

Study of the structure of the thylakoid membrane-bound chloroplast coupling factor CF1.

The structure of thylakoid membrane-bound chloroplast coupling factor CF1 was studied by limited proteolysis followed by sodium dodecylsulfate polyacrylamide gel electrophoresis and N-terminal sequence analysis. The N-terminal fragment of the alpha-subunit was shown to have an exposed area including the peptide bond R21-E22. The cleavage of this peptide bond caused the alphaK24-V25 bond to be exposed to the outside. In the N-terminal fragment of the beta-subunit, the L14-E15 bond was identified and found to be subject to trypsinolysis. Also, the alphaR140-S141, alphaG160-R161, and betaG102-G103 bonds were accessible to the proteolytic attack. In general, the beta-subunit of membrane-bound CF1 is more sensitive to proteolysis than that of solubilized CF1. The products of proteolysis of the alpha-subunit did not contain the polypeptides typical of the reaction of cleavage of the alphaE17-G18 and alphaE22-V23 bonds in isolated CF1. These results suggest a significant structural difference between soluble and membrane-bound CF1. A number of peptide bonds, alphaG160-R161 in particular, were shown to be shielded from proteolytic attack by papain in illuminated thylakoid membranes, probably as a result of membrane energization. In contrast, the light-induced reduction of the gamma-subunit caused an increase in the accessibility of some peptide bonds to this protease, including the alphaG160-R161 bond.     

ATP binding to noncatalytic sites of chloroplast coupling factor CF1

A kinetic analysis of ATP binding to noncatalytic sites of chloroplast coupling factor CF₁ was made. The ATP binding proved to be unaffected by reduction of the disulfide bridge of the CF₁ -subunit. The first-order equation describing nucleotide binding to noncatalytic sites allowed for two vacant nucleotide binding sites different in their kinetics. As suggested by nucleotide concentration dependence of the rate of nucleotide binding, the tight binding was preceded by rapid reversible binding of nucleotides. Preincubation of CF₁ with Mg²⁺ resulted in a decreased rate of ATP binding. ATP dissociation from noncatalytic sites was described by the first order equation for similar sites with a dissociation rate constant k _d (ATP) 10^–3 min^–1. Noncatalytic sites of CF₁ were shown to be not homogeneous. One of them retained the major part of endogenous ADP after precipitation of CF₁ with ammonium sulfate. Its two other sites differed in kinetic parameters and affinity for ATP. Anions of phosphate, sulfite, and especially, pyrophosphate inhibited the interaction between ATP and the noncatalytic sites.