SY7 - L1
INTERACTION OF TENTOXIN AND DERIVATIVES WITH THE CATALYTIC SECTOR OF CHLOROPLAST ATP-SYNTHASE
Santolini J., Sigalat C., André F., He X., Haraux F.
Section de Bioénergétique, CEA Saclay, F91191 Gif-sur-Yvette Cedex, France
This lecture focuses on the mechanism by which the cyclic tetrapeptide tentoxin (TTX) inhibits and reactivates the F1 part of CF0CF1. Our studies use natural TTX as well as synthetic derivatives, radioactive or not. Two binding sites of different affinities were found to be enough to describe inhibition and reactivation. Some features of the tight site (inhibitory) could be deduced from the comparative efficiency of different TTX analogues. These analogues also allowed to discriminate binding on the loose site and reactivatory effect. We have shown that the interaction of TTX with the tight site does not depend on its dynamic state, whereas interaction of TTX with the loose site does depends on its dynamic state. A minimal model has been developed to describe the complex ATP-dependent interaction of TTX with the loose site. We are also interested in the cooperativity between the two TTX-binding sites and have shown that: 1) toxin binding on the tight site modifies the affinity of toxin for the loose site; 2) The activity of ternary complexes CF1·toxin1·toxin2 depends on the nature of toxins bound on both sites. We have studied the effect of TTX on membrane-bound CF0CF1 and have shown that the enzyme bearing two TTX molecules is competent for proton pumping. We are trying to identify the aminoacids of the binding site(s) of TTX in CF1. Our results will be discussed in the context of the rotatory mechanism of ATP synthesis.
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SY7 - L2
PROTON-TRANSLOCATING TRANSHYDROGENASE FROM PHOTOSYNTHETIC BACTERIA
Jackson J.B., Quirk P.G., Cotton N.P.J., Venning J.D., Gupta S., Peake S.J., Jeeves M., Smith K.J.
School of Biochemistry, University of Birmingham, UK
Transhydrogenase, which uses the energy of Dp to drive the reduction of NADP+ by NADH, has a tripartite structure. Domain I and domain III, which have the binding sites for NAD(H) and NADP(H), respectively, protrude from the membrane. Domain II spans the membrane. Stopped-flow spectroscopy on isolated, recombinant domains I and III shows that hydride transfer proceeds directly between the two nucleotides without intervention of any redox intermediates. A mobile loop region on the surface of domain I folds down and makes contact with the adenosine part of NADH when the nucleotide binds to the protein. Mutants in the loop inhibit the hydride transfer reaction (by 98% in Tyr235Asn). From NMR spectroscopy amino acid residues involved in the interaction between domains I and III are identified. These will be discussed with reference to our understanding of the three-dimensional structure of the proteins.
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SY7 - L3
THE ROLE OF e-g INTERACTIONS IN REGULATING THE ACTIVITY OF THE CHLOROPLAST ATP SYNTHASE
Richter M.
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SY7 - L4
KINETIC ANALYSIS OF ROTARY F0F1-ATP SYNTHASE
Rumberg B., Pänke O.
Max-Volmer-Institut, Technische Universität Berlin, Str. d. 17. Juni 35, D-10623 Berlin
Detailed kinetic modeling of ATP synthase from spinach is presented which pays regard to latest information on H+ stoichiometry, molecular structure and motoric property. The main reaction path is completed by dead end and slip routes. Satisfactory fit to the following experimental results is obtained:
- rates of ATP synthesis and hydrolysis in dependence on ADP, P, ATP and internal H+;
- rate of proton slip in the absence of nucleotides in dependence on internal H+;
- rate of inactivation in the absence of transmembrane pH difference in dependence on ADP, P, ATP.
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SY7 - L5
APPROACHES TO THE UNDERSTANDING OF THE DµH+-LINKED ACTIVATION OF THE CHLOROPLAST ATP SYNTHASE
Strotmann H.,
Ponomarenko S.,
Schwarz O.
Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf
Universitätsstr. 1, D-40225 Düsseldorf, Germany
Membrane energization enhances the detachment of CF1 from thylakoids by chaotropic salts. This effect is explained by protonations at the interface between CF0 and CF1 which attenuates the electrostatic interaction and is related to the DµH+-dependent activation of CF0CF1. Detailed investigation of the process of deactivation following thylakoid deenergization and reactivation
following energization reveals basically two different interconvertible active enzyme forms, one which is highly active and unstable and another one which is less active but metastable. The transition from the inactive to the highest active form proceeds in two steps which depend on the extent of the proton gradient. The apparent pK values of the protonable groups available from the thylakoid lumen side were determined. The Hill coefficients with regard to the intrathylakoidal proton suggest the cooperation of two protons for each step. The results are discussed with regard to the current concepts of the mechanism of the ATP synthase.
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SY7 - L6
COMPARISON OF THE STRUCTURE AND FUNCTION OF CF1 e SUBUNIT FROM DIFFERENT PLANT CHLOROPLASTS
Wei J.-M.,
Shi J.,
Gu Y.,
Yang Y.-Z.,
Shen Y.-K.
Shanghai Institute of Plant Physiology, the Chinese Academy of Sciences. Shanghai 200032, China
Previously, it was found in our lab that changing the CF1 between broad bean and spinach chloroplasts showed enhancement in photophosphorylation activities. Now in comparative study of structure and function of chloroplast-ATPase from spinach and broad bean, we found that the activity of Mg2+-ATPase of chloroplasts activated by methanol from broad bean was higher than that from spinach, but the Mg2+-ATPase activity of spinach chloroplasts activated by DTT + light was higher than that from broad bean. The SDS-PAGE map showed that the molecular weight of e subunit of broad bean CF1 was smaller than that of spinach CF1.
We cloned atpE gene of chloroplast of broad bean and determined its DNA sequence. The amino acid sequence of e subunit of broad bean CF1 induced from DNA sequence, shows 84% and 74.4% homology respectively when compared with that of spinach sequence and maize sequence, but the degree of inhibition by adding e subunit of maize CF1 on the ATPase activity of spinach CF1(-e) was higher than that of broad bean.
After changing Thr-42 of e subunit to Cys, Arg, Ile and Pro by site-directed mutagenesis, which were named mutant eT42C, mutant eT42R, mutant eT42I, mutant eT42P respectively, the mutant eT42I showed much stronger inhibition on the Ca2+-ATPase activity than that of wild-type e subunit, while mutant eT42C and eT42R only slight inhibition.
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