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W2068774323 abstract "The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) embedded in the phosphoribulokinase (PRK)·GAPDH·CP12 complex was increased 2–3-fold by reducing agents. This occurred by interaction with PRK as the cysteinyl sulfhydryls (4 SH/subunit) of GAPDH within the complex were unchanged whatever the redox state of the complex. But isolated GAPDH was not activated. Alkylation plus mass spectrometry also showed that PRK had one disulfide bridge and three SH groups per monomer in the active oxidized complex. Reduction disrupted this disulfide bridge to give 2 more SH groups and a much more active enzyme. We assessed the kinetics and dynamics of the interactions between PRK and GAPDH/CP12 using biosensors to measure complex formation in real time. The apparent equilibrium binding constant for GAPDH/CP12 and PRK was 14 ± 1.6 nm for oxidized PRK and 62 ± 10 nmfor reduced PRK. These interactions were neither pH- nor temperature-dependent. Thus, the dynamics of PRK·GAPDH·CP12 complex formation and GAPDH activity are modulated by the redox state of PRK. The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) embedded in the phosphoribulokinase (PRK)·GAPDH·CP12 complex was increased 2–3-fold by reducing agents. This occurred by interaction with PRK as the cysteinyl sulfhydryls (4 SH/subunit) of GAPDH within the complex were unchanged whatever the redox state of the complex. But isolated GAPDH was not activated. Alkylation plus mass spectrometry also showed that PRK had one disulfide bridge and three SH groups per monomer in the active oxidized complex. Reduction disrupted this disulfide bridge to give 2 more SH groups and a much more active enzyme. We assessed the kinetics and dynamics of the interactions between PRK and GAPDH/CP12 using biosensors to measure complex formation in real time. The apparent equilibrium binding constant for GAPDH/CP12 and PRK was 14 ± 1.6 nm for oxidized PRK and 62 ± 10 nmfor reduced PRK. These interactions were neither pH- nor temperature-dependent. Thus, the dynamics of PRK·GAPDH·CP12 complex formation and GAPDH activity are modulated by the redox state of PRK. phosphoribulokinase glyceraldehyde-3-phosphate dehydrogenase dithiothreitol 1,3-biphosphoglyceric acid surface plasmon resonance matrix-assisted laser desorption ionization time-of-flight Phosphoribulokinase (PRK)1 (EC 2.7.1.19) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.13) form multienzyme complexes. Several multienzyme complexes with different compositions have been isolated from chloroplasts (1Müller B. Z. Naturforsch. Sect. B Chem. Sci. 1972; 27: 925-932Google Scholar, 2Sainis J.K. Harris G.C. Biochem. Biophys. Res. Commun. 1986; 139: 947-954Google Scholar, 3Sainis J.K. Jawali N. Indian J. Biochem. Biophys. 1994; 31: 215-220Google Scholar, 4Sainis J.K. Merriam K. Harris G.C. Plant Physiol. 1989; 89: 368-374Google Scholar, 5Giudici-Orticoni M.T. Gontero B. Rault M. Ricard J. C.R. Acad. Sci. Paris t. 1992; (314 477483, serie III)Google Scholar, 6Gontero B. Cardenas M.L. Ricard J. Eur. J. Biochem. 1988; 173: 437-443Google Scholar, 7Gontero B. Giudici-Orticoni M.T. Ricard J. Eur. J. Biochem. 1994; 226: 999-1006Google Scholar, 8Gontero B. Mulliert G. Rault M. Giudici-Orticoni M.T. Ricard J. Eur. J. Biochem. 1993; 217: 1075-1082Google Scholar, 9Avilan L. Gontero B. Lebreton S. Ricard J. Eur. J. Biochem. 1997; 250: 296-302Google Scholar, 10Avilan L. Gontero B. Lebreton S. Ricard J. Eur. J. Biochem. 1997; 246: 78-84Google Scholar, 11Avilan L. Lebreton S. Gontero B. J. Biol. Chem. 2000; 275: 9447-9451Google Scholar, 12Lebreton S. Gontero B. J. Biol. Chem. 1999; 274: 20879-20884Google Scholar, 13Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 250: 286-295Google Scholar, 14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar, 15Clasper S. Chelvarajan R.E. Easterby J.S. Powls R. Biochim. Biophys. Acta. 1994; 1209: 101-106Google Scholar, 16Clasper S. Easterby J.S. Powls R. Eur. J. Biochem. 1991; 202: 1239-1246Google Scholar, 17Nicholson S. Easterby J.S. Powls R. Eur. J. Biochem. 1987; 162: 423-431Google Scholar, 18Süss K.H. Arkona C. Manteuffel R. Adler K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5514-5518Google Scholar, 19Süss K.H. Prokhorenko I. Adler K. Plant Physiol. 1995; 107: 1387-1397Google Scholar, 20Wedel N. Soll J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9699-9704Google Scholar, 21Wedel N. Soll J. Paap B.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10479-10484Google Scholar).The PRK·GAPDH core complex is linked to photosynthesis, as these two enzymes are part of the Benson-Calvin cycle and use ATP and NADPH produced by the primary reactions of photosynthesis. PRK catalyzes the ATP-dependent phosphorylation of ribulose 5-phosphate to form ribulose 1,5-bisphosphate, the CO2 acceptor in photosynthetic organisms, and GAPDH catalyzes the reversible reduction and dephosphorylation of 1,3-bisphosphoglycerate (BPGA) to glyceraldehyde 3-phosphate using NADPH.We have purified a complex from the green alga Chlamydomonas reinhardtii (10Avilan L. Gontero B. Lebreton S. Ricard J. Eur. J. Biochem. 1997; 246: 78-84Google Scholar) that is made up of two dimeric PRK and two tetrameric GAPDH. The protein CP12 (20Wedel N. Soll J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9699-9704Google Scholar, 21Wedel N. Soll J. Paap B.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10479-10484Google Scholar, 22Pohlmeyer K. Paap B.K. Soll J. Wedel N. Plant Mol. Biol. 1996; 32: 969-978Google Scholar) was found recently to be associated with this supramolecular edifice (23Graciet E. Lebreton S. Camadro J.M. Gontero B. Eur. J. Biochem. 2003; 270: 129-136Google Scholar). The two enzymes, PRK and GAPDH, may each be obtained in a free independent state. When they are not associated with each other they form dimers (PRK) or tetramers (GAPDH). CP12 is tightly associated with GAPDH (23Graciet E. Lebreton S. Camadro J.M. Gontero B. Eur. J. Biochem. 2003; 270: 129-136Google Scholar). The complex can be dissociated by harsh reduction and reversed by oxidizing conditions, because the oxidized partners can spontaneously reform a complexin vitro that is quite similar to the native state (10Avilan L. Gontero B. Lebreton S. Ricard J. Eur. J. Biochem. 1997; 246: 78-84Google Scholar). In only a few cases has it been possible to assemble particles from their separate parts in vitro that resemble the native complexes (24Reed L.J. Pettit F.H. Eley M.H. Hamilton L. Collins J.H. Oliver R.M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3068-3072Google Scholar, 25Fasshauer D. Bruns D. Shen B. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 4582-4590Google Scholar). The association of these two enzymes also gives rise to new regulatory properties. PRK and GAPDH within the complex are regulated by NADP(H) rather than by NAD(H), whereas the independent stable enzymes are not (26Graciet E. Lebreton S. Camadro J.M. Gontero B. J. Biol. Chem. 2002; 277: 12697-12702Google Scholar). Oxidized PRK may be active when associated with GAPDH or when dissociated from the complex upon dilution (14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar). We have also shown that the complex may exist under mild reducing conditions (12Lebreton S. Gontero B. J. Biol. Chem. 1999; 274: 20879-20884Google Scholar) even if it is dissociated by severe reducing conditions (DTT concentrations up to 20 mm). But it dissociates faster upon dilution, as reduction weakens the complex.Whereas plant enzymes are heterotetrameric (A2B2), algal GAPDHs are homotetrameric and made up of only A subunits (27Cerff R. Eur. J. Biochem. 1979; 94: 243-247Google Scholar, 28Qi J. Isupov M.N. Littlechild J.A. Anderson L.E. J. Biol. Chem. 2001; 276: 35247-35252Google Scholar). The B subunit has a C-terminal extension that contains two cysteine residues believed to be involved in the regulation of the enzyme (29Baalmann E. Scheibe R. Cerff R. Martin W. Plant Mol. Biol. 1996; 32: 505-513Google Scholar, 30Scheibe R. Baalmann E. Backhausen J.E. Rak C. Vetter S. Biochim. Biophys. Acta. 1996; 1296: 228-234Google Scholar, 31Sparla F. Pupillo P. Trost P. J. Biol. Chem. 2002; 277: 44946-44952Google Scholar). Nonetheless, studies on crude extracts of Chlamydomonas indicate that the algal enzyme that lacks these 28 amino acid residues can be activated reductively by light (32Li A.D. Stevens F.J. Huppe H.C. Kersanach R. Anderson L.E. Photosyn. Res. 1997; 51: 167-177Google Scholar). We have studied the regulation of this enzyme upon reduction or oxidation in its isolated state or within the PRK·GAPDH·CP12 complex to see if this reductive light activation is explained by the interaction of GAPDH with its protein partners. The same study was performed on the other partner, PRK. In previous studies (14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar) we have fully characterized the activity of isolated PRK and PRK in the complex. PRK in the so-called oxidized complex was active as mentioned above, but this result has been disputed (33Miziorko H.M. Adv. Enzymol. Relat. Areas Mol. Biol. 2000; 74: 95-127Google Scholar), as the cysteinyl sulfhydryls of PRK in the complex have never been directly tested. We have therefore used mass spectrometry coupled with protein chemistry to analyze the cysteinyl sulfhydryl contents of PRK, GAPDH, and CP12 in the complex, whatever its redox state.Finally, we have used a biosensor to study the interaction between the two enzymes depending on their redox states, as cellular redox signaling contributes to the control of the Benson-Calvin cycle and many other physiological processes (34Cooper C.E. Patel R.P. Brookes P.S. Darley-Usmar V.M. Trends Biochem. Sci. 2002; 27: 489-492Google Scholar). The qualitative and quantitative aspects of these interactions have been analyzed.DISCUSSIONMany enzymes belonging to the Benson-Calvin cycle are regulated by dark-light transitions via thioredoxins in vivo (38Balmer Y. Buchanan B.B. Trends Plant Sci. 2002; 7: 191-193Google Scholar) (or dithiothreitol in vitro). In particular, thioredoxin (39Geck M.K. Larimer F.W. Hartman F.C. J. Biol. Chem. 1996; 271: 24736-24740Google Scholar,40Geck M.K. Hartman F.C. J. Biol. Chem. 2000; 275: 18034-18039Google Scholar) reduces the disulfide between Cys55 and Cys16 of inactive oxidized spinach PRK (41Brandes H.K. Larimer F.W. Hartman F.C. J. Biol. Chem. 1996; 271: 3333-3335Google Scholar, 42Milanez S. Mural R.J. Hartman F.C. J. Biol. Chem. 1991; 266: 10694-10699Google Scholar).We have shown (14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar) that oxidized PRK from C. reinhardtii may have some activity when it is associated with GAPDH, contrary to common belief, but no direct evidence of the regulatory disulfide bridge between Cys16 and Cys55 present in oxidized PRK, was given. We have now used alkylation of the so-called oxidized complex to show that there is one disulfide bridge in the PRK monomer. The activity of PRK is greatly increased when it is reduced, indicating that the Cys16–Cys55 bond is the target.Another troubling aspect of the activity of oxidized PRK concerns the P-loop. This contains residues that interact with the ॆ- and γ-phosphoryls of Mg2+-ATP (43Runquist J.A. Rios S.E. Vinarov D.A. Miziorko H.M. Biochemistry. 2001; 40: 14530-14537Google Scholar). This loop should a priori be free to move for efficient catalysis. Some data for bacterial PRK (33Miziorko H.M. Adv. Enzymol. Relat. Areas Mol. Biol. 2000; 74: 95-127Google Scholar, 44Harrison D.H. Runquist J.A. Holub A. Miziorko H.M. Biochemistry. 1998; 37: 5074-5085Google Scholar) suggest that the Cys16–Cys55 disulfide bridge found only in the eukaryotic enzymes could immobilize the P-loop, thereby preventing catalytic turnover. However, it is worthwhile to pinpoint that there are insertions and deletions in the plant and algal enzymes, such as an insertion of 15 residues after the P-loop of the eukaryotic enzyme that may modify its mobility even in the presence of the Cys16–Cys55 disulfide bridge. These sulfhydryl groups are also not essential for catalysis, as a Cys16–Cys55 double mutant of spinach PRK still has some activity (42Milanez S. Mural R.J. Hartman F.C. J. Biol. Chem. 1991; 266: 10694-10699Google Scholar, 45Brandes H.K. Hartman F.C. Lu T.Y. Larimer F.W. J. Biol. Chem. 1996; 271: 6490-6496Google Scholar). The conformational constraint imposed by this bond is more likely to be responsible for the decreased PRK activity than the formation of the disulfide bridge itself (45Brandes H.K. Hartman F.C. Lu T.Y. Larimer F.W. J. Biol. Chem. 1996; 271: 6490-6496Google Scholar). Thus, although the bacterial PRK structure is a useful model, it may not be wise to extrapolate data obtained with it to eukaryotic PRKs, especially the eukaryotic enzyme in a multienzyme complex. Our results indicate that the conformational constraint near the P-loop could be attenuated when the PRK is within the complex, allowing oxidized PRK to be active. The interaction of PRK with GAPDH modifies the conformation of this enzyme (46Mouche F. Gontero B. Callebaut I. Mornon J.P. Boisset N. J. Biol. Chem. 2002; 277: 6743-6749Google Scholar) and hence its kinetic properties (12Lebreton S. Gontero B. J. Biol. Chem. 1999; 274: 20879-20884Google Scholar, 14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar).There is now considerable published evidence from NMR studies that supramolecular edifices (protein-protein or DNA-protein complexes) appear to be flexible (47Finerty Jr., P.J. Muhandiram R. Forman-Kay J.D. J. Mol. Biol. 2002; 322: 605-620Google Scholar, 48Yu L. Zhu C.X. Tse-Dinh Y.C. Fesik S.W. Biochemistry. 1996; 35: 9661-9666Google Scholar). The entropic cost of the decrease in conformational freedom must be offset, to some extent, by preserving the flexibility of other regions. These reports support the assertion made above by the authors, but only structural data will clarify this point. Our direct evaluation of the redox status of the regulatory cysteine residues now clearly shows that oxidized PRK may be catalytically active. This activity is, however, lower than the activity of the released metastable form, as the PRK-GAPDH association hampers the overall flexibility of these enzymes. The released PRK with decreased conformational constraint around the P-loop, because of a memory effect, and with greater overall flexibility is thus a better catalyst than the enzyme in the complex.Whereas each PRK monomer in the oxidized complex has one disulfide bridge, all the cysteinyl sulfhydryl groups of GAPDH are free and CP12 has two disulfide bridges. We found five cysteinyl sulfhydryl groups per PRK subunit by alkylation after reduction of the oxidized complex. The GAPDH content of thiol groups obviously does not change, but two or four thiol groups become titrated per CP12 monomer. Reduction of the PRK disulfide bridge is followed by an increase in enzyme activity, as mentioned above.Chlamydomonas GAPDH is an A4 homotetramer; the regulatory cysteine residues are thus absent (28Qi J. Isupov M.N. Littlechild J.A. Anderson L.E. J. Biol. Chem. 2001; 276: 35247-35252Google Scholar, 31Sparla F. Pupillo P. Trost P. J. Biol. Chem. 2002; 277: 44946-44952Google Scholar). Nonetheless, incubation of a crude extract with DTT increased its enzyme activity 3-fold (32Li A.D. Stevens F.J. Huppe H.C. Kersanach R. Anderson L.E. Photosyn. Res. 1997; 51: 167-177Google Scholar). Somewhat surprisingly, we saw the same increase in NADPH-dependent activity for GAPDH when it was part of the complex, but not for the isolated enzyme. We therefore proposed (49Heineke D. Burgi R. Heike G. Hoferichter P. Peter U. Flügge U.I. Heldt H.W. Plant Physiol. 1991; 95: 1131-1137Google Scholar) that this modulation had a physiological role, as the NADPH concentrations are increased in the light. Our present results also show that reducing the complex leads to a decrease in the use of NADH.As titration of the thiol groups in GAPDH in the complex reveals 4 SH groups whatever the redox state of the complex, the modulation of GAPDH activity is not linked to disulfide reduction. Our present finding also indicates that these effects are linked to heterologous interactions within the complex as there is no longer an effect once these interactions are weakened or broken. This supports the idea mentioned above that new properties may emerge as a consequence of conformation change within multienzyme complexes. It also shows that the regulation of an enzyme like PRK may modulate the regulation of another (GAPDH) via a 舠domino-like舡 effect.We have tested the effect of oxidizing agents on PRK and GAPDH activities, as oxidized thioredoxin causes the oxidation of the light-reduced enzymes. Treatment with oxidants caused the activity of reduced PRK isolated or released from the complex (e.g.using cystine) to decrease. This effect was reversed by reduction, as expected. The situation is quite different for GAPDH, as the loss of activity by the isolated enzyme that follows incubation with oxidized glutathione is very likely because of a modification of the active site cysteine (Cys156), as it is well documented for other GAPDHs (50Harris J.I. Waters M. Boyer P.D. The Enzymes. 13. Academic Press, New York1976: 1-49Google Scholar). This is also supported by protection experiments using BPGA and by the fact that oxidized thioredoxins have no effect on GAPDH activity. The inactivation is not reversed by a reducing agent. It has been suggested that the oxidation of glycolytic enzymes may introduce an element of strain resulting from the formation of a disulfide bond, which then causes an irreversible conformational change, perhaps with displacement of an essential residue from the active site. GAPDH is not significantly inactivated by oxidized glutathione even after dissociation of the complex, like PRK. This may be because the cysteinyl sulfhydryl in this molecule is less exposed than is that in the isolated GAPDH. We checked this by measuring the enzyme kinetics of the isolated GAPDH and of the released form from the complex. The differences observed with the pseudo-affinity constants for BPGA clearly support the assertion made above. Thus, GAPDH retains the conformation it had within the complex (imprinting) even after the physical separation of PRK and GAPDH, so that the sulhydryl group of the Cys156 is poorly accessible to oxidized glutathione. This corroborates the imprinting effect previously reported (12Lebreton S. Gontero B. J. Biol. Chem. 1999; 274: 20879-20884Google Scholar, 13Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 250: 286-295Google Scholar,26Graciet E. Lebreton S. Camadro J.M. Gontero B. J. Biol. Chem. 2002; 277: 12697-12702Google Scholar).Finally, the complex we have purified is a suitable model with which to study protein-protein interactions, but most of the information on it stems from in vitro reconstitution experiments. This report, for the first time, describes the use of Biosensor technology to determine the dynamics of these interactions between PRK and the GAPDH·CP12 subcomplex under different redox states. The dissociation constants between the PRK and GAPDH·CP12 subcomplex are rather low (nm range), with the lowest value (14 ± 1.6 nm) being obtained with oxidized PRK. The probable conformational change caused by the chemical modification of GAPDH by oxidation has no influence on the binding of GAPDH to PRK andvice versa. The SPR results indicate that these enzymes (PRK and GAPDH/CP12) may reconstitute the complex under reducing conditions with a low dissociation constant (62 ± 10 nm) even if complex formation is sensitive to the redox state of PRK. Our results also suggest that this complex may form at pH 7, under oxidizing conditions (dark conditions) or at pH 8, under mild reducing conditions (light conditions).To conclude, thiol/disulfide exchange influences the state of activation of chloroplast PRK, in its isolated form and when it is part of a complex. On the other hand, the modulation of PRK activity influences the state of activation of GAPDH via protein-protein interactions, at least in the alga C. reinhardtii. A new mode of light regulation could emerge that involves a 舠domino effect舡 and this could well apply to enzymes that are not direct targets of light. Phosphoribulokinase (PRK)1 (EC 2.7.1.19) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.13) form multienzyme complexes. Several multienzyme complexes with different compositions have been isolated from chloroplasts (1Müller B. Z. Naturforsch. Sect. B Chem. Sci. 1972; 27: 925-932Google Scholar, 2Sainis J.K. Harris G.C. Biochem. Biophys. Res. Commun. 1986; 139: 947-954Google Scholar, 3Sainis J.K. Jawali N. Indian J. Biochem. Biophys. 1994; 31: 215-220Google Scholar, 4Sainis J.K. Merriam K. Harris G.C. Plant Physiol. 1989; 89: 368-374Google Scholar, 5Giudici-Orticoni M.T. Gontero B. Rault M. Ricard J. C.R. Acad. Sci. Paris t. 1992; (314 477483, serie III)Google Scholar, 6Gontero B. Cardenas M.L. Ricard J. Eur. J. Biochem. 1988; 173: 437-443Google Scholar, 7Gontero B. Giudici-Orticoni M.T. Ricard J. Eur. J. Biochem. 1994; 226: 999-1006Google Scholar, 8Gontero B. Mulliert G. Rault M. Giudici-Orticoni M.T. Ricard J. Eur. J. Biochem. 1993; 217: 1075-1082Google Scholar, 9Avilan L. Gontero B. Lebreton S. Ricard J. Eur. J. Biochem. 1997; 250: 296-302Google Scholar, 10Avilan L. Gontero B. Lebreton S. Ricard J. Eur. J. Biochem. 1997; 246: 78-84Google Scholar, 11Avilan L. Lebreton S. Gontero B. J. Biol. Chem. 2000; 275: 9447-9451Google Scholar, 12Lebreton S. Gontero B. J. Biol. Chem. 1999; 274: 20879-20884Google Scholar, 13Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 250: 286-295Google Scholar, 14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar, 15Clasper S. Chelvarajan R.E. Easterby J.S. Powls R. Biochim. Biophys. Acta. 1994; 1209: 101-106Google Scholar, 16Clasper S. Easterby J.S. Powls R. Eur. J. Biochem. 1991; 202: 1239-1246Google Scholar, 17Nicholson S. Easterby J.S. Powls R. Eur. J. Biochem. 1987; 162: 423-431Google Scholar, 18Süss K.H. Arkona C. Manteuffel R. Adler K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5514-5518Google Scholar, 19Süss K.H. Prokhorenko I. Adler K. Plant Physiol. 1995; 107: 1387-1397Google Scholar, 20Wedel N. Soll J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9699-9704Google Scholar, 21Wedel N. Soll J. Paap B.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10479-10484Google Scholar). The PRK·GAPDH core complex is linked to photosynthesis, as these two enzymes are part of the Benson-Calvin cycle and use ATP and NADPH produced by the primary reactions of photosynthesis. PRK catalyzes the ATP-dependent phosphorylation of ribulose 5-phosphate to form ribulose 1,5-bisphosphate, the CO2 acceptor in photosynthetic organisms, and GAPDH catalyzes the reversible reduction and dephosphorylation of 1,3-bisphosphoglycerate (BPGA) to glyceraldehyde 3-phosphate using NADPH. We have purified a complex from the green alga Chlamydomonas reinhardtii (10Avilan L. Gontero B. Lebreton S. Ricard J. Eur. J. Biochem. 1997; 246: 78-84Google Scholar) that is made up of two dimeric PRK and two tetrameric GAPDH. The protein CP12 (20Wedel N. Soll J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9699-9704Google Scholar, 21Wedel N. Soll J. Paap B.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10479-10484Google Scholar, 22Pohlmeyer K. Paap B.K. Soll J. Wedel N. Plant Mol. Biol. 1996; 32: 969-978Google Scholar) was found recently to be associated with this supramolecular edifice (23Graciet E. Lebreton S. Camadro J.M. Gontero B. Eur. J. Biochem. 2003; 270: 129-136Google Scholar). The two enzymes, PRK and GAPDH, may each be obtained in a free independent state. When they are not associated with each other they form dimers (PRK) or tetramers (GAPDH). CP12 is tightly associated with GAPDH (23Graciet E. Lebreton S. Camadro J.M. Gontero B. Eur. J. Biochem. 2003; 270: 129-136Google Scholar). The complex can be dissociated by harsh reduction and reversed by oxidizing conditions, because the oxidized partners can spontaneously reform a complexin vitro that is quite similar to the native state (10Avilan L. Gontero B. Lebreton S. Ricard J. Eur. J. Biochem. 1997; 246: 78-84Google Scholar). In only a few cases has it been possible to assemble particles from their separate parts in vitro that resemble the native complexes (24Reed L.J. Pettit F.H. Eley M.H. Hamilton L. Collins J.H. Oliver R.M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3068-3072Google Scholar, 25Fasshauer D. Bruns D. Shen B. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 4582-4590Google Scholar). The association of these two enzymes also gives rise to new regulatory properties. PRK and GAPDH within the complex are regulated by NADP(H) rather than by NAD(H), whereas the independent stable enzymes are not (26Graciet E. Lebreton S. Camadro J.M. Gontero B. J. Biol. Chem. 2002; 277: 12697-12702Google Scholar). Oxidized PRK may be active when associated with GAPDH or when dissociated from the complex upon dilution (14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar). We have also shown that the complex may exist under mild reducing conditions (12Lebreton S. Gontero B. J. Biol. Chem. 1999; 274: 20879-20884Google Scholar) even if it is dissociated by severe reducing conditions (DTT concentrations up to 20 mm). But it dissociates faster upon dilution, as reduction weakens the complex. Whereas plant enzymes are heterotetrameric (A2B2), algal GAPDHs are homotetrameric and made up of only A subunits (27Cerff R. Eur. J. Biochem. 1979; 94: 243-247Google Scholar, 28Qi J. Isupov M.N. Littlechild J.A. Anderson L.E. J. Biol. Chem. 2001; 276: 35247-35252Google Scholar). The B subunit has a C-terminal extension that contains two cysteine residues believed to be involved in the regulation of the enzyme (29Baalmann E. Scheibe R. Cerff R. Martin W. Plant Mol. Biol. 1996; 32: 505-513Google Scholar, 30Scheibe R. Baalmann E. Backhausen J.E. Rak C. Vetter S. Biochim. Biophys. Acta. 1996; 1296: 228-234Google Scholar, 31Sparla F. Pupillo P. Trost P. J. Biol. Chem. 2002; 277: 44946-44952Google Scholar). Nonetheless, studies on crude extracts of Chlamydomonas indicate that the algal enzyme that lacks these 28 amino acid residues can be activated reductively by light (32Li A.D. Stevens F.J. Huppe H.C. Kersanach R. Anderson L.E. Photosyn. Res. 1997; 51: 167-177Google Scholar). We have studied the regulation of this enzyme upon reduction or oxidation in its isolated state or within the PRK·GAPDH·CP12 complex to see if this reductive light activation is explained by the interaction of GAPDH with its protein partners. The same study was performed on the other partner, PRK. In previous studies (14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar) we have fully characterized the activity of isolated PRK and PRK in the complex. PRK in the so-called oxidized complex was active as mentioned above, but this result has been disputed (33Miziorko H.M. Adv. Enzymol. Relat. Areas Mol. Biol. 2000; 74: 95-127Google Scholar), as the cysteinyl sulfhydryls of PRK in the complex have never been directly tested. We have therefore used mass spectrometry coupled with protein chemistry to analyze the cysteinyl sulfhydryl contents of PRK, GAPDH, and CP12 in the complex, whatever its redox state. Finally, we have used a biosensor to study the interaction between the two enzymes depending on their redox states, as cellular redox signaling contributes to the control of the Benson-Calvin cycle and many other physiological processes (34Cooper C.E. Patel R.P. Brookes P.S. Darley-Usmar V.M. Trends Biochem. Sci. 2002; 27: 489-492Google Scholar). The qualitative and quantitative aspects of these interactions have been analyzed. DISCUSSIONMany enzymes belonging to the Benson-Calvin cycle are regulated by dark-light transitions via thioredoxins in vivo (38Balmer Y. Buchanan B.B. Trends Plant Sci. 2002; 7: 191-193Google Scholar) (or dithiothreitol in vitro). In particular, thioredoxin (39Geck M.K. Larimer F.W. Hartman F.C. J. Biol. Chem. 1996; 271: 24736-24740Google Scholar,40Geck M.K. Hartman F.C. J. Biol. Chem. 2000; 275: 18034-18039Google Scholar) reduces the disulfide between Cys55 and Cys16 of inactive oxidized spinach PRK (41Brandes H.K. Larimer F.W. Hartman F.C. J. Biol. Chem. 1996; 271: 3333-3335Google Scholar, 42Milanez S. Mural R.J. Hartman F.C. J. Biol. Chem. 1991; 266: 10694-10699Google Scholar).We have shown (14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar) that oxidized PRK from C. reinhardtii may have some activity when it is associated with GAPDH, contrary to common belief, but no direct evidence of the regulatory disulfide bridge between Cys16 and Cys55 present in oxidized PRK, was given. We have now used alkylation of the so-called oxidized complex to show that there is one disulfide bridge in the PRK monomer. The activity of PRK is greatly increased when it is reduced, indicating that the Cys16–Cys55 bond is the target.Another troubling aspect of the activity of oxidized PRK concerns the P-loop. This contains residues that interact with the ॆ- and γ-phosphoryls of Mg2+-ATP (43Runquist J.A. Rios S.E. Vinarov D.A. Miziorko H.M. Biochemistry. 2001; 40: 14530-14537Google Scholar). This loop should a priori be free to move for efficient catalysis. Some data for bacterial PRK (33Miziorko H.M. Adv. Enzymol. Relat. Areas Mol. Biol. 2000; 74: 95-127Google Scholar, 44Harrison D.H. Runquist J.A. Holub A. Miziorko H.M. Biochemistry. 1998; 37: 5074-5085Google Scholar) suggest that the Cys16–Cys55 disulfide bridge found only in the eukaryotic enzymes could immobilize the P-loop, thereby preventing catalytic turnover. However, it is worthwhile to pinpoint that there are insertions and deletions in the plant and algal enzymes, such as an insertion of 15 residues after the P-loop of the eukaryotic enzyme that may modify its mobility even in the presence of the Cys16–Cys55 disulfide bridge. These sulfhydryl groups are also not essential for catalysis, as a Cys16–Cys55 double mutant of spinach PRK still has some activity (42Milanez S. Mural R.J. Hartman F.C. J. Biol. Chem. 1991; 266: 10694-10699Google Scholar, 45Brandes H.K. Hartman F.C. Lu T.Y. Larimer F.W. J. Biol. Chem. 1996; 271: 6490-6496Google Scholar). The conformational constraint imposed by this bond is more likely to be responsible for the decreased PRK activity than the formation of the disulfide bridge itself (45Brandes H.K. Hartman F.C. Lu T.Y. Larimer F.W. J. Biol. Chem. 1996; 271: 6490-6496Google Scholar). Thus, although the bacterial PRK structure is a useful model, it may not be wise to extrapolate data obtained with it to eukaryotic PRKs, especially the eukaryotic enzyme in a multienzyme complex. Our results indicate that the conformational constraint near the P-loop could be attenuated when the PRK is within the complex, allowing oxidized PRK to be active. The interaction of PRK with GAPDH modifies the conformation of this enzyme (46Mouche F. Gontero B. Callebaut I. Mornon J.P. Boisset N. J. Biol. Chem. 2002; 277: 6743-6749Google Scholar) and hence its kinetic properties (12Lebreton S. Gontero B. J. Biol. Chem. 1999; 274: 20879-20884Google Scholar, 14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar).There is now considerable published evidence from NMR studies that supramolecular edifices (protein-protein or DNA-protein complexes) appear to be flexible (47Finerty Jr., P.J. Muhandiram R. Forman-Kay J.D. J. Mol. Biol. 2002; 322: 605-620Google Scholar, 48Yu L. Zhu C.X. Tse-Dinh Y.C. Fesik S.W. Biochemistry. 1996; 35: 9661-9666Google Scholar). The entropic cost of the decrease in conformational freedom must be offset, to some extent, by preserving the flexibility of other regions. These reports support the assertion made above by the authors, but only structural data will clarify this point. Our direct evaluation of the redox status of the regulatory cysteine residues now clearly shows that oxidized PRK may be catalytically active. This activity is, however, lower than the activity of the released metastable form, as the PRK-GAPDH association hampers the overall flexibility of these enzymes. The released PRK with decreased conformational constraint around the P-loop, because of a memory effect, and with greater overall flexibility is thus a better catalyst than the enzyme in the complex.Whereas each PRK monomer in the oxidized complex has one disulfide bridge, all the cysteinyl sulfhydryl groups of GAPDH are free and CP12 has two disulfide bridges. We found five cysteinyl sulfhydryl groups per PRK subunit by alkylation after reduction of the oxidized complex. The GAPDH content of thiol groups obviously does not change, but two or four thiol groups become titrated per CP12 monomer. Reduction of the PRK disulfide bridge is followed by an increase in enzyme activity, as mentioned above.Chlamydomonas GAPDH is an A4 homotetramer; the regulatory cysteine residues are thus absent (28Qi J. Isupov M.N. Littlechild J.A. Anderson L.E. J. Biol. Chem. 2001; 276: 35247-35252Google Scholar, 31Sparla F. Pupillo P. Trost P. J. Biol. Chem. 2002; 277: 44946-44952Google Scholar). Nonetheless, incubation of a crude extract with DTT increased its enzyme activity 3-fold (32Li A.D. Stevens F.J. Huppe H.C. Kersanach R. Anderson L.E. Photosyn. Res. 1997; 51: 167-177Google Scholar). Somewhat surprisingly, we saw the same increase in NADPH-dependent activity for GAPDH when it was part of the complex, but not for the isolated enzyme. We therefore proposed (49Heineke D. Burgi R. Heike G. Hoferichter P. Peter U. Flügge U.I. Heldt H.W. Plant Physiol. 1991; 95: 1131-1137Google Scholar) that this modulation had a physiological role, as the NADPH concentrations are increased in the light. Our present results also show that reducing the complex leads to a decrease in the use of NADH.As titration of the thiol groups in GAPDH in the complex reveals 4 SH groups whatever the redox state of the complex, the modulation of GAPDH activity is not linked to disulfide reduction. Our present finding also indicates that these effects are linked to heterologous interactions within the complex as there is no longer an effect once these interactions are weakened or broken. This supports the idea mentioned above that new properties may emerge as a consequence of conformation change within multienzyme complexes. It also shows that the regulation of an enzyme like PRK may modulate the regulation of another (GAPDH) via a 舠domino-like舡 effect.We have tested the effect of oxidizing agents on PRK and GAPDH activities, as oxidized thioredoxin causes the oxidation of the light-reduced enzymes. Treatment with oxidants caused the activity of reduced PRK isolated or released from the complex (e.g.using cystine) to decrease. This effect was reversed by reduction, as expected. The situation is quite different for GAPDH, as the loss of activity by the isolated enzyme that follows incubation with oxidized glutathione is very likely because of a modification of the active site cysteine (Cys156), as it is well documented for other GAPDHs (50Harris J.I. Waters M. Boyer P.D. The Enzymes. 13. Academic Press, New York1976: 1-49Google Scholar). This is also supported by protection experiments using BPGA and by the fact that oxidized thioredoxins have no effect on GAPDH activity. The inactivation is not reversed by a reducing agent. It has been suggested that the oxidation of glycolytic enzymes may introduce an element of strain resulting from the formation of a disulfide bond, which then causes an irreversible conformational change, perhaps with displacement of an essential residue from the active site. GAPDH is not significantly inactivated by oxidized glutathione even after dissociation of the complex, like PRK. This may be because the cysteinyl sulfhydryl in this molecule is less exposed than is that in the isolated GAPDH. We checked this by measuring the enzyme kinetics of the isolated GAPDH and of the released form from the complex. The differences observed with the pseudo-affinity constants for BPGA clearly support the assertion made above. Thus, GAPDH retains the conformation it had within the complex (imprinting) even after the physical separation of PRK and GAPDH, so that the sulhydryl group of the Cys156 is poorly accessible to oxidized glutathione. This corroborates the imprinting effect previously reported (12Lebreton S. Gontero B. J. Biol. Chem. 1999; 274: 20879-20884Google Scholar, 13Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 250: 286-295Google Scholar,26Graciet E. Lebreton S. Camadro J.M. Gontero B. J. Biol. Chem. 2002; 277: 12697-12702Google Scholar).Finally, the complex we have purified is a suitable model with which to study protein-protein interactions, but most of the information on it stems from in vitro reconstitution experiments. This report, for the first time, describes the use of Biosensor technology to determine the dynamics of these interactions between PRK and the GAPDH·CP12 subcomplex under different redox states. The dissociation constants between the PRK and GAPDH·CP12 subcomplex are rather low (nm range), with the lowest value (14 ± 1.6 nm) being obtained with oxidized PRK. The probable conformational change caused by the chemical modification of GAPDH by oxidation has no influence on the binding of GAPDH to PRK andvice versa. The SPR results indicate that these enzymes (PRK and GAPDH/CP12) may reconstitute the complex under reducing conditions with a low dissociation constant (62 ± 10 nm) even if complex formation is sensitive to the redox state of PRK. Our results also suggest that this complex may form at pH 7, under oxidizing conditions (dark conditions) or at pH 8, under mild reducing conditions (light conditions).To conclude, thiol/disulfide exchange influences the state of activation of chloroplast PRK, in its isolated form and when it is part of a complex. On the other hand, the modulation of PRK activity influences the state of activation of GAPDH via protein-protein interactions, at least in the alga C. reinhardtii. A new mode of light regulation could emerge that involves a 舠domino effect舡 and this could well apply to enzymes that are not direct targets of light. Many enzymes belonging to the Benson-Calvin cycle are regulated by dark-light transitions via thioredoxins in vivo (38Balmer Y. Buchanan B.B. Trends Plant Sci. 2002; 7: 191-193Google Scholar) (or dithiothreitol in vitro). In particular, thioredoxin (39Geck M.K. Larimer F.W. Hartman F.C. J. Biol. Chem. 1996; 271: 24736-24740Google Scholar,40Geck M.K. Hartman F.C. J. Biol. Chem. 2000; 275: 18034-18039Google Scholar) reduces the disulfide between Cys55 and Cys16 of inactive oxidized spinach PRK (41Brandes H.K. Larimer F.W. Hartman F.C. J. Biol. Chem. 1996; 271: 3333-3335Google Scholar, 42Milanez S. Mural R.J. Hartman F.C. J. Biol. Chem. 1991; 266: 10694-10699Google Scholar). We have shown (14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar) that oxidized PRK from C. reinhardtii may have some activity when it is associated with GAPDH, contrary to common belief, but no direct evidence of the regulatory disulfide bridge between Cys16 and Cys55 present in oxidized PRK, was given. We have now used alkylation of the so-called oxidized complex to show that there is one disulfide bridge in the PRK monomer. The activity of PRK is greatly increased when it is reduced, indicating that the Cys16–Cys55 bond is the target. Another troubling aspect of the activity of oxidized PRK concerns the P-loop. This contains residues that interact with the ॆ- and γ-phosphoryls of Mg2+-ATP (43Runquist J.A. Rios S.E. Vinarov D.A. Miziorko H.M. Biochemistry. 2001; 40: 14530-14537Google Scholar). This loop should a priori be free to move for efficient catalysis. Some data for bacterial PRK (33Miziorko H.M. Adv. Enzymol. Relat. Areas Mol. Biol. 2000; 74: 95-127Google Scholar, 44Harrison D.H. Runquist J.A. Holub A. Miziorko H.M. Biochemistry. 1998; 37: 5074-5085Google Scholar) suggest that the Cys16–Cys55 disulfide bridge found only in the eukaryotic enzymes could immobilize the P-loop, thereby preventing catalytic turnover. However, it is worthwhile to pinpoint that there are insertions and deletions in the plant and algal enzymes, such as an insertion of 15 residues after the P-loop of the eukaryotic enzyme that may modify its mobility even in the presence of the Cys16–Cys55 disulfide bridge. These sulfhydryl groups are also not essential for catalysis, as a Cys16–Cys55 double mutant of spinach PRK still has some activity (42Milanez S. Mural R.J. Hartman F.C. J. Biol. Chem. 1991; 266: 10694-10699Google Scholar, 45Brandes H.K. Hartman F.C. Lu T.Y. Larimer F.W. J. Biol. Chem. 1996; 271: 6490-6496Google Scholar). The conformational constraint imposed by this bond is more likely to be responsible for the decreased PRK activity than the formation of the disulfide bridge itself (45Brandes H.K. Hartman F.C. Lu T.Y. Larimer F.W. J. Biol. Chem. 1996; 271: 6490-6496Google Scholar). Thus, although the bacterial PRK structure is a useful model, it may not be wise to extrapolate data obtained with it to eukaryotic PRKs, especially the eukaryotic enzyme in a multienzyme complex. Our results indicate that the conformational constraint near the P-loop could be attenuated when the PRK is within the complex, allowing oxidized PRK to be active. The interaction of PRK with GAPDH modifies the conformation of this enzyme (46Mouche F. Gontero B. Callebaut I. Mornon J.P. Boisset N. J. Biol. Chem. 2002; 277: 6743-6749Google Scholar) and hence its kinetic properties (12Lebreton S. Gontero B. J. Biol. Chem. 1999; 274: 20879-20884Google Scholar, 14Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 246: 85-91Google Scholar). There is now considerable published evidence from NMR studies that supramolecular edifices (protein-protein or DNA-protein complexes) appear to be flexible (47Finerty Jr., P.J. Muhandiram R. Forman-Kay J.D. J. Mol. Biol. 2002; 322: 605-620Google Scholar, 48Yu L. Zhu C.X. Tse-Dinh Y.C. Fesik S.W. Biochemistry. 1996; 35: 9661-9666Google Scholar). The entropic cost of the decrease in conformational freedom must be offset, to some extent, by preserving the flexibility of other regions. These reports support the assertion made above by the authors, but only structural data will clarify this point. Our direct evaluation of the redox status of the regulatory cysteine residues now clearly shows that oxidized PRK may be catalytically active. This activity is, however, lower than the activity of the released metastable form, as the PRK-GAPDH association hampers the overall flexibility of these enzymes. The released PRK with decreased conformational constraint around the P-loop, because of a memory effect, and with greater overall flexibility is thus a better catalyst than the enzyme in the complex. Whereas each PRK monomer in the oxidized complex has one disulfide bridge, all the cysteinyl sulfhydryl groups of GAPDH are free and CP12 has two disulfide bridges. We found five cysteinyl sulfhydryl groups per PRK subunit by alkylation after reduction of the oxidized complex. The GAPDH content of thiol groups obviously does not change, but two or four thiol groups become titrated per CP12 monomer. Reduction of the PRK disulfide bridge is followed by an increase in enzyme activity, as mentioned above. Chlamydomonas GAPDH is an A4 homotetramer; the regulatory cysteine residues are thus absent (28Qi J. Isupov M.N. Littlechild J.A. Anderson L.E. J. Biol. Chem. 2001; 276: 35247-35252Google Scholar, 31Sparla F. Pupillo P. Trost P. J. Biol. Chem. 2002; 277: 44946-44952Google Scholar). Nonetheless, incubation of a crude extract with DTT increased its enzyme activity 3-fold (32Li A.D. Stevens F.J. Huppe H.C. Kersanach R. Anderson L.E. Photosyn. Res. 1997; 51: 167-177Google Scholar). Somewhat surprisingly, we saw the same increase in NADPH-dependent activity for GAPDH when it was part of the complex, but not for the isolated enzyme. We therefore proposed (49Heineke D. Burgi R. Heike G. Hoferichter P. Peter U. Flügge U.I. Heldt H.W. Plant Physiol. 1991; 95: 1131-1137Google Scholar) that this modulation had a physiological role, as the NADPH concentrations are increased in the light. Our present results also show that reducing the complex leads to a decrease in the use of NADH. As titration of the thiol groups in GAPDH in the complex reveals 4 SH groups whatever the redox state of the complex, the modulation of GAPDH activity is not linked to disulfide reduction. Our present finding also indicates that these effects are linked to heterologous interactions within the complex as there is no longer an effect once these interactions are weakened or broken. This supports the idea mentioned above that new properties may emerge as a consequence of conformation change within multienzyme complexes. It also shows that the regulation of an enzyme like PRK may modulate the regulation of another (GAPDH) via a 舠domino-like舡 effect. We have tested the effect of oxidizing agents on PRK and GAPDH activities, as oxidized thioredoxin causes the oxidation of the light-reduced enzymes. Treatment with oxidants caused the activity of reduced PRK isolated or released from the complex (e.g.using cystine) to decrease. This effect was reversed by reduction, as expected. The situation is quite different for GAPDH, as the loss of activity by the isolated enzyme that follows incubation with oxidized glutathione is very likely because of a modification of the active site cysteine (Cys156), as it is well documented for other GAPDHs (50Harris J.I. Waters M. Boyer P.D. The Enzymes. 13. Academic Press, New York1976: 1-49Google Scholar). This is also supported by protection experiments using BPGA and by the fact that oxidized thioredoxins have no effect on GAPDH activity. The inactivation is not reversed by a reducing agent. It has been suggested that the oxidation of glycolytic enzymes may introduce an element of strain resulting from the formation of a disulfide bond, which then causes an irreversible conformational change, perhaps with displacement of an essential residue from the active site. GAPDH is not significantly inactivated by oxidized glutathione even after dissociation of the complex, like PRK. This may be because the cysteinyl sulfhydryl in this molecule is less exposed than is that in the isolated GAPDH. We checked this by measuring the enzyme kinetics of the isolated GAPDH and of the released form from the complex. The differences observed with the pseudo-affinity constants for BPGA clearly support the assertion made above. Thus, GAPDH retains the conformation it had within the complex (imprinting) even after the physical separation of PRK and GAPDH, so that the sulhydryl group of the Cys156 is poorly accessible to oxidized glutathione. This corroborates the imprinting effect previously reported (12Lebreton S. Gontero B. J. Biol. Chem. 1999; 274: 20879-20884Google Scholar, 13Lebreton S. Gontero B. Avilan L. Ricard J. Eur. J. Biochem. 1997; 250: 286-295Google Scholar,26Graciet E. Lebreton S. Camadro J.M. Gontero B. J. Biol. Chem. 2002; 277: 12697-12702Google Scholar). Finally, the complex we have purified is a suitable model with which to study protein-protein interactions, but most of the information on it stems from in vitro reconstitution experiments. This report, for the first time, describes the use of Biosensor technology to determine the dynamics of these interactions between PRK and the GAPDH·CP12 subcomplex under different redox states. The dissociation constants between the PRK and GAPDH·CP12 subcomplex are rather low (nm range), with the lowest value (14 ± 1.6 nm) being obtained with oxidized PRK. The probable conformational change caused by the chemical modification of GAPDH by oxidation has no influence on the binding of GAPDH to PRK andvice versa. The SPR results indicate that these enzymes (PRK and GAPDH/CP12) may reconstitute the complex under reducing conditions with a low dissociation constant (62 ± 10 nm) even if complex formation is sensitive to the redox state of PRK. Our results also suggest that this complex may form at pH 7, under oxidizing conditions (dark conditions) or at pH 8, under mild reducing conditions (light conditions). To conclude, thiol/disulfide exchange influences the state of activation of chloroplast PRK, in its isolated form and when it is part of a complex. On the other hand, the modulation of PRK activity influences the state of activation of GAPDH via protein-protein interactions, at least in the alga C. reinhardtii. A new mode of light regulation could emerge that involves a 舠domino effect舡 and this could well apply to enzymes that are not direct targets of light. We are grateful to Monique Haquet for technical assistance in preparing enzymes and media, Jean-Jacques Montagne for mass spectrometry advice, and Owen Parkes for editing the manuscript." @default.
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W2068774323 title "Modulation, via Protein-Protein Interactions, of Glyceraldehyde-3-phosphate Dehydrogenase Activity through Redox Phosphoribulokinase Regulation" @default.
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