Cloning and sequencing of cDNA encoding the mature form of phosphoribulokinase from spinach

Milanez, Sylvia; Mural, Richard J.

doi:10.1016/0378-1119(88)90224-7

Cited by 47 publications

(30 citation statements)

References 35 publications

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“…PRK, a homodimer with a subunit molecular weight of 39,232 (16,17), is representative of plant enzymes that are regulated during light-dark transitions (18). Photon flux drives the reduction of thioredoxin (as mediated by ferredoxin-thioredoxin reductase), which in turn reduces the regulatory disulfide of target proteins (18 -20).…”

Section: Resultsmentioning

confidence: 99%

The Molecular Pathway for the Regulation of Phosphoribulokinase by Thioredoxin f

Brandes

Larimer

Hartman

1996

Journal of Biological Chemistry

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Thioredoxins are small (12-14 kDa) ubiquitous proteins that control the redox state of diverse target proteins by the mediation of thiol-disulfide exchanges (1, 2). The redox-active sulfhydryls of thioredoxin are located in the highly conserved active-site sequence -Trp-Cys-Gly-Pro-Cys-. The pathway for the reduction of a protein disulfide by thioredoxin entails nucleophilic attack by one of the active-site sulfhydryls to form a protein-protein mixed disulfide followed by intramolecular displacement of the reduced target protein with concomitant formation of oxidized thioredoxin (Fig. 1). As the mixed disulfide is disfavored thermodynamically, its direct characterization has not been possible. However, the active-site cysteinyl residue of thioredoxin that is nearest the N terminus (Cys-32 of the Escherichia coli protein) is well established as the primary nucleophile. Originally, this assignment was deduced from the high chemical reactivity and the low pK a (6.7) of Cys-32 (3-6) and was subsequently supported by the three-dimensional structure of E. coli thioredoxin, in which Cys-32 is solventaccessible and Cys-35 is inaccessible (7). More recently, Cys-32 of E. coli thioredoxin, the corresponding residue of human thioredoxin, and the corresponding residue of the closely related protein glutaredoxin were shown to engage in mixed disulfide bond formation with low molecular weight thiols or peptides (8 -11). Finally, the C49S mutant of chloroplastic thioredoxin f (Trx) 1 retains the capacity to activate chloroplastic fructose-1,6-bisphosphatase, whereas the C46S mutant is totally inactive in this regard. Thus, Cys-46 of Trx (analogous to Cys-32 of E. coli thioredoxin) is verified as the primary nucleophile in the reduction of in vivo target proteins, thereby minimizing the possibility that protein-protein interactions might alter the relative accessibility and reactivity of the active-site sulfhydryls of thioredoxin.In contrast to the rigorous proof that Cys-46 of Trx participates in intermolecular disulfide bond formation, the identity of the pairing residue in any target protein has not been established heretofore. We have addressed this issue with PRK by examining the potential of site-directed mutants, which lack either one of the two redox-active sulfhydryls (Cys-16 or Cys-55), to form stable mixed disulfides with the C49S mutant of Trx. This approach, which should be applicable to other target proteins, clearly identifies Cys-55 as the participant in the intermolecular mixed disulfide. EXPERIMENTAL PROCEDURESMaterials-The following chemicals and biologicals were procured from the indicated vendors: Bicine and DTT, Research Organics, Inc.; components for kinase assays, Sigma; cupric sulfate, Fisher Scientific; DTNB, Pierce. D-Ribulose 5-phosphate was prepared from D-ribose 5-phosphate by the action of phosphoribose isomerase, and its concen-

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Section: Resultsmentioning

confidence: 99%

The Molecular Pathway for the Regulation of Phosphoribulokinase by Thioredoxin f

Brandes

Larimer

Hartman

1996

Journal of Biological Chemistry

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“…Our results lead to the conclusion that both forms of the kinase (isolated or integrated in the complex) are activated by disulfide reagents, such as dithiothreitol or thioredoxin, although the enzyme inserted in the complex exhibited a much greater sensitivity towards thioredoxin f, as compared to that of the isolated kinase. Thus, the interaction of the multi-Moreover, Milanez et al [20] have recently reported that there is only one gene coding for phosphoribulokinase in spinach. We conclude, therefore, that these two enzyme forms cannot correspond to different isoforms of phosphoribulokinase.…”

Section: Discussionmentioning

confidence: 99%

Thioredoxin activation of phosphoribulokinase in a chloroplast multi‐enzyme complex

Rault¹,

Gontero²,

Ricard³

1991

European Journal of Biochemistry

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The activation process of spinach phosphoribulokinase by thioredoxin f has been studied with the enzyme in a free, isolated state, or integrated in a multi-enzyme complex. The time periods required for enzyme activation are always smaller and the maximal enzyme velocities are always greater when chloroplast phosphoribulokinase is included in the multi-enzyme complex than when it is in the isolated state. Comparative kinetic studies show that phosphoribulokinase extracted from the complex behaves exactly as in the isolated state. The reduced form of the kinase, whatever it has been included in the complex or isolated from the chloroplasts, are deactivated by oxidized thioredoxins. In the absence of thioredoxin f however, the reduced form of the isolated enzyme undergoes spontaneous oxidation whereas the reduced kinase included in the multi-enzyme complex is stable. 'Unspecific' proteins such as bovine serum albumin do not provide any protection of the kinase against autooxidation, whereas 'homologous' specific proteins such as ribulose-1 ,j-bisphosphate carboxylase/oxygenase dramatically decrease the rate of this autooxidation process. These results therefore support the view that interactions between phosphoribulokinase and the other components of the multi-enzyme complex play an important role in the modulation of the activity of this enzyme. The possible part of these interactions in the control of the Calvin cycle is discussed.It is now well established that in the chloroplast of plant cells the activities of several enzymes of the Calvin cycle (i.e. the reductive pentose phosphate pathway) are regulated by light [l -31. This control seems to be effected through lightinduced modifications of the levels of substrates and cofactors, and/or of the local pH [4, 51. Besides this type of control, several studies have suggested that activation of chloroplast enzymes by light also involves the reduction of enzyme disulfide bridges, in a process that is thought to be mediated by small protein factors, the chloroplast thioredoxins [6 -81. This overall process includes at least the following main reactions : (a) generation of reduced thioredoxin from the corresponding oxidized molecule and photochemically reduced ferredoxin, as catalyzed by ferredoxin -thioredoxin reductase [9], and (b) reduction, mediated by reduced thioredoxin, of disulfide bonds within the light-activated enzymes, resulting in promotion of their full enzyme activity [lo]. Among the enzymes constituting the Calvin cycle, phosphoribulokinase (an enzyme specific to this pathway) appears to be regulated by the variation of stromal metabolites [ll, 121, as well as through the thioredoxin-modulated reduction of a disulfide bond of the enzyme [13, 141. The binding site for thioredoxin on phosCorrespondence to J.

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“…6). The presence of this residue in bacterial phosphoribulokinase is especially noteworthy, because the bacterial enzyme possesses <15% sequence identity (10) in comparison with the Chlamydomonas and higher plant enzymes (14,23,25,26). The conserved arginine is located in one of five sequence motifs present in phosphoribulokinase of both prokaryotes and eukaryotes (10).…”

Section: Discussionmentioning

confidence: 99%

Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase

Roesler¹,

Marcotte²,

Ogren³

1992

Plant Physiol.

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Cloning and sequencing of cDNA encoding the mature form of phosphoribulokinase from spinach

Cited by 47 publications

References 35 publications

The Molecular Pathway for the Regulation of Phosphoribulokinase by Thioredoxin f

The Molecular Pathway for the Regulation of Phosphoribulokinase by Thioredoxin f

Thioredoxin activation of phosphoribulokinase in a chloroplast multi‐enzyme complex

Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase

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