A functional five‐enzyme complex of chloroplasts involved in the Calvin cycle

Abstract

A complex of five different enzymes: ribose-phosphate isomerase, phosphoribulokinase, ribulose-bisphosphate carboxylase/oxygenase, phosphoglycerate kinase and glyceraldehyde-phosphate dehydrogenase, has been purified from spinach chloroplasts. These enzymes catalyse five consecutive reactions of the Calvin cycle, of which the two reactions catalysed by phosphoribulokinase and ribulose-bisphosphate carboxylase/oxygenase are unique to this cycle. The five-enzyme complex has been purified by successive chromatographies on DEAE-Trisacryl, Sephadex G-200 and hydroxyapatite. The homogeneity of the complex has been tested by analytical centrifugation and electrophoresis. Depending on the technique used to estimate its molecular mass, the value obtained varies between 520 kDa and 536 kDa. In addition to the five enzymes mentioned above, the complex contains a 65-kDa polypeptide. The quaternary structure of these enzymes in the complex appears to be different from what has been described for the individual enzymes in the 'noncomplexed state'.The five-enzyme complex is functional as glyceraldehyde 3-phosphate is formed from ribose 5-phosphate. Preliminary experiments suggest that channelling of reaction intermediates occurs within the complex.

Proteomic Characterization of A Triton-Insoluble Fraction from Chloroplasts Defines A Novel Group of Proteins Associated with Macromolecular Structures

“…[59][60][61] Furthermore, RuBisCO was previously observed to form a complex with four other enzymes of the Calvin cycle. 62,63 Interestingly, two of those enzymes were also observed in this investigation, phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GPDH). Surprisingly, both forms of GPDH were observed and were the major proteins in bands 24 and 26, representing approximately 4% of the total protein.…”

“…Indeed, these data support the previous proposal of a large, multi-enzyme assembly to facilitate metabolite channeling of reductive pentose phosphate pathway intermediates. 63 Despite rigorous de-lipidation with Triton X-100 detergent, sixteen envelope membrane proteins were also identified suggesting domains of this membrane system are recalcitrant to Triton extraction. Surprisingly, 32 proteins were classified as unknown or hypothetical proteins, most matching to Arabidopsis ORFs.…”

Properties to two high‐molecular‐mass forms of glyceraldehyde‐3‐phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity

“…Co-purification of PRK with other enzymes of the reductive pentose phosphate cycle has been suggested to be due to the formation of multienzyme complexes [22,23]. Of these only that of Gontero et al [23] contains both G3PDH and PRK.…”

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants

“…Complete dissociation of the complex was also achieved by incubation with 50 mM DTT for 1 h at 30°C (conditions as in ref. 10), most probably due to destruction of the peptide loops of CP12 by reduction of its disulfide bonds (data not shown). Incubation with 2.5 mM ATP or ADP resulted in the same pattern, as in the water control and therefore they appear to be not the effectors for complex dissociation (data not shown).…”

“…Further data suggest that GAPDH and PRK could be associated in a complex together with Rubisco (9). These three enzymes together were then proposed to be members of a 600-kDa multienzyme complex, in addition of ribose-5-P isomerase, and 3-phosphoglycerate kinase (PGK) (10,11). Another multienzyme complex thought to be associated with thylakoids, was proposed to contain SBPase instead of PGK and therefore organized in a stable 900-kDa protein complex, together with ferredoxin-NADP ϩ reductase (12).…”