Fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species occurs near Gly-329.
The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in the illuminated lysates of wheat (Triticum aestivum L.) chloroplasts is broken down by reactive oxygen radicals into 37- and 16-kDa polypeptides. Analysis of the terminal amino acid residues of both fragments revealed that the C terminus of the 37-kDa fragment was Ser-328 and the N terminus of the 16-kDa fragment was Thr-330. Gly-329, which links the two fragments, was missing, suggesting that the fragmentation of the LSU in the lysates driven by oxygen-free radicals occurs at Gly-329. Purified rubisco, exposed to a hydroxyl radical-generating system, was also cleaved at the same site of the LSU. The cleavage site was positioned at the N-terminal end of the flexible loop (loop 6) within the beta/alpha-barrel domain, constituting the catalytic site of rubisco. The binding of a reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate, to the active form of rubisco completely protected the enzyme from the fragmentation. The fragmentation was differentially affected by CO2, Mg2+, ribulose 1, 5-bisphosphate, or 2-carboxyarabinitol 1,5-bisphosphate. All these results indicate that the conformation of the catalytic site of the enzyme is involved as an important factor determining the breakdown of rubisco by reactive oxygen species. Reactive oxygen species generated at its catalytic site by a Fenton-type reaction may trigger the site-specific degradation of the LSU in the lysates of chloroplasts in the light.
Ishida
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H
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai 981-8555, Japan.
Makino
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A
Mae
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T
D000595:Amino Acid Sequence; D002245:Carbon Dioxide / Q000737:chemistry; D020134:Catalytic Domain; D005998:Glycine / Q000737:chemistry*; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D010428:Pentosephosphates / Q000737:chemistry; D017382:Reactive Oxygen Species; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry*; D012274:Ribulosephosphates / Q000737:chemistry; D013402:Sugar Alcohols / Q000737:chemistry; D014908:Triticum / Q000201:enzymology