Ribulose bisphosphate-induced, slow conformational changes of spinach ribulose bisphosphate carboxylase cause the two types of inflections in the course of its carboxylase reaction.
The cause of the inflection in the course of the carboxylase reaction and the changes in the functioning form of spinach ribulose bisphosphate carboxylase (RuBisCO) during the reaction were elucidated by relating the activity to the protein conformation of RuBisCO using a fluorescence probe, 2-p-toluidinylnaphthalene sulfonate. The activity of RuBisCO in the linear phase was 50 to 60% of that in the initial burst at 0.5 to 1.0 mM ribulose bisphosphate (RuBP) and 65 to 80% at 2 to 5 mM RuBP. The amount and the progress of the decrease in the activity during the reaction had a close relationship to a change in the protein conformation of RuBisCO. The enzyme, the substrate binding sites of which were masked beforehand with carboxyarabinitol bisphosphate, still showed a change of its protein conformation upon addition of RuBP, suggesting that RuBisCO has two (substrate and regulatory) RuBP-binding sites per RuBisCO promoter. RuBisCO required over 2 mM RuBP for binding on the regulatory sites. Both sites also bound 6-phosphogluconate. When both sites were masked with 6-phosphogluconate beforehand, the course of the subsequent carboxylase reaction was linear with time. From these results, I propose that the inflection in the course of the reaction of spinach RuBisCO is a hysteretic response of the enzyme to RuBP bound to both substrate and regulatory sites.
Yokota
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A
Department of Agricultural Chemistry, University of Osaka Prefecture.
D000818:Animals; D005055:Euglena / Q000201:enzymology; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates / Q000737:chemistry; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D012274:Ribulosephosphates / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D012274:Ribulosephosphates; C001933:ribulose-1,5 diphosphate; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D012273:Ribulose-Bisphosphate Carboxylase