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Articles

Regulation of Ribulose 1,5-Diphosphate Carboxylase by Substrates and Other Metabolites

Further Evidence for Several Types of Binding Sites ¹

^a Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720

Ribulose 1,5-diphosphate carboxylase (RuDPCase, EC 4.1.1.39) isolated from spinach leaves is metabolically regulated at 10 mM Mg²⁺ and low CO₂ concentrations by its substrates (RuDP and CO₂) and by effectors which include 6-phosphogluconate (6-PGluA), NADPH, and fructose 1,6-diphosphate (FDP), but not fructose 6-phosphate. Physiological concentrations of RuDP severely inhibit the enzyme activity when the enzyme has not been preincubated with HCO₃^– and Mg^2–, and this inactivity persists for 20 minutes or longer after 1 mM HCO₃^– and 10 mM Mg²⁺ are added. Maximum activity requires that the preincubation mixture also include either 0.01 mM 6-PGluA or 0.5 mM NADPH.

When the enzyme, following preincubation with HCO₃^– and Mg²⁺, is presented with RuDP plus either 6-PGluA or FDP, competitive inhibition is observed with respect to RuDP. The Ki value for 6-PGluA is 0.02 mM and the Ki value for FDP is 190 µM. NADPH or 3-phosphoglycerate (PGA) at physiological concentrations does not have any effect when presented simultaneously with RuDP. Other studies on the order of addition of substrates and effectors, concentration effects, and kinetics provide additional information that serves as a basis for a proposed model of allosteric regulation combined with competitive inhibition.

In this model, there are catalytic sites at which the substrates and 6-PGluA and FDP can bind, and at least four allosteric regulatory sites, which we designate I, A₁, A₂, and A₃. RuDP binds very tightly to site I (in the absence of Mg²⁺ or HCO₃^–), causing a conformational change in the protein to an inactive form which persists for as long as 20 minutes in the subsequent presence of Mg²⁺ and 1 mM HCO₃^–. Mg²⁺ and HCO₃^– (or CO₂) bind to site A₃ (in the absence of RuDP), holding the enzyme in an active form which has a much lower affinity for RuDP at site I, so that when physiological levels of RuDP are then added, only part of the enzyme activity is lost. This active form of the enzyme can bind 6-PGluA or FDP at site A₁ and NADPH at site A₂ during preincubation with Mg²⁺ and HCO₃^–. With optimal levels of bound effectors, 6-PGluA or NADPH, enzyme activity is fully maintained, even when RuDP is subsequently added. Without one of these effectors present, addition of RuDP following preincubation reduces enzyme activity to about 40% at the levels of substrates and effectors studied. FDP is a much poorer effector, and this is ascribed to a possible binding of FDP at site I, as well as at site A₁.

The physiological role of this regulation is discussed, particularly with respect to protection of "C-3" plants against oxidation of RuDP to phosphoglycolate.

¹ This research was supported by the United States Atomic Energy Commission.

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