Activation of Ribulose 1,5-Diphosphate Carboxylase by Nicotinamide Adenine Dinucleotide Phosphate and Other Chloroplast Metabolites

Abstract

Ribulose 1, 5-diphosphate carboxylase, when activated by preincubation with 10 mM MgCl2 and 1 mM bicarbonate in the absence of ribulose 1,5-diphosphate, can be further activated about 170% with 0.5 mM NADPH present in the preincubation mixture. NADP+, NADH, and NAD' are ineffective. The activation by NADPH is comparable to that previously seen with 0.05 to 0.10 mM 6-phosphogluconate in that these specific preincubation conditions are required, but the effects of NADPH and 6-phosphogluconate are not additive. Moreover, where higher concentrations of 6-phosphogluconate inhibited the enzyme, higher concentrations of NADPH give a greater activation, saturating at about 1 mM and 200%. Under the specified conditions of preincubation, fructose 1, 6-diphosphate has an activation curve similar to that of 6-phosphogluconate, peaking at 0.1 mM and 70 %. Above this level, activation decreases, and inhibition is seen at still higher concentrations. Other metabolites tested produced smaller or no effects on the enzyme activity assayed under these conditions. When either reduced NADP or 6-phosphogluconate are present in the preincubation mixture, it becomes possible to determine the Km for bicarbonate using a Lineweaver-Burk plot, and the Km for bicarbonate under these conditions is 2.8 mM, corresponding to 0.3 % C02 at pH 7.8 and 25 C.

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

“…Although RuDPCase has been known for 20 years and has been extensively studied and its properties reviewed (12,15,24), the specific type of allosteric behavior described in this and our two previous reports (10,11) has largely escaped notice. Probably this is due to the fact that, although Pon et al.…”

“…Smaller activations (about 50%) are seen if 0.1 mm FDP or 0.5 mm PGA are included in the preincubation and assay mixtures (11). None of these activations is additive when the concentrations giving the maximum effects are present.…”

Effects of illumination of etiolated leaves on the redox state of NADP in the plastids

“…Thus, it is becoming increasingly apparent that photosynthetically produced reductants, e.g., reduced dithiols, ferredoxin or NADPH can activate chloroplast enzymes associated with anabolic activities, e.g., carboxydismutase [9] , NADPH-linked The methods employed were as in table 2 except that only a brief 2 min illumination was given to the plants glyceraldehyde-3-phosphate dehydrogenase [lo] and sedoheptulose [ 1 l] and fructose diphosphate phosphatases [ 121 while serving to depress the activities of catabolic, oxidative enzymes, e.g., glucose 6-phosphate dehydrogenase [ 13,141. In darkness, due to the cessation of photosynthesis the reverse situation exists with conditions now favouring the oxidative enzymes and enzymes of the reductive pathway being largely inhibited. Illumination of dark grown plants activates various metabolic processes which co-operatively results in chloroplast development.…”

Comments on the structure and function of the large subunit of the enzyme ribulose bisphosphate carboxylase-oxygenase

“…Certain carbohydrate metabolites and cofactors of carbohydrate-metabolism interact with the activities of the enzyme. The most important of these are 6-phosphogluconate (6PGIuA), fructose-1,5-bisphosphate (FBP) and NADPH (3,7,8,27,28,49). A reaction scheme for the enzyme employing the formulas of the involved molecules is shown in Figure 3, which also contains the formulas of relevant inhibitors.…”

“…A reaction scheme for the enzyme employing the formulas of the involved molecules is shown in Figure 3, which also contains the formulas of relevant inhibitors. Whereas NADPH, 6PGluA and FBP earlier were considered also to bind to atlosteric sites (7,8) of the carboxylase, recent enzyme kinetic studies (3) indicate that these effectors exhibit their function at the catalytic site only. Stimulation or inhibition by these effectors is secondary to activation by CO2 and Mg 2+ .…”