Ribulose 1, 5-diphosphate carboxylase (RuDPCase, EC 4.1.1.39) isolated from spinach leaves is metabolically regulated at 10 mM Mg2+ and low C02 concentrations by its substrates (RuDP and C02) 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 Mg2-, and this inactivity persists for 20 minutes or longer after 1 mM HCO,-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 HC03-and Mg2+, is presented with RuDP plus either 6-PGluA or FDP, competitive inhibition is observed with respect to RuDP.
ABSTRACT6-PGluA: 6-phosphogluconate; GlcuA-1-P: glucuronic acid 1-phosphate.Berkeley Laboratory, University of California, Berkeley, metabolites such as fructose 1 ,6-diphosphate, which can also inhibit this enzyme (5, 6). In our previous report of this inhibition of RuDPCase by 6-PGluA (6), the reaction was initiated by adding the enzyme to the otherwise complete reaction mixture. It had been reported that preincubation of the enzyme with bicarbonate and Mg`+ ions increased the activity of the enzyme when assayed for 5 min (11,17). In the course of further studies of the effects of 6-PGluA on the activity of RuDPCase, we have found that preincubation of the enzyme with Mg`+ and bicarbonate produces a long lasting activation of the enzyme, and that inclusion of 6-PGluA in the preincubation mixture causes a large additional activation (rather than inhibition) of the enzyme. However, when the enzyme is activated by preincubation with Mg'+ and bicarbonate and the reaction is initiated with addition of RuDP, subsequent addition of 6-PGluA causes inhibition. These and other results in this study suggest a complex regulatory mechanism, involving inactive and active forms of the enzyme, which respond differently to 6-PGluA, with the response further modified by presence or absence of RuDP, concentration of bicarbonate and Mg'+, and other factors.
MATERIALS AND METHODSMaterials. In addition to the materials used in a previous report (6), glucuronic acid 1-phosphate, the potassium salt, was purchased from Sigma Chemical Co. The sodium salt of 6-PGluA was used in the present studies.Enzyme Isolation. The enzyme was isolated from spinach leaves. The isolation method was the same as the one previously described (6)
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.
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