Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720.
Plant Physiol. 1974 Oct;54(4):556-9. doi: 10.1104/pp.54.4.556.
Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 10 mm MgCl(2) 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% CO(2) at pH 7.8 and 25 C.
核酮糖 1,5-二磷酸羧化酶，经预孵育，用 10mM MgCl2 和 1mM 碳酸氢盐激活，在预孵育混合物中存在 0.5mM NADPH 时，可进一步激活约 170%。NADP(+)、NADH 和 NAD(+)无效。NADPH 的激活与之前用 0.05 至 0.10mM 6-磷酸葡萄糖酸观察到的激活相当，因为这些特定的预孵育条件是必需的，但 NADPH 和 6-磷酸葡萄糖酸的作用不是累加的。此外，在较高浓度的 6-磷酸葡萄糖酸抑制酶的情况下，较高浓度的 NADPH 会产生更大的激活作用，在约 1mM 和 200%时达到饱和。在预孵育的指定条件下，果糖 1,6-二磷酸的激活曲线与 6-磷酸葡萄糖酸相似，在 0.1mM 和 70%处达到峰值。在此水平之上，激活降低，并且在更高的浓度下观察到抑制。在这些条件下测定酶活性时，其他测试的代谢物对酶活性的影响较小或没有。当还原型 NADP 或 6-磷酸葡萄糖酸存在于预孵育混合物中时，就可以使用 Lineweaver-Burk 图来确定碳酸氢盐的 Km 值，并且在这些条件下碳酸氢盐的 Km 值为 2.8mM，相当于 pH 7.8 和 25°C 下的 0.3%CO2。
