Department of Biochemistry, University of California, Riverside, California 92521.
Plant Physiol. 1986 Dec;82(4):985-90. doi: 10.1104/pp.82.4.985.
Phosphoenolpyruvate carboxylase partially purified from leaves of Crassula and rendered insensitive to malate by storage without adjuvants can be altered to the form sensitive to malate inhibition by brief, 5-minute preincubation with 5 millimolar malate. The induction of malate sensitivity is reversible by lowering the malate(2-) concentration. Of the reaction components only HCO(3) (-) increases the sensitivity to malate in subsequent assay. Phosphoenolpyruvate (PEP), which itself tends to lower sensitivity to subsequent malate inhibition, also reduces the effect of malate in the assay, as does glucose-6-phosphate. PEP isotherms showed that the insensitive or unpreincubated enzyme, responds to the presence of 5 millimolar malate during assay with a 3-fold increase in K(m), but no effect on V(max). Enzyme preincubated with malate shows the same effect of malate on K(m), but in addition V(max) is inhibited 72%. It thus appears that both sensitive and insensitive forms of PEP carboxylase are subject to K-type inhibition by malate, but only the sensitive form also shows V-type inhibition. Preincubation with malate at different pH values showed that at pH 6.15, the inhibition by malate in subsequent assay at pH 7 was much lower than at pH 7 or 8. When the reaction is prerun for 30 minutes with increasing concentrations of PEP, subsequent assay with malate shows progressively less inhibition due to malate. When 0.3 millimolar PEP either alone or with 0.1 millimolar ATP and 0.3 millimolar NaF is present during preincubation, the effect of malate in a following assay is to activate the reaction. These results may indicate an effect of phosphorylation of the enzyme on sensitivity to malate.
从景天科植物叶片中部分纯化的磷酸烯醇丙酮酸羧激酶，在没有助剂的情况下储存使其对苹果酸不敏感，可通过与 5mmol/L 苹果酸短暂孵育 5 分钟，转化为对苹果酸抑制敏感的形式。通过降低苹果酸（2-）浓度可使苹果酸敏感性逆转。在随后的测定中，只有 HCO₃⁻（碳酸氢根）增加了对苹果酸的敏感性。本身倾向于降低随后对苹果酸抑制敏感性的磷酸烯醇丙酮酸（PEP）也降低了测定中的苹果酸效应，葡萄糖-6-磷酸也是如此。PEP 等压线表明，在测定中，不敏感或未孵育的酶对 5mmol/L 苹果酸的存在的响应是 K m增加了 3 倍，但对 V max没有影响。用苹果酸预孵育的酶对 K m表现出相同的苹果酸效应，但此外 V max被抑制了 72%。因此，似乎敏感和不敏感形式的 PEP 羧激酶都受到苹果酸的 K 型抑制，但只有敏感形式也表现出 V 型抑制。在不同 pH 值下用苹果酸预孵育表明，在 pH 6.15 下，在 pH 7 下随后测定中的苹果酸抑制作用比在 pH 7 或 8 下低得多。当反应在不断增加的 PEP 浓度下预运行 30 分钟时，随后用苹果酸进行测定显示，由于苹果酸而导致的抑制作用逐渐减少。当 0.3mmol/L 的 PEP 单独或与 0.1mmol/L 的 ATP 和 0.3mmol/L 的 NaF 一起存在于预孵育中时，苹果酸在随后的测定中的作用是激活反应。这些结果可能表明酶的磷酸化对苹果酸敏感性的影响。
