Sygusch J, Beaudry D, Allaire M
Département de biochimie, Faculté de Médecine, Université de Sherbrooke, Fleurimont, Québec, Canada.
Arch Biochem Biophys. 1990 Dec;283(2):227-33. doi: 10.1016/0003-9861(90)90636-d.
Rabbit skeletal muscle and liver fructose 1,6-diphosphate aldolases autophosphorylate in the presence of inorganic phosphate at physiological and alkaline pH. ATP as well as nonhydrolyzable ATP analogues inhibits autophosphorylation. Autophosphorylation of aldolases abolishes catalytic activity, which is restored upon treatment with alkaline phosphatase. Limited proteolysis of aldolase preferentially hydrolyzes the COOH terminus and liberates a phosphorylated peptide. Treatment of rabbit aldolases with carboxypeptidase, which liberates the COOH terminal residue Tyr 363, although modifying catalytic activity does not affect autophosphorylation. Amino acid analyses are consistent with results of autophosphorylation of the COOH terminus showing residue His 361 in muscle aldolase and Tyr 361 in liver aldolase. Phosphate lability in acid pH by phosphorylated muscle aldolase but not by phosphorylated liver aldolase corroborates the amino acid assignment. Autophosphorylation of the aldolases in the crystalline state is consistent with an intramolecular mechanism. The pH dependence of autophosphorylation being dependent on the enzyme's physical state (soluble or crystalline) is not inconsistent with crystallization stabilizing a conformer having different amino acid pka values and/or reactivities than those of the soluble state.
兔骨骼肌和肝脏中的果糖1,6 - 二磷酸醛缩酶在生理pH值和碱性pH值条件下，于无机磷酸盐存在时会发生自身磷酸化。ATP以及不可水解的ATP类似物会抑制自身磷酸化。醛缩酶的自身磷酸化会使催化活性丧失，而用碱性磷酸酶处理后活性得以恢复。醛缩酶的有限蛋白水解优先水解COOH末端并释放出一个磷酸化肽段。用羧肽酶处理兔醛缩酶，该酶会释放COOH末端残基Tyr 363，虽然会改变催化活性，但不影响自身磷酸化。氨基酸分析结果与COOH末端自身磷酸化的结果一致，显示肌肉醛缩酶中的残基为His 361，肝脏醛缩酶中的残基为Tyr 361。磷酸化的肌肉醛缩酶在酸性pH值下对磷酸盐不稳定，而磷酸化的肝脏醛缩酶则不然，这证实了氨基酸的归属。结晶态醛缩酶的自身磷酸化与分子内机制一致。自身磷酸化对pH值的依赖性取决于酶的物理状态（可溶或结晶），这与结晶使一种构象体稳定的情况并不矛盾，该构象体具有与可溶状态不同的氨基酸pKa值和/或反应活性。
