Kurland I J, Pilkis S J
Department of Physiology, State University of New York at Stony Brook 11794-8661, USA.
Protein Sci. 1995 Jun;4(6):1023-37. doi: 10.1002/pro.5560040601.
The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.
肝脏双功能酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶（6PF-2-K/Fru-2,6-P2ase），酶编号E.C. 2.7-1-105/E.C. 3-1-3-46，是一类独特的双功能蛋白家族的成员之一，该家族催化调节性代谢物果糖-2,6-二磷酸（Fru-2,6-P2）的合成与降解。Fru-2,6-P2是糖酵解酶6-磷酸果糖-1-激酶的强效激活剂，也是糖异生酶果糖-1,6-二磷酸酶的抑制剂，它为肝脏碳水化合物代谢的这两条相反途径之间提供了一种切换机制。肝脏6PF-2-K/Fru-2,6-P2ase同工型的活性通过环磷酸腺苷依赖性蛋白激酶（cAPK）催化的单个NH2末端残基Ser-32的磷酸化进行相互调节。Ser-32的磷酸化抑制激酶并激活双磷酸酶，部分是通过静电机制。用天冬氨酸替代Ser-32模拟了cAPK催化的磷酸化作用。在去磷酸化的同型二聚体中，NH2和COOH末端尾部区域也与同一亚基上各自的活性位点相互作用，对双磷酸酶产生自动调节抑制并激活激酶。为支持这一假说，删除NH2或COOH末端尾部区域，或两个区域都删除，会导致这些相互作用的破坏，并使双磷酸酶最大程度激活。在NH2截短形式中观察到激酶受到抑制，其中cAPK磷酸化也减少，从而降低了对果糖-6-磷酸的米氏常数（Km）。cAPK对双功能酶的磷酸化破坏了这些自动调节相互作用，导致激酶受到抑制和双磷酸酶被激活。因此，环磷酸腺苷依赖性磷酸化的作用是由静电和自动调节控制机制共同介导的。
