果糖-2,6-二磷酸对番茄果实PFP酶活化效应的巯基依赖性

在无二硫苏糖醇(DTT)存在下得到部份纯化的氧化型PFP酶,在广泛的pH范围内(pH6.0～9.0)失去其大部分对果糖2,6-二磷酸的敏感性。活化效应可藉与DTT保温得到恢复而不改变其最适pH值。在与DTT保温过程中,酶对果糖2,6-二磷酸的亲和力逐步增加。氧化型酶的K_a值(对果糖2,6-二磷酸)在酶与DTT保温(pH8)1h之后从1400nmol/L下降到约50nmol/L。 在DTT存在下纯化的酶(还原型)经低浓度5,5′-二硫代双(2-硝基苯甲酸)(DTNB)处理,在使酶活性迅速失活的同时引起酶对果糖2,6-二磷酸脱敏。这一过程可为DTT处理所回复。从小麦胚中纯化的硫氧还蛋白h在恢复酶活性和酶的果糖2,6-二磷酸敏感性的效应中表明,细胞内的氧化还原状态可能藉以改变酶对果糖2,6-二磷酸的亲和力而调节PFP酶的活性。

The Regulation of Inorganic Pyruphosphate D-Fructose 6-Phosphate 1-Phosphotransferase (PFP) from Tomato Fruits by the Thio-dependent Fru-2, 6-P_2 Activation

The thio-dependent activation of Fru-2, 6-P_2 to tomato fruit PFP has been investigated. The enzyme partially purified in the absence of dithiothreitol almost lost its sensitivity to Fru-2, 6-P_2 over the range from pH 6.0 to 9.0 (Fig. 2). The activation of this oxidized form PFP could be regained by incubating the enzyme with dithiothreitol without changing its optimum pH for the activation. The affinity of the enzyme to Fru-2, 6-P_2 increased during the incubation. K_a value of oxidized form PFP for Fru-2, 6-P_2 decreased from 1400 nmol/L, to 50 nmol/L after 1 hour incubation with DTT at pH 8 (Fig. 3, Fig. 4).The enzyme purified in the presence of DTT-(reduced form) was rapidly inactivated and desensitized with respect to Fru-2, 6-P_2 at pH 8 by low concentrations of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (Fig. 5). Its substrates, F-6-P and pyrophosphate, protected the enzyme against DTNB inactivation (Fig. 6). The inactivation could be reversed by DTT treatment. Effects of thioredoxin h (purified from wheat germ) on the restoration of enzyme activity and affinity to Fru-2,6-P_2 have been shown in Fig. 7. It is suggested that the redox status of enzyme may be of significance in determining the PFP activity by changing the affinity of the enzyme to Fru-2,6-P_2 in vivo (Fig. 8, Fig. 9).