THE PHOSPHORYLATION SITE OF SNAKE MUSCLE FRUCTOSE 1,6-BISPHOSPHATASE AND ITS PHOSPHORYLATION CONDITIONS

From the tryptic digests of phosphorylated snake muscle FruP_(2ase), a phosphoryl peptide has beenisolated, its amino acid sequence was Gly-Ala-Gly-Ser-Arg and the phosphorylation site was consideredto be on the serine residue. Effectors of the enzyme such as FruP_2, F6P and AMP did not affect thephosphorylation. The effect of pH on phosphorylation was consistent with that on the activity of theenzyme. The activity of phosphorylated enzyme was slightly lower than that of the native enzyme, thisdifference in activities between the two forms of the enzyme increased with decreasing the substrateconcentration. Results further support that a phosphorylated intermediate is involved in the catalytic reac-tion of FruP_(2ase).     

蛇肌果糖1,6-二磷酸酯酶与腺苷—磷酸的相互作用和抑制的机制——紫外差光谱的研究

本文证明别构抑制剂AMP和dAMP与蛇肌果糖1,6-二磷酸酯酶结合的差光谱,都有270,280和288nm正峰,此外dAMP尚有275nm正峰。计算得到两者结合常数分别为2.0×10~6M~(-1)和1.2×10~6M~(-1)。两者抑制行为不同和AMP的核糖上的2′-羟基与酶的别构部位相互作用有关。胰蛋白酶的限制性酶解引起该酶活力增加,同时受AMP抑制作用丧失,在差光谱上表现为280和288nm负峰,其特征和酶的脲素差光谱一致。说明高活性酶是处于结构松散状态,受AMP抑制的酶则处于紧凑状态,此二种状态均伴随着酪氨酸残基的微环境的改变。     

鸡肝果糖-6-磷酸-2-激酶的巯基与碱性氨基酸残基的研究

本文报道半胱氨酸、赖氨酸和精氨酸等残基在鸡肝果糖-6-磷酸-2-激酶的作用。DTNB修饰的结果表明鸡肝PFK-2每亚基具有9个巯基。其中,在中性pH下可滴定的巯基数目为6个;在Fru6P存在下的可滴定巯基数为4个;而在ATP存在下的可滴定巯基数为7个。在修饰的过程中酶的活力随着被滴定的巯基数目的增加先是提高后为下降,但最后的酶仍不低于初始活力。NEMI对该酶活力的影响与DTNB的相似,它的修饰首先使酶活力升高,当巯基完全被修饰后的活力仍不低于初始活力。但是PCMB和碘代乙酸对该酶的活力影响很小。pH动力学结果显示该酶有1个pK~a为9.0的必需基团,它很可能是赖氨酸残基。PLP和苯乙二醛修饰PFK-2皆可导致该酶的失活。Fru6P对PLP的作用有强烈的保护,Fru2,6P2和Pi也有一定的保护作用,而ATP则稍有增加PLP对该酶的失活作用;苯乙二醛修饰的结果和PLP不同,Fru2,6P2有较强的保护作用,ATP有部分保护作用,Fru6P则对失活完全无保护作用。以上结果表明赖氨酸残基是鸡肝PFK-2与底物Fru2,6P2结合有关的必需残基,而精氨酸可能是该酶与产物Fru2,6P2结合有关的残基。     

小鼠肝果糖6-磷酸,2-激酶的纯化及动力学研究

经过超离心,聚乙二醇分级沉淀及DEAE-Sephadex,Blue-Sepbarose。磷酸纤维素三种柱层析等一系列过程,纯化了小白鼠肝脏的果糖6-磷酸,2-激酶。经凝胶过滤和SDS聚丙烯酰胺电泳方法测出该酶是由两个相同亚基构成的分子量为110000的蛋白。Mg~(2+)是其活性所必需的,活化方式呈正协同性。酶与底物的结合方式,对果糖6-磷酸表现正协同性,对ATP无协同性,其Km值随果糖6-磷酸的浓度减小而增大,表明酶的作用为顺序机制。活性中心有一个必需氨基酸残基与结合ATP有关,结合后,其侧链pKa由9.5移至9.8。     

蛇肌果糖1,6-二磷酸酯酶的限制性酶解

蛇肌果糖1.6-二磷酸酯酶被枯草杆菌蛋白酶或胰蛋白酶限制性酶解,可分别产生29000和7000或32000和4000的肽段。酶解产物在pH7.5和pH9.2分别有2倍和4—5倍的激活,pH7.5的激活作用,仅可用无机磷测活法观察到。5′AMP对该产物的别构抑制作用部分或全部丧失。本文与文献[1]的结果使我们提出了天然状态的蛇肌酶的催化部位的结构是不完善的看法。被限制性酶解的蛇肌酶受6-磷酸果糖激活,同时又受6-磷酸葡萄糖酸,6-磷酸葡萄糖和还原型辅酶Ⅱ的抑制。因此限制性酶解作用可能在蛇肌中有一定生理功能。     

蛇肌果糖1,6-二磷酸酯酶的磷酰化部位与磷酰化条件

本文报道了从磷酰化的果糖1,6-二磷酸酯酶的胰蛋白酶水解物中分离得到了一个磷酰化的肽段及该肽段的氨基酸排列顺序和磷酰化的部位是在丝氨酸残基上。果糖1,6-二磷酸,果糖6磷酸和腺嘌呤核苷-磷酸等效应剂不影响该酶被无机正磷酸磷酰化。pH对磷酰化程度的影响和对酶活力的影响一致。磷酰化酶的活力比非磷酰化状态的酶活力稍低,活力的差别随着底物浓度的减小而增大。磷酰化的酶与Mg~(2+)和果糖1,6-二磷酸一起保温能脱磷酰化,本文结果进一步支持了蛇肌果糖1,6-二磷酸酯酶在催化果糖1,6-二磷酸水解的过程中,包含了形成磷酰化酶中间物这一过程。     

PURIFICATION AND KINETICS OF MOUSE LIVER FRUCTOSE 6-PHOSPHATE, 2-KINASE

The mouse liver fructose 6-phosphate, 2-kinase was purified by ultracentrifugation, polyethylene glycol precipitation, and subsequently by chromatography on DEAE-Sephadex, Blue-Sepharose and phasphocellulose columnS. Gel filtration and SDS polyacrylamide electrophorcsis showed that the enzyme has a molecular weight of 110,000 with two identical subunits. Mg~(2+) is essential for its activity. The activation of the enzyme by Mg~(2+) showed a positive cooperativity. The substrate saturation curve for fructose 6-phosphate was sigmoidal and for ATP was hyperbolic. The K_m's for ATP increased with decrease in concentrations of fructose 6-phosphate indicating that the sequence for the substrates binding was in an ordered mechanism with respect to fructose 6-phosphate prior to ATP. An ionizable residue at the active site with pKa 9.5 was essential for the ATP binding and the pKa shifted to 9.8 after the binding of ATP.     

蛇肌果糖1,6-二磷酸酯酶的逆反应中的磷酰化作用

本文证明在蛇肌果糖1,6-二磷酸酯酶的反应体系中,若有非水磷酸受体存在,其底物果糖1,6-二磷酸的1位磷酰基能转移到这些受体上形成有机磷化合物,导致F6P的释放速度快于无机磷的释放。用~(32)P-无机正磷酸和蛇肌酶一起保温,可分离出被无机磷共价磷酰化的酶中间物。这个磷酰化中间物可能和酶的正反应催化过程有关。     

鸡肝果糖-6-磷酸-2-激酶的性质研究

本文经过匀浆、聚乙二醇分级沉淀和DEAE-Sephadex A-50柱层析分离步骤,进一步用蓝色三嗪染料琼脂糖柱层析分离,得到了电泳均一的鸡肝果糖-6-磷酸-2-激酶(EC2.7.2.105)。该酶的性质为:(1)对果糖-6-磷酸的底物饱和曲线为双曲线型,无机磷降低果糖-6-磷酸的Km,而不影响V_(max);(2)对底物ATP的结合具有负协同性,Hill系数为0.56;(3)受低浓度Mg~(2+)的激活,但受高浓度Mg~(2+)的抑制,在EDTA存在下不表现活力;(4)在30℃内较稳定,高于40℃下易失活;(5)在pH7—9范围内活性比较平稳,pH高于9.0时,活性上升,而在大于9.5后,活性很快下降;(6)对胰蛋白酶较敏感,但ATP对其有显著的保护作用。     

PHOSPHORYLATED REACTION OF SNAKE MUSCLE FRUCTOSE 1,6-BISPHOSPHATASE IN ITS REVERSE REACTION

A phosphoryl enzyme intermediate was observed in the hydrolysis of FruP_2 catalyzed by snake muscleFruP_(2ase) based on the fact that the formation rate of F6P is faster than that of inorganic phosphatewhen the reaction mixture contains a phosphoryl acceptor other than water. A covalently bound phosphateon the enzyme was isolated from the reverse reaction. This intermediate may relate to the process of theforward catalytic reaction by the enzyme.