油菜籽焦磷酸:果糖-6-磷酸1-磷酸转移酶的动力学研究

Kinetics of Pyrophosphate:Fructose-6-phosphate Phosphotransferase from Brassica napus

关键词:  油菜  焦磷酸:果糖-6-磷酸1-磷酸转移酶  分离纯化  动力学

Key words:  Brassica napus  pyrophosphate:fructose-6-phosphate 1-phosphotransferase  purification  kinetics

摘要:

采用DEAE-Sephadex A-50及磷酸纤维素柱层析,用底物亲和洗脱法从萌发油菜(Brassica napus)种子中分离纯化了焦磷酸:果糖-6-磷酸1-磷酸转移酶(PFP)。纯化倍数679.7倍,比活力为21.75单位/毫克·蛋白,活力回收率22.9%。酶的最适pH值为7.5,Mg²⁺和M_n²⁺对酶有激活作用。进行酶的初级动力学及稳态动力学研究,对果糖-6-磷酸(F6P)表现为典型的米氏规律,K_m值为3.33mmol/L;对焦磷酸(PP_i)的活力变化,在PP_i浓度小于1.0mmol/L时具有部分米氏酶特点(K_m=1.0mmol/L),大于1.0mmol/L时,PP_i对酶有抑制作用。从两底物F6P和PP_i的相互作用以及产物磷酸(P_i)与底物(F6P和PP_i)的相互关系分析,初步推断油菜籽PFP的催化反应为双底物双产物的有序机制。

Abstract:

The pyrophosphate:fructose-6-phosphate phosphotransferase(PFP) was isolated from germinating Brassica napus seeds mainly by DEAE-Sephadex A-50 chromatography and then by substrate affinity elution from phosphocellulose colume.The multiple of purification is 679.7.The specific activity is 21.75 units/mg·protein.The yield of activity is 22.9%.From the analysis of polyacrylamide gel electrophoresis in sodium dodecyl sulfate,the enzyme consists of two subunits which molecular mass value is 60KD and 68 KD respectively.The optimum pH value is 7.5. The divalent metal ions of Mg²⁺ and Mn²⁺ could activate the enzyme.The prelimary kinetics and steady-state kinetics of the PFP from Brassica napus seeds were researched.For Fructose-6-phosphate(F6P),the enzyme exhibits a typical Michaelis Menten hyperbolic kinetics and its K_m value is 3.33 mmol/L.For pyrophosphate(PP_i),when the concentration of PP_i is lower than 1.0 mmol/L,the enzyme displays partly the properties of Michaelis enzyme(K_m=1.0 mmol/L).But when the concentration of PP_i is higher than 1.0 mmol/L,PP_i inhibits the activity of the enzyme.From the research of the reaction between two substrates(F6P and PP_i) and the relationship between product(P_i) and substrates(F6P and PP_i),a sequential enzyme mechanism with two substrates and two products are proposed preliminarily for the PFP from Brassica napus.