核盘菌5-烯醇丙酮酰莽草酸-3-磷酸合酶的酶学性质(英文)  

Characterization of 5-Enolpyruvylshikimate-3-phosphate Synthase from Sclerotinia sclerotiorum

作　　者：于寒颖[1] 杨谦[1] 李琳[1] 

机构地区：[1]哈尔滨工业大学生命科学与工程系

出　　处：《中国生物化学与分子生物学报》2006年第4期301-307,共7页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：核盘菌5-烯醇丙酮酰莽草酸-3-磷酸合酶(EPSP合酶)是AROM多功能酶的活性之一.该酶催化莽草酸磷酸(S3P)和磷酸烯醇式丙酮酸(PEP)产生5-烯醇丙酮酰莽草酸-3-磷酸和无机磷酸的可逆反应,受除草剂草甘膦(N-(膦羧甲基)甘氨酸)抑制.纯化了核盘菌AROM蛋白并对EPSP合酶进行了酶学特征研究.结果显示,该酶反应的最适pH值为7·2 ,最适温度为30℃.热失活反应活化能是69·62 kJ/mol .底物S3P和PEP浓度分别高于1 mmol/L和2 mmol/L时,对EPSP合酶反应产生抑制作用.用双底物反应恒态动力学Dalziel方程求得的Km(PEP)为140·98μmol/L,Km(S3P)为139·58μmol/L.酶动力学模型遵循顺序反应机制.草甘膦是该酶反应底物PEP的竞争性抑制剂(Ki为0·32μmol/L)和S3P的非竞争性抑制剂.正向反应受K+激活.当[K+]增加时,Km(PEP)随之降低,Km(S3P)不规律变化,而Ki(PEP)随[K+]增加而提高.The 5-enolpyruvylshikimate-3-phosphate （EPSP） synthase activity of Sclerotinia sclerotiorum is one of the multifunctional enzyme AROM activities, which catalyzes a reversible conversion of shikimate 3-phosphate （S3P） and phosphoenolpyruvate （PEP） to EPSP and inorganic phosphate, and is inhibited by the herbicide glyphosate （N-phosphonomethyl glycine）. AROM protein has been purified from Sclerotinia sclerotiorum and the EPSP synthase has been analyzed. The results indicated that the optimal pH and temperature of EPSP synthase were 7.2 and 30℃ respectively. The activation energy of the heat-deactivated reaction of the enzyme was found to be 69.62 kJ/mol. Both of the substrates, S3P and PEP, were showed to inhibit the reaction rate when their concentrations exceeded 1 mmol/L and 2 mmol/L respectively. The Km of 140.98 μmol/L for PEP and 139.58 μmol/L for S3P were obtained by Dalziel equation which was a steadystate kinetic equation of the enzymatic reaction with the double substrates. The kinetic pattern of the enzyme was consistent with a sequential mechanism. Inhibition of the EPSP synthase reaction by glyphosate was competitive with respect to PEP, with the Ki 0. 32 μmol/L, and noncompetitive with regard to S3P. Activation by [ K＋ ] was observed in the forward reaction. The Km （PEP） was lowered by increasing [ K＋ ], while the Km （S3P） changed irregularly and the Ki （PEP） was enhanced.

关 键 词：EPSP合酶 莽草酸途径 稳态动力学 核盘菌 草甘膦 

分 类 号：Q55[生物学—生物化学]