不同抑制剂对谷氨酸棒状杆菌磷酸烯醇式丙酮酸羧化酶酶活的影响

Phosphoenolpyruvate Carboxylase from Corynebacterium glutamicum Inhibited by Diverse Biochemicals

磷酸烯醇式丙酮酸羧化酶(Phosphoenolpyruvate carboxylase,PPC)是生物体碳代谢四碳回补途径的重要酶,是很多生物基化学品合成途径中的关键反应。为揭示谷氨酸棒状杆菌的PPC酶受体内代谢物影响的情况,对谷氨酸棒状杆菌Corynebacterium glutamicumATCC13032的ppc基因进行了PCR克隆,成功地在大肠杆菌中诱导表达为可溶性蛋白。利用Ni柱亲和层析纯化了该蛋白,酶活性测定表明其具有磷酸烯醇式丙酮酸羧化酶活性。细致研究了各种抑制剂,如柠檬酸、异柠檬酸、富马酸、琥珀酸、苹果酸、谷氨酸、天冬氨酸等对PPC酶活的影响,证实天冬氨酸和苹果酸是谷氨酸棒杆菌PPC的强抑制剂,并首次发现PPC也受到N-乙酰谷氨酰胺的抑制作用。

Phosphoenolpyruvate earboxylase （ PPC ） , an enzyme involved in replenishment of the four-carbon diearboxylie acid intermediate pool of the TCA cycle, plays an.important role in both biological and biotechnological synthesis pathways. To discover the effect of diverse inhibitors on the activity of PPC, the full-length sequence of the ppc gene from Corynebacterium glutamicum ATCC13032 was cloned and expressed as soluble form in Escherichia coll. The recombinant PPC was purified with Ni-Column. The purified enzyme showed high phosphoenolpyruvate carboxylase activity, and was inhibited by diverse inhibitors such as citrate, isocitrate, fumarate, glutamate, aspartate, malate etc. Aspartate and malate were the strongest inhibitors. For the first time, the inhibition of PPC activity by N-acetyl glutamine was clearly demonstrated in a glutamate-producing strain of C. glutamicum.