Annexation of a High-Activity Enzyme in a Synthetic Three-Enzyme Complex Greatly Decreases the Degree of Substrate Channeling
Annexation of a High-Activity Enzyme in a Synthetic Three-Enzyme Complex Greatly Decreases the Degree of Substrate Channeling
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DOI：
10.1021/sb4000993
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发表时间：
2014-06-01
影响因子：
4.7
通讯作者：
Zhang, Y. -H. Percival
中科院分区：
文献类型：
--
作者：
You, Chun;Zhang, Y. -H. Percival
通过含有三种不同的cohesins和三种含dockerin的酶的mini-scaffoldin构建了含有磷酸丙糖异构酶（TIM）、醛缩酶（ALD）和果糖1，6-二磷酸酶（FBP）的自组装三酶复合物。这种酶复合物表现出1个数量级以上的初始反应速率比非复合的三种酶的混合物。在该酶级联反应中，ALD介导的反应是限速步骤。为了深入了解限速酶ALD在影响合成酶复合物的底物通道效应中的作用，用从嗜热栖热菌分离的类似大小的ALD代替来自海栖热袍菌的低活性ALD，当后者含有5个以上的人工合成的三酶复合物，并附加有低活性或高活性的ALD时，其初始反应速率比两者的混合物高-酶复合物（TIM-FBP）和非结合的低活性或高活性ALD在相同的酶浓度。还发现，在合成酶复合物中更高活性的ALD的兼并使底物通道的程度从7.5急剧降低到1.5。这些结果表明，在合成的酶复合物的底物通道的程度取决于酶的选择。这一研究表明，在合成级联途径中构建合成酶可能是一个非常重要的工具，以加速由低活性酶控制的限速步骤。
The self-assembled three-enzyme complex containing triosephosphate isomerase (TIM), aldolase (ALD), and fructose 1,6-biphosphatase (FBP) was constructed via a mini-scaffoldin containing three different cohesins and the three dockerin-containing enzymes. This enzyme complex exhibited 1 order of magnitude higher initial reaction rates than the mixture of noncomplexed three enzymes. In this enzyme cascade reactions, the reaction mediated by ALD was the rate-limiting step. To understand the in-depth role of the rate-limiting enzyme ALD in influencing the substrate channeling effect of synthetic enzyme complexes, low-activity ALD from Thermotoga maritima was replaced with a similarsize ALD isolated from Thermus therrnophilus, where the latter had more than 5 synthetic three-enzyme complexes annexed with either low-activity or high-activity ALDs exhibited higher initial reaction rates than the mixtures of the two-enzyme complex (TIM-FBP) and the nonbound low-activity or high activity ALD at the same enzyme concentration. It was also found that the annexation of more high-activity ALD in the synthetic enzyme complexes drastically decreased the degree of substrate channeling from 7.5 to 1.5. These results suggested that the degree of substrate channeling in synthetic enzyme complexes depended on the enzyme choice. This study implied that the construction of synthetic enzyme enzymes in synthetic cascade pathways could be a very important tool to accrelerate rate-limiting steps controlled by low-activity enzymes.