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首页> 外文期刊>Chemtracts >Engineering Protein Allostery: 1.05 A Resolution Structure and Enzymatic Properties of aNa+-Activated Trypsin
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Engineering Protein Allostery: 1.05 A Resolution Structure and Enzymatic Properties of aNa+-Activated Trypsin

机译：工程蛋白的变构：1.05 aNa +激活胰蛋白酶的解析结构和酶学性质。

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摘要

Some trypsin-like proteases, especially those members of the complementsystem and participants in blood coagulation, are known to be allostericallyactivated by Na~+binding. In this study, an attempt was made to introducesimilar allosteric activation into Streptomyces griseus trypsin (SGT) byswapping its 170-, 186- and 200-loops with those of factor Xa (FXa), a Nat-activated protease involved in blood coagulation. Because of the poor pro-tease activity of the SGT derivative, designated FX170 (< 1% compared towild-type SGT), it was further modified by inclusion of five amino acid sub-stitutions to introduce the entire 99-loop of FXa. The new construct, FX99,carried a total of 19 amino acid replacements and the entire structural deter-minants for Na~+binding and substrate site found in FXa. Kineticmeasurements of k_(cat)/K_mrevealed twofold higher affinity of FX99 for Na+(K_a= 6.1 ± 0.1 M~(-1))compared to that of FX170, and almost comparable tothat of FXa (Fig. 1). The activity of FX99 toward chromogenic substrateswas also significantly higher than that of FX170 and comparable to FXa.The X-ray crystal structure of FX99 solved at 1.05 A resolution defined theengineered Na~+ site and active site loops. Efficient Na~+ activation wasbelieved to be linked to the acquisition of new substrate selectivity, similarto that of FXa.
机译：已知一些胰蛋白酶样蛋白酶，特别是那些补体系统成员和凝血参与者，被Na +结合变构激活。在这项研究中，尝试通过将其170-，186-和200-环与因子Xa（FXa）（一种参与血液凝固的Nat活化蛋白酶）交换而将相似的变构激活引入灰链霉菌胰蛋白酶（SGT）中。由于命名为FX170的SGT衍生物的蛋白酶活性差（与野生型SGT相比，<1％），因此通过包含5个氨基酸取代基对其进行了进一步修饰，以引入FXa的整个99个环。新的构建体FX99携带了总共19个氨基酸置换，以及FXa中Na〜+结合和底物位点的整个结构决定因素。 k_（cat）/ K_m的动力学测定表明，FX99对Na +的亲和力比FX170高两倍，K_a = 6.1±0.1 M〜（-1），几乎与FXa相当（图1）。 FX99对发色底物的活性也显着高于FX170，与FXa相当。FX99的X射线晶体结构以1.05 A的分辨率解析，定义了工程化的Na〜+位点和活性位点环。认为有效的Na〜+活化与获得新的底物选择性有关，类似于FXa。

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