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首页> 外文期刊>Biochemistry >Investigation of the DsbA mechanism through the synthesis and analysis of an irreversible enzyme-ligand complex.
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Investigation of the DsbA mechanism through the synthesis and analysis of an irreversible enzyme-ligand complex.

机译：通过合成和不可逆酶-配体复合物的分析研究DsbA机制。

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Approaching the molecular mechanism of some enzymes is hindered by the difficulty of obtaining suitable protein-ligand complexes for structural characterization. DsbA, the major disulfide oxidase in the bacterial periplasm, is such an enzyme. Its structure has been well characterized in both its oxidized and its reduced states, but structural data about DsbA-peptide complexes are still missing. We report herein an original, straightforward, and versatile strategy for making a stable covalent complex with a cysteine-homoalanine thioether bond instead of the labile cystine disulfide bond which normally forms between the enzyme and polypeptides during the catalytic cycle of DsbA. We substituted a bromohomoalanine for the cysteine in a model 14-mer peptide derived from DsbB (PID-Br), the membrane partner of DsbA. When incubated in the presence of the enzyme, a selective nucleophilic substitution of the bromine by the thiolate of the DsbA Cys(30) occurred. The major advantage of this strategy is that it enables the direct use of the wild-type form of the enzyme, which is the most relevant to obtain unbiased information on the enzymatic mechanism. Numerous intermolecular NOEs between DsbA and PID could be observed by NMR, indicating the presence of preferential noncovalent interactions between the two partners. The thermodynamic properties of the DsbA-PID complex were measured by differential scanning calorimetry. In the complex, the values for both denaturation temperature and variation in enthalpy associated with thermal unfolding were between those of oxidized and reduced forms of DsbA. This progressive increase in stability along the DsbA catalytic pathway strongly supports the model of a thermodynamically driven mechanism.
机译：由于难以获得合适的蛋白质-配体复合物进行结构表征，因此阻碍了某些酶的分子机理的研究。 DsbA是细菌周质中的主要二硫化物氧化酶，就是这种酶。它的结构在其氧化态和还原态都得到了很好的表征，但是有关DsbA-肽复合物的结构数据仍然缺失。我们在此报告了一种新颖，简单，通用的策略，用于制备稳定的共价复合物，该复合物具有半胱氨酸-高丙氨酸硫醚键，而不是通常在DsbA催化周期内在酶和多肽之间形成的不稳定的胱氨酸二硫键。我们用衍生自DsbA的膜伴侣DsbB（PID-Br）的14-mer模型肽中的半胱氨酸替换了溴高丙氨酸。当在酶的存在下孵育时，溴被DsbA Cys（30）的硫醇盐选择性地亲核取代。该策略的主要优势在于，它可以直接使用酶的野生型形式，这与获取有关酶机制的公正信息最相关。通过NMR可以观察到DsbA和PID之间的许多分子间NOE，表明这两个伙伴之间存在优先的非共价相互作用。 DsbA-PID复合物的热力学性质通过差示扫描量热法测量。在该复合物中，变性温度和与热展开相关的焓变化值都介于氧化形式和还原形式的DsbA之间。沿着DsbA催化途径的稳定性的这种逐步提高，有力地支持了热力学驱动机制的模型。

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