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首页> 外文期刊>Chemistry: A European journal >3-Fluoroaspartate and pyruvoyl-dependant aspartate decarboxylase: Exploiting the unique characteristics of fluorine to probe reactivity and binding
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3-Fluoroaspartate and pyruvoyl-dependant aspartate decarboxylase: Exploiting the unique characteristics of fluorine to probe reactivity and binding

机译：3-氟天门冬氨酸和依赖于丙酮酰的天冬氨酸脱羧酶：利用氟的独特特性来探测反应性和结合

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摘要

Fluorine-containing amino acids have been used with great success as mechanism-based inhibitors of pyridoxal phosphate (PLP)-dependent enzymes, and the influence of fluorine on the conformation of molecules has also been extensively studied and practically exploited. In this study, we sought to use these unique characteristics to probe the reactivity and binding of aspartate decarboxylase (ADC) enzymes, which are members of the small class of pyruvoyl-dependant decarboxylases. Since ADC activity has been shown to be essential to the virulence of Mycobacterium tuberculosis, information gained in this manner could be used for the development of inhibitors that selectively target pyruvoyl-dependent enzymes such as ADC, without affecting PLP-dependent enzymes in the host. For this purpose, we synthesized the L-erythro and L-threo isomers of 3-fluoroaspartate and tested their ability to act as substrates and/or inhibitors of the M. tuberculosis and Escherichia coli ADC enzymes. Trapping and MS-based binding analysis was additionally used to confirm that both isomers enter the enzymes' active sites. Our studies show that both isomers undergo single turnover decarboxylation and fluorine elimination reactions to give enamine products that can be trapped within the active site. Interestingly, the enamine/ADC complex that forms from the lerythro (but not the L-threo) isomer is sufficiently stable that it can be observed even without any trapping. This finding suggests that the two 3-fluoroaspartates maintain different conformations within the ADC active site, which leads to the enamine products with configurations of different stabilities. Taken together, our results provide new insights for the development of cofactor-specific inhibitors, and confirm the utility of fluorine as a unique tool for probing reactivity and binding profiles within enzymes.
机译：含氟氨基酸已被用作磷酸吡ido醛（PLP）依赖性酶的基于机理的抑制剂，并且对氟对分子构象的影响也进行了广泛的研究和实际开发。在这项研究中，我们试图利用这些独特的特征来探测天冬氨酸脱羧酶（ADC）酶的反应性和结合，天冬氨酸脱羧酶是一类丙酮酰依赖性脱羧酶的成员。由于已证明ADC活性对于结核分枝杆菌的毒力至关重要，因此以这种方式获得的信息可用于开发选择性靶向丙酮酰依赖性酶（例如ADC）的抑制剂，而不会影响宿主中PLP依赖性酶。为此，我们合成了3-氟天冬氨酸的L-赤型和L-苏式异构体，并测试了它们充当结核分枝杆菌和大肠杆菌ADC酶的底物和/或抑制剂的能力。还使用了诱捕和基于MS的结合分析来确认两种异构体都进入了酶的活性位点。我们的研究表明，两种异构体均经过一次脱羧和氟消除反应，生成烯胺产物，该产物可被捕获在活性位点内。有趣的是，由莱索罗（但不是L-苏氨酸）异构体形成的烯胺/ ADC络合物足够稳定，即使没有任何捕获也可以观察到。该发现表明，两个3-氟天冬氨酸在ADC活性位点内保持不同的构象，这导致烯胺产物具有不同稳定性的构型。综上所述，我们的研究结果为辅因子特异性抑制剂的开发提供了新见识，并证实了氟作为探测酶反应性和结合特性的独特工具的实用性。

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