Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa.
Chemistry. 2010 Sep 3;16(33):10030-41. doi: 10.1002/chem.201000622.
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 L-erythro (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.
含氟氨基酸已被成功用作吡啶醛磷酸 (PLP) 依赖性酶的基于机制的抑制剂，并且氟对分子构象的影响也得到了广泛的研究和实际应用。在这项研究中，我们试图利用这些独特的特性来研究天冬氨酸脱羧酶（ADC）酶的反应性和结合，ADC 酶是一类小型的依赖丙酮酸的脱羧酶的成员。由于 ADC 活性已被证明对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抑制剂，这些抑制剂选择性地靶向依赖丙酮酸的酶，如 ADC，而不影响宿主中的依赖 PLP 的酶。为此，我们合成了 3-氟天冬氨酸的 L-赤式和 L-苏式异构体，并测试了它们作为结核分枝杆菌和大肠杆菌 ADC 酶的底物和/或抑制剂的能力。此外，还使用捕获和基于 MS 的结合分析来确认两种异构体都进入了酶的活性部位。我们的研究表明，两种异构体都经历了单轮脱羧和氟消除反应，生成可以在活性部位捕获的烯胺产物。有趣的是，从 L-赤式（而不是 L-苏式）异构体形成的烯胺/ADC 复合物足够稳定，即使没有任何捕获也可以观察到。这一发现表明，两种 3-氟天冬氨酸在 ADC 活性部位保持不同的构象，导致烯胺产物具有不同稳定性的构型。总之，我们的结果为开发辅助因子特异性抑制剂提供了新的见解，并证实了氟作为一种独特工具用于探测酶内反应性和结合特性的实用性。
