Berg Alexander K, Yu Qingfeng, Qian Steven Y, Haldar Manas K, Srivastava D K
Department of Chemistry, Biochemistry and Molecular Biology, North Dakota State University, Fargo, ND 58102, USA.
Biochim Biophys Acta. 2010 Apr;1804(4):704-13. doi: 10.1016/j.bbapap.2009.11.006. Epub 2009 Nov 14.
Due to its potential as an antibiotic target, E. coli peptide deformylase (PDF(Ec)) serves as a model enzyme system for inhibitor design. While investigating the structural-functional and inhibitory features of this enzyme, we unexpectedly discovered that 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) served as a slow-binding inhibitor of PDF(Ec) when the above compound was dissolved only in dimethylformamide (DMF), but not in any other solvent, and allowed to age. The time dependent inhibitory potency of the DMF-dissolved AMT was correlated with the broadening of the inhibitor's 295 nm spectral band toward the visible region, concomitant with the increase in the mass of the parent compound by about 2-fold. These data led to the suggestion that DMF facilitated the slow dimerization of AMT (via the formation of a disulfide bond), and that the dimeric form of AMT served as an inhibitor for PDF(Ec). The latter is not caused by the simple oxidation of sulfhydryl groups by oxidizing agents such as H(2)O(2). Newly synthesized dimeric/dithiolated form of AMT ("bis-AMT") exhibited similar spectral and inhibitory features as given by the parent compound when incubated with DMF. The computer graphic modeling data revealed that bis-AMT could be reliably accommodated within the active site pocket of PDF(Ec), and the above enzyme-ligand interaction involves coordination with the enzyme resident Ni(2+) cofactor. The mechanism of the DMF-assisted activation of AMT (generating bis-AMT), the overall microscopic pathway for the slow-binding inhibition of PDF(Ec) by bis-AMT, and the potential of bis-AMT to serve as a new class of antibiotic agent are presented.
由于其作为抗生素靶点的潜力，大肠杆菌肽脱甲酰基酶（PDF(Ec)）成为抑制剂设计的模型酶系统。在研究该酶的结构功能和抑制特性时，我们意外发现，当2-氨基-5-巯基-1,3,4-噻二唑（AMT）仅溶解于二甲基甲酰胺（DMF）而非其他任何溶剂中并放置老化时，它可作为PDF(Ec)的慢结合抑制剂。DMF溶解的AMT的时间依赖性抑制效力与抑制剂在295 nm光谱带向可见光区域的展宽相关，同时母体化合物的质量增加约2倍。这些数据表明，DMF促进了AMT的缓慢二聚化（通过形成二硫键），并且AMT的二聚体形式作为PDF(Ec)的抑制剂。后者并非由诸如H(2)O(2)等氧化剂对巯基的简单氧化所致。新合成的AMT二聚体/二硫醇化形式（“双AMT”）与母体化合物在DMF中孵育时表现出相似的光谱和抑制特性。计算机图形建模数据显示，双AMT可可靠地容纳在PDF(Ec)的活性位点口袋内，并且上述酶-配体相互作用涉及与酶中的Ni(2+)辅因子配位。本文介绍了DMF辅助激活AMT（生成双AMT）的机制、双AMT对PDF(Ec)慢结合抑制的整体微观途径以及双AMT作为新型抗生素的潜力。
