机械的调查UDP-galactopyranose变位酶的大肠杆菌利用2 -和3-fluorinated UDP-galactofuranose探针。
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刘张问,H
机械的调查UDP-galactopyranose变位酶的大肠杆菌利用2 -和3-fluorinated UDP-galactofuranose探针。
J是化学Soc。2001年7月18日,123 (28):6756 - 66。
- PubMed ID
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11448178 (在PubMed]
- 文摘
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的galactofuranose一半发现在许多表面成分的微生物来源于UDP-D-galactopyranose (UDP-Galp)通过一个独特的环收缩UDP-Galp变位酶催化的反应。这种酶,分离出一些细菌来源是黄素蛋白。研究这种催化作用,克隆的大肠杆菌变位酶纯化和两个氟化类似物,UDP - [2 - f] Galf(9)和UDP - [3 f] Galf(10),化学合成。这两个化合物被发现是底物的减少UDP-Galp变位酶的Km值确定为65年和861年microM 9日和10日,分别对应kcat值估计为0.033和5.7 (1)。由于氟原子取代基氧化还原惰性,氧化的机制由2哦或3-OH半乳糖一部分因此可以坚定地排除。此外,9和10都穷比UDP-Galf基质,和9的降息尤其重要。这一发现可能归因于2 - f取代基的诱导效应,立即毗邻异头中心,并与涉及oxocarbenium中间体的形成机制是一致的在营业额或过渡状态。nonreducing条件下,有趣的是,化合物9和10不是底物,而是变位酶的抑制剂。10的失活时间、active-site-directed和不可逆的K (I) 270 microM和K(非活性的)0.19分钟(1)。自K(我)值类似于公里,观察到的失活不太可能紧密结合的结果。 To our surprise, the inactivated enzyme could be regenerated in the presence of dithionite, and the reduced enzyme is resistant to inactivation by these fluorinated analogues. It is possible that reduction of the enzyme-bound FAD may induce a conformational change that facilitates the breakdown of the putative covalent enzyme-inhibitor adduct to reactivate the enzyme. It is also conceivable that the reduced flavin bears a higher electron density at N-1, which may play a role in preventing the formation of the covalent adduct or facilitating its breakdown by charge stabilization of the oxocarbenium intermediates/transition states. Clearly, this study has led to the identification of a potent inactivator (10) for this enzyme, and study of its inactivation has also shed light on the possible mechanism of this mutase.