Eliminations in the reactions catalyzed by UDP-N-acetylglucosamine 2-epimerase
Eliminations in the reactions catalyzed by UDP-N-acetylglucosamine 2-epimerase
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DOI：
10.1021/ja971718q
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发表时间：
1997-10-29
影响因子：
15
通讯作者：
Tanner, ME
中科院分区：
文献类型：
--
作者：
Morgan, PM;Sala, RF;Tanner, ME
已经对细菌酶UDP-N-乙酰葡糖胺2-差向异构酶进行了机理研究，该酶催化UDP-N-乙酰葡糖胺（UDP-GlcNAc）和UDP-N-乙酰甘露糖胺（UDP-ManNAc）的相互转化。这种酶是有趣的，因为它差向异构化不携带酸性质子的立体中心，因此它不能利用简单的去质子化/再质子化机制。偶联酶测定采用UDP-ManNAc脱氢酶已开发。发现D2 O中的差向异构化伴随着氘掺入两种差向异构体的C-2“位置，支持最终涉及在该位置的质子转移的机制。[2“”-H-2]UDP-GlcNAc的差向异构化被一级动力学同位素效应减慢，表明在反应的速率决定步骤期间发生C-H键裂解。位置同位素交换（PM）实验表明，O-18标签在糖UDP桥接位置将在酶差向异构化过程中进入非桥接二磷酸位置。这些观察结果与通过异头C-O键裂解进行的机制一致，2-乙酰胺基葡萄糖醛和UDP作为酶结合的中间体。在不寻常的观察中发现了这种机制的额外证据，即在长时间孵育期间，中间体逐渐从酶中释放并在溶液中积累。这些中间体通过UDP从UDP-GlcNAc的反消除和UDP从UDP-ManNAc的顺消除形成。这是可能的，通过oxocarbenium中间体的EL样消除参与反应。进一步的实验表明，3“-脱氧-UDP-GlcNAc不是酶的底物，并且酶不含紧密结合的NAD（+）辅因子。
Mechanistic studies have been carried out on the bacterial enzyme UDP-N-acetylglucosamine 2-epimerase, which catalyzes the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This enzyme is interesting because it epimerizes a stereocenter that does not bear an acidic proton, and therefore it cannot utilize a simple deprotonation/reprotonation mechanism. A coupled enzyme assay employing UDP-ManNAc dehydrogenase has been developed. The epimerization in D2O is found to be accompanied by the incorporation of deuterium into the C-2 '' position of both epimers, supporting a mechanism that ultimately involves a proton transfer at this position. The epimerization of [2 ''-H-2]UDP-GlcNAc is slowed by a primary kinetic isotope effect indicating that C-H bond cleavage is occurring during a rate-determining step of the reaction. A positional isotope exchange (PM) experiment shows that an O-18 label in the sugar-UDP bridging position will scramble into nonbridging diphosphate positions during enzymatic epimerization. These observations are consistent with a mechanism that proceeds via cleavage of the anomeric C-O bond, with 2-acetamidoglucal and UDP as enzyme-bound intermediates. Additional evidence for this mechanism is found in the unusual observation that during extended incubations, the intermediates are gradually released from the enzyme and accumulate in solution. These intermediates are formed by an anti elimination of UDP from UDP-GlcNAc and a syn elimination of UDP from UDP-ManNAc. It is likely that El-like eliminations via oxocarbenium intermediates are involved in the reaction. Further experiments show that 3 ''-deoxy-UDP-GlcNAc is not a substrate for the enzyme and that the enzyme does not contain a tightly bound NAD(+) cofactor.