Multiple active site histidine protonation states in Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase detected by REDOR NMR.
Multiple active site histidine protonation states in Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase detected by REDOR NMR.
复制标题
通过 REDOR NMR 检测醋酸杆菌 N5-羧氨基咪唑核糖核苷酸变位酶中的多个活性位点组氨酸质子化状态。
DOI：
10.1021/bi700899q
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Kappock,TJoseph
中科院分区：
文献类型：
--
作者：
Schaefer,Jacob;Jiang,Hong;Ransome,AaronE;Kappock,TJoseph
I类纯品(N5-羧氨基咪唑变位酶)催化化学上独特的变位酶反应。一种工作机制假说涉及组氨酸(在Escherichia coliPurE中是His45)作为一种通用酸，但没有获得多个质子化状态的证据。对于这项任务来说，溶液核磁共振是一个无与伦比的工具，但对酶的应用有限，因为大多数酶的分子大小超过了它的有效分子大小限制。固态核磁共振不受这一限制。对均一的15N标记的醋酸杆菌(AaPurE)和活性部位配体[6-13C]柠檬酸(AaPurE His59)的151 kDa复合体的REDOR核磁共振研究探索了与关键组氨酸(AaPurE His59)相关的单一电离平衡。在AaPurE络合物中，柠檬酸中心羧酸C613C峰前移，表明负电荷减少，并扩大，表明不均匀。组氨酸15N化学位移表明，His59以大约等摩尔量的Nδ非质子化(类吡啶)形式和Nδ质子化(类吡咯)形式存在，每一种形式都是柠檬酸盐C6的∼4？光谱数据与涉及His59Nδ的质子转移是一致的，这是在I类纯机制中调用的。
Class I PurE (N5-carboxyaminoimidazole mutase) catalyzes a chemically unique mutase reaction. A working mechanistic hypothesis involves a histidine (His45 inEscherichia coliPurE) functioning as a general acid, but no evidence for multiple protonation states has been obtained. Solution NMR is a peerless tool for this task but has had limited application to enzymes, most of which are larger than its effective molecular size limit. Solid-state NMR is not subject to this limit. REDOR NMR studies of a 151 kDa complex of uniformly15N-labeledAcetobacter acetiPurE (AaPurE) and the active site ligand [6-13C]citrate probed a single ionization equilibrium associated with the key histidine (AaPurE His59). In theAaPurE complex, the citrate central carboxylate C613C peak moves upfield, indicating diminution of negative charge, and broadens, indicating heterogeneity. Histidine15N chemical shifts indicate His59 exists in approximately equimolar amounts of an Nδ-unprotonated (pyridine-like) form and an Nδ-protonated (pyrrole-like) form, each of which is ∼4 Å from citrate C6. The spectroscopic data are consistent with proton transfers involving His59 Nδthat are invoked in the class I PurE mechanism.