多个功能角色的活性部位组氨酸在催化和大肠杆菌葡萄糖胺6-phosphate脱氨酶的变构调节。
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多个功能角色的活性部位组氨酸在催化和大肠杆菌葡萄糖胺6-phosphate脱氨酶的变构调节。
生物化学。2001年8月28日,40 (34):10187 - 96。
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11513596 (在PubMed]
- 文摘
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glucosamine-6-phosphate脱氨酶的活性部位(EC 3.5.99.6,原名5.3.1.10)从大肠杆菌首次为特征的基础上,酶的晶体结构到绑定的竞争抑制剂2-amino-2-deoxy-glucitol - 6 -磷酸。结构对应于R变构酶的状态;它显示了侧链His143接近O5原子的抑制剂。这种安排表明His143可能作用的催化开环步的氨基葡萄糖6-phosphate alpha-anomer才是真正的衬底。活性部位组氨酸的咪唑组联系人的羧基组Glu148 Asp141,通过其Ndelta1原子(奥利瓦et al .(1995)结构3、1323 - 1332年)。这些交互T的变化状态,因为侧链Glu148朝着别构部位,离开活动现场二分体Asp141-His143 [Horjales et al。(1999)结构7,527 - 536]。在这个研究中,一种双重方法使用定点诱变和控制化学改性的组氨酸残基被用于调查的作用活性部位组氨酸。我们的研究结果支持His143的多功能作用;在正向反应,参与底物的催化开环的一步,氨基葡萄糖6 p。逆反应,衬底果糖6 p在开放链结合,羰基的形式。 The role of His143 in the binding of both glucosamine 6-P and reaction intermediates in their extended-chain forms was demonstrated by binding experiments using the reaction intermediate analogue, 2-amino-2-deoxy-D-glucitol 6-phosphate. His143 was also shown to be a critical residue for the conformational coupling between active and allosteric sites. From the pH dependence of the reactivity of the active site histidine to diethyl dicarbonate, we observed a pK(a) change of 1.2 units to the acid side when the enzyme undergoes the allosteric T to R transition during which the side chain of Glu148 moves toward the active site. The kinetic study of the Glu148-Gln mutant deaminase shows that the loss of the carboxy group and its replacement with the corresponding amide modifies the k(cat) versus pH profile of the enzyme, suggesting that the catalytic step requiring the participation of His143 has become rate-limiting. This, in turn, indicates that the interaction Glu148-His143 in the wild-type enzyme in the R state contributes to make the enzyme functional over a wide pH range.