动能和x射线结构突变的研究大肠杆菌碱性磷酸酶(- 412——> Gln)的锌结合位点。
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Kantrowitz马L, ER
动能和x射线结构突变的研究大肠杆菌碱性磷酸酶(- 412——> Gln)的锌结合位点。
生物化学。1996年2月20日,35 (7):2394 - 402。
- PubMed ID
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8652582 (在PubMed]
- 文摘
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位点突变被用来代替他与谷氨酰胺- 412在大肠杆菌碱性磷酸酶。在野生型酶- 412是直接配体催化地重要的锌原子(Zn1)活性部位。突变体酶(H412Q)表现出相同的k (cat),但增加50倍k (m)beplayapp与相应的野生型酶的动力学参数。此外,H412Q酶较低锌含量比野生型酶。与野生型酶相比,三少有效的转移酶反应,显著抑制H412Q酶的水解反应。添加锌的突变酶增加了k (cat)的价值高于野生型酶,部分恢复疲软的衬底和磷酸盐绑定,同时缓解三的抑制。H412Q酶的结构也由x射线晶体学。H412Q酶的总体结构非常相似的野生型酶;唯一的α碳位移超过埃观察附近的突变。H412Q结构没有磷酸结合在酶的活性部位; however, two water molecules were observed where phosphate normally binds in the wild-type enzyme. Close examination of the active site of the H412Q structure revealed structural changes in Ser-102 as well as at the mutation site. For example, the carbonyl oxygen of the side chain of Gln-412 rotated away from the position of His-412 in the wild-type structure, although too far away (3.2 angstroms) to coordinate to Zn1. Studies on the H412Q enzyme, and a comparison of the H412Q and H412N structures, suggest that the structure and electostatics of the imidazole ring of histidine are critical for its function as a zinc ligand in alkaline phosphatase.