Human carbonic anhydrase III: Structural and kinetic study of catalysis and proton transfer
Human carbonic anhydrase III: Structural and kinetic study of catalysis and proton transfer
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DOI：
10.1021/bi050610h
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发表时间：
2005-08-02
期刊：
影响因子：
2.9
通讯作者：
McKenna, R
中科院分区：
文献类型：
--
作者：
Duda, DM;Tu, CK;McKenna, R
与 HCA II 和在此位点具有亮氨酸的其他同工酶相比，残基苯丙氨酸 198 (Phe 198) 是人碳酸酐酶 III (HCA III) 活性较低的主要原因。我们报告了 HCA III 和定点突变体 F198L HCA III 的晶体结构，分辨率均为 2.1 埃，并且通过含有咪唑环的外源质子供体增强了催化活性。两种酶在羧基末端都有一个六组氨酸延伸，用于帮助纯化，其在结合在相邻对称相关酶的活性位点空腔中的晶体结构中是有序的。这一观察结果使我们能够评论咪唑及其衍生物在 HCA III 催化中作为外源质子供体/受体的许多可能的结合位点。动力学和结构证据表明，HCA III 中 Phe 198 的苯基侧链距锌约 5 埃，是活性位点中的空间收缩，可能会导致锌结合溶剂的相互作用发生改变，并且是组氨酰组氨酸催化激活的结合位点。这表明碳酸酐酶中质子转移途径的激活位点比以前的研究中认为的更接近锌。
The residue phenylalanine 198 (Phe 198) is a prominent cause of the lower activity of human carbonic anhydrase III (HCA III) compared with HCA II and other isozymes which have leucine at this site. We report the crystal structures of HCA III and the site-directed mutant F198L HCA III, both at 2.1 angstrom resolution, and the enhancement of catalytic activity by exogenous proton donors containing imidazole rings. Both enzymes had a hexahistidine extension at the carboxy-terminal end, used to aid in purification, that was ordered in the crystal structures bound in the active site cavity of an adjacent symmetry-related enzyme. This observation allowed us to comment on a number of possible binding sites for imidazole and derivatives as exogenous proton donors/acceptors in catalysis by HCA III. Kinetic and structural evidence indicates that the phenyl side chain of Phe 198 in HCA III, about 5 angstrom from the zinc, is a steric constriction in the active site, may cause altered interactions at the zinc-bound solvent, and is a binding site for the activation of catalysis by histidylhistidine. This suggests that sites of activation of the proton-transfer pathway in carbonic anhydrase are closer to the zinc than considered in previous studies.