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首页> 外文期刊>Biochemistry >Histidine --> carboxamide ligand substitutions in the zinc binding site of carbonic anhydrase II alter metal coordination geometry but retain catalytic activity
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Histidine --> carboxamide ligand substitutions in the zinc binding site of carbonic anhydrase II alter metal coordination geometry but retain catalytic activity

机译：组氨酸->碳酸酐酶II锌结合位点上的羧酰胺配体取代改变金属配位几何形状但保留催化活性

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    摘要

    The catalytic zinc ion of human carbonic anhydrase II (CAII) is coordinated by three histidine ligands (H94, H96, and H119) and a hydroxide ion with tetrahedral geometry. Structural and functional analysis of variants in which the zinc ligands H94 and H119 are substituted with asparagine and glutamine, and comparison with results obtained with aspartate and glutamate substitutions indicate that the neutral ligand field provided by the protein optimizes the electrostatic environment for the catalytic function of the metal ion, including stabilization of bound anions. This is demonstrated by catalytic activity measurements for ester hydrolysis and CO2 hydration, as well as sulfonamide inhibitor affinity assays. High-resolution X-ray crystal structure determinations of H94N, H119N, and H119Q CAIIs reveal that the engineered carboxamide side chains coordinate to zinc with optimal stereochemistry. However, zinc coordination geometry remains tetrahedral only in H119Q CAII. Metal geometry changes to trigonal bipyramidal in H119N CAII due to the addition of a second water molecule to the zinc coordination polyhedron and also in H94N CAII due to the displacement of zinc-bound hydroxide by the bidentate coordination of a Tris molecule. Possibly, the bulky histidine imidazole ligands of the native enzyme play a role in disfavoring trigonal bipyramidal coordination geometry for zinc. Protein-metal affinity is significantly compromised by all histidine --> carboxamide ligand substitutions. Diminished affinity may result from significant movements (up to 1 A) of the metal ion from its position in the wild-type enzyme, as well as the associated, minor conformational changes of metal ligands and their neighboring residues.
    机译：人碳酸酐酶II（CAII）的催化锌离子由三个组氨酸配体（H94，H96和H119）和具有四面体几何形状的氢氧根离子配位。锌配体H94和H119被天冬酰胺和谷氨酰胺取代的变体的结构和功能分析，并与天冬氨酸和谷氨酸取代获得的结果进行比较表明，该蛋白质提供的中性配体场优化了静电环境，从而促进了其催化功能金属离子，包括稳定的结合阴离子。酯水解和CO2水合的催化活性测量以及磺酰胺抑制剂亲和力测定证明了这一点。 H94N，H119N和H119Q CAII的高分辨率X射线晶体结构测定表明，工程羧酰胺侧链与锌的配位具有最佳立体化学。但是，锌配位几何仅在H119Q CAII中保持四面体。在H119N CAII中，由于向锌配位多面体中添加了第二个水分子，因此在H119N CAII中，金属几何形状变为三角双锥体，在H94N CAII中，由于Tris分子的双齿配位取代了与锌结合的氢氧化物，金属几何形状也发生了变化。可能的是，天然酶的庞大的组氨酸咪唑配体在不利于锌的三角双锥体配位几何结构中起作用。所有组氨酸->羧酰胺配体取代都会严重损害蛋白质-金属的亲和力。亲和力降低可能是由于金属离子从其在野生型酶中的位置发生明显移动（最多1 A），以及金属配体及其邻近残基的相关，较小的构象变化所致。

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