Cox J D, Hunt J A, Compher K M, Fierke C A, Christianson D W
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.
Biochemistry. 2000 Nov 14;39(45):13687-94. doi: 10.1021/bi001649j.
Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) influence metal ion binding in the active site. Residues F93, F95, and W97 are contained in a beta-strand that also contains two zinc ligands, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities in the hydrophobic core, compensatory movements of surrounding side chains, and the incorporation of buried water molecules; nevertheless, the enzyme maintains tetrahedral zinc coordination geometry. However, a conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.
人碳酸酐酶II（CAII）疏水核心中的芳香族残基影响活性位点的金属离子结合。F93、F95和W97残基位于一条β链中，该链还包含两个锌配体H94和H96。这些芳香族氨基酸有助于CAII具有高锌亲和力和缓慢的锌解离速率常数[亨特，J.A.，和菲尔克，C.A.（1997）《生物化学杂志》272，20364 - 20372]。用侧链较小的氨基酸取代这些芳香族氨基酸会增强Cu(2+)亲和力，同时降低Co(2+)和Zn(2+)亲和力[亨特，J.A.，马希丁，A.，& 菲尔克，C.A.（1999）《生物化学》38，9054 - 9062]。在这里，锌结合的F93I/F95M/W97V和F93S/F95L/W97M CAII的X射线晶体结构揭示了疏水核心中出现了新的空洞、周围侧链的补偿性移动以及埋藏水分子的纳入；然而，该酶仍保持四面体锌配位几何结构。然而，在F93I/F95M/W97V和F93S/F95L/W97M CAII中，直接金属配体H94以及间接（即“第二壳层”）配体Q92的构象变化都伴随着金属释放，从而消除了脱辅基酶中组氨酸配体以四面体几何结构的预取向。只有一种钴结合变体，F93I/F95M/W97V CAII，保持四面体金属配位几何结构；F93S/F95L/W97M CAII以三角双锥配位几何结构结合Co(2+)，这是由于在金属配位多面体中添加了叠氮阴离子。由于在金属配位多面体中添加了第二个溶剂分子，铜结合变体呈现出四方锥或三角双锥金属配位几何结构。这项工作的关键发现是，芳香族核心残基起到锚的作用，有助于使直接和第二壳层配体预取向，以优化锌结合几何结构并使其他几何结构不稳定。与小分子螯合剂相比，这些几何限制可能是CAII增强的锌/铜特异性的主要决定因素。
