Hunt J A, Fierke C A
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1997 Aug 15;272(33):20364-72. doi: 10.1074/jbc.272.33.20364.
In all metalloenzymes, hydrophobic residues surround the metal binding site. In carbonic anhydrase II (CAII) residues Phe93, Phe95, and Trp97 flank two of the three histidines that coordinate zinc to form a hydrophobic cluster beneath the zinc binding site. A library of CAII variants differing in these hydrophobic amino acids was prepared using cassette mutagenesis, then displayed on filamentous phage, and screened for proteins retaining high zinc affinity. Wild-type CAII was enriched 20-fold by selection, and consensus residues at each position were identified from the enriched CAII variants (Ile, Phe, Leu, and Met at position 93; Ile, Leu, and Met at position 95; and Trp and Val at position 97). Highly selected variants have zinc affinity and catalytic activity nearly equal to that of wild-type CAII, indicating that the aromatic residues are not absolutely essential. However, the zinc dissociation rate constant and catalytic activity of the variants correlate with the volume of the amino acids at positions 93, 95, and 97. In summary, metalloenzyme variants displayed on phage can be selected on the basis of metal affinity; such methods will be useful for optimization of metal ion biosensors.
在所有金属酶中，疏水残基围绕着金属结合位点。在碳酸酐酶II（CAII）中，苯丙氨酸93、苯丙氨酸95和色氨酸97位于与锌配位的三个组氨酸中的两个组氨酸两侧，在锌结合位点下方形成一个疏水簇。利用盒式诱变制备了这些疏水氨基酸不同的CAII变体文库，然后展示在丝状噬菌体上，并筛选保留高锌亲和力的蛋白质。通过选择，野生型CAII富集了20倍，并从富集的CAII变体中鉴定出每个位置的共有残基（93位为异亮氨酸、苯丙氨酸、亮氨酸和甲硫氨酸；95位为异亮氨酸、亮氨酸和甲硫氨酸；97位为色氨酸和缬氨酸）。高度选择的变体具有与野生型CAII几乎相等的锌亲和力和催化活性，表明芳香族残基并非绝对必需。然而，变体的锌解离速率常数和催化活性与93、95和97位氨基酸的体积相关。总之，展示在噬菌体上的金属酶变体可以根据金属亲和力进行选择；此类方法将有助于优化金属离子生物传感器。
