Kiefer L L, Krebs J F, Paterno S A, Fierke C A
Biochemistry Department, Duke University Medical Center, Durham, North Carolina 27710.
Biochemistry. 1993 Sep 28;32(38):9896-900. doi: 10.1021/bi00089a004.
Substitution of cysteine for threonine-199, the amino acid which hydrogen bonds with zinc-bound hydroxide in wild-type carbonic anhydrase II (CAII), leads to the formation of a new His3Cys zinc coordination polyhedron. The optical absorption spectrum of the Co(2+)-substituted threonine-199-->cysteine (T199C) variant and the three-dimensional structure [Ippolito, J. A., & Christianson, D. W. (1993) Biochemistry (following paper in this issue)] indicate that the new thiolate side chain coordinates to the metal ion, displacing the metal-bound solvent molecule. The engineered thiolate ligand increases zinc binding (4-fold) and decreases catalytic activity substantially (approximately 10(3)-fold) but not completely. However, this residual activity is due to an active species containing a zinc-bound solvent ligand with the cysteine-199 side chain occupying an alternate conformation. The equilibrium between these conformers reflects the energetic balance between the formation of the zinc-thiolate bond and structural rearrangements in the Ser-197-->Cys-206 loop necessary to achieve this metal coordination. This designed His3Cys metal polyhedron may mimic the zinc binding site in the matrix metalloproteinase prostromelysin.
将野生型碳酸酐酶II（CAII）中与锌结合的氢氧化物形成氢键的苏氨酸-199替换为半胱氨酸，会导致形成一种新的His3Cys锌配位多面体。Co(2+)取代的苏氨酸-199→半胱氨酸（T199C）变体的光吸收光谱以及三维结构[Ippolito, J. A., & Christianson, D. W. (1993) Biochemistry（本期后续论文）]表明，新的硫醇盐侧链与金属离子配位，取代了与金属结合的溶剂分子。工程化的硫醇盐配体增加了锌的结合（4倍），并大幅降低了催化活性（约10(3)倍），但并未完全消除。然而，这种残余活性是由于一种活性物种，其中含有一个与锌结合的溶剂配体，半胱氨酸-199侧链处于另一种构象。这些构象异构体之间的平衡反映了锌-硫醇盐键形成与Ser-197→Cys-206环中实现这种金属配位所需的结构重排之间的能量平衡。这种设计的His3Cys金属多面体可能模拟了基质金属蛋白酶原溶基质素中的锌结合位点。
