Alexander R S, Nair S K, Christianson D W
Department of Chemistry, University of Pennsylvania, Philadelphia 19104-6323.
Biochemistry. 1991 Nov 19;30(46):11064-72. doi: 10.1021/bi00110a008.
Wild-type and mutant human carbonic anhydrases II, where mutations have been made in the hydrophobic pocket of the active site, have been studied by X-ray crystallographic methods. Specifically, mutations at Val-143 (the base of the pocket) lead to significant changes in catalytic activity and protein structure. The obliteration of a well-defined pocket in the Val-143----Phe and Val-143----Tyr mutants results in significantly diminished enzyme activity [(5 x 10(4))-fold and (3 x 10(5))-fold, respectively]; however, the activity of the Val-143----His mutant is diminished less (10(2)-fold), and deepening the pocket in the Val-143----Gly mutant results in only a 2-fold decrease in activity [Fierke et al., 1991 (preceding paper in this issue)]. These results indicate that the hydrophobic pocket is important for substrate association with the enzyme, but there are probably several catalytically acceptable substrate trajectories through this region of the enzyme structure. Additionally, each mutant protein exhibits long-range (ca. 10-15 A) compensatory structural changes which accommodate the Val-143 substitution. As such, the genetic-structural approach represented in this work serves as a three-dimensional paradigm for the redesign of specificity pockets in other protein catalysts.
利用X射线晶体学方法对野生型和突变型人碳酸酐酶II进行了研究，这些突变发生在活性位点的疏水口袋中。具体而言，位于口袋底部的缬氨酸-143发生突变会导致催化活性和蛋白质结构发生显著变化。缬氨酸-143突变为苯丙氨酸和缬氨酸-143突变为酪氨酸的突变体中，原本明确的口袋消失，导致酶活性显著降低（分别降低了(5×10⁴)倍和(3×10⁵)倍）；然而，缬氨酸-143突变为组氨酸的突变体活性降低较少（降低了10²倍），而缬氨酸-143突变为甘氨酸的突变体中口袋加深，仅导致活性降低2倍[菲克等人，1991年（本期前文）]。这些结果表明，疏水口袋对于底物与酶的结合很重要，但在酶结构的这一区域可能存在几种催化上可接受的底物轨迹。此外，每个突变蛋白都表现出长程（约10 - 15埃）的补偿性结构变化，以适应缬氨酸-143的取代。因此，这项工作中所代表的基因 - 结构方法为重新设计其他蛋白质催化剂中的特异性口袋提供了一个三维范例。
