Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, United States.
Biochemistry. 2012 Nov 13;51(45):9156-63. doi: 10.1021/bi301155z. Epub 2012 Nov 2.
This work examines the effect of perturbing the position of bound CO(2) in the active site of human carbonic anhydrase II (HCA II) on catalysis. Variants of HCA II in which Val143 was replaced with hydrophobic residues Ile, Leu, and Ala were examined. The efficiency of catalysis in the hydration of CO(2) for these variants was characterized by (18)O exchange mass spectrometry, and their structures were determined by X-ray crystallography at 1.7-1.5 Å resolution. The most hydrophobic substitutions, V143I and V143L, showed decreases in the level of catalysis, as much as 20-fold, while the replacement by the smaller V143A mutation showed an only moderate 2-fold decrease in activity. Structural data for all three variants show no significant change in the overall position of amino acid side chains in the active site compared with the wild type. However, V143A HCA II showed additional ordered water molecules in the active site compared to the number for the wild type. To further investigate the decrease in the catalytic efficiency of V143I HCA II, an X-ray crystallographic CO(2) entrapment experiment was performed to 0.93 Å resolution. This structure revealed an unexpected shift in the CO(2) substrate toward the zinc-bound solvent, placing it ~0.3 Ǻ closer than previously observed in the wild type in conjunction with the observed dual occupancy of the product bicarbonate, presumably formed during the acquisition of data. These data suggest that the Ile substitution at position 143 reduced the catalytic efficiency, which is likely due to steric crowding resulting in destabilization of the transition state for conversion of CO(2) into bicarbonate and a decreased product dissociation rate.
这项工作研究了扰动人碳酸酐酶 II（HCA II）活性部位结合的 CO2 位置对催化的影响。研究了用疏水性残基异亮氨酸、亮氨酸和丙氨酸取代 Val143 的 HCA II 变体。通过（18）O 交换质谱法对这些变体催化 CO2 水合的效率进行了表征，并通过 X 射线晶体学在 1.7-1.5 Å分辨率下确定了它们的结构。疏水性最大的取代物 V143I 和 V143L 的催化水平下降了 20 倍，而 V143A 突变的取代物活性仅下降了 2 倍。所有三种变体的结构数据表明，与野生型相比，活性部位氨基酸侧链的整体位置没有明显变化。然而，与野生型相比，V143A HCA II 在活性部位显示出更多的有序水分子。为了进一步研究 V143I HCA II 催化效率的降低，进行了 X 射线晶体学 CO2 捕获实验，分辨率达到 0.93 Å。该结构揭示了 CO2 底物向锌结合溶剂的意外移动，使其比野生型中以前观察到的距离近 0.3 Å，同时观察到产物碳酸氢盐的双重占据，推测是在数据获取过程中形成的。这些数据表明，143 位的异亮氨酸取代降低了催化效率，这可能是由于空间位阻导致 CO2 转化为碳酸氢盐的过渡态不稳定，以及产物解离速率降低。
