Mutation of active site residues Asn67 to Ile, Gln92 to Val and Leu204 to Ser in human carbonic anhydrase II: Influences on the catalytic activity and affinity for inhibitors

详细信息	查看全文 | 推荐本文 |

• 作者：Sumeyye Turkoglu^a ; Alfonso Maresca^b ; Meltem Alper^a ; Feray Kockar^a ; Semra I艧谋k^b ; ^c ; Selma Sinan^a ; Ozen Ozensoy^b ; ^d ; Oktay Arslan^c ; Claudiu T. Supuran^b ; ^{claudiu.supuran@unifi.it}
• 关键词：Carbonic anhydrase ; Site directed mutagenesis ; Active site ; Sulfonamide ; Sulfamate ; Enzyme inhibitor ; Inhibition mechanism
• 刊名：Bioorganic and Medicinal Chemistry
• 出版年：2012
• 期刊代码：9_09680896
• 类别：ch
• 出版时间：1 April, 2012
• 卷：20
• 期：7
• 页码：2208-2213
• 文件大小：336 K

摘要

Site-directed mutagenesis has been used to change three amino acid residues involved in the binding of inhibitors (Asn67Ile; Gln92Val and Leu204Ser) within the active site of human carbonic anhydrase (CA, EC 4.2.1.1) II (hCA II). Residues 67, 92 and 204 were changed from hydrophobic to hydrophilic ones, and vice versa. The Asn67Ile and Leu204Ser mutants showed similar k_cat/K_M values compared to the wild type (wt) enzyme, whereas the Gln92Val mutant was around 30%less active as a catalyst for CO₂ hydration to bicarbonate compared to the wt protein. Affinity for sulfonamides/sulfamates was decreased in all three mutants compared to wt hCA II. The effect was stronger for the Asn67Ile mutant (the closest residue to the zinc ion), followed by the Gln92Val mutant (residue situated in the middle of the active site) and weakest for the Leu204Ser mutant, an amino acid situated far away from the catalytic metal ion, at the entrance of the cavity. This study shows that small perturbations within the active site architecture have influences on the catalytic efficiency but dramatically change affinity for inhibitors among the CA enzymes, especially when the mutated amino acid residues are nearby the catalytic metal ion.