3DV7

Role of Hydrophilic Residues in Proton Transfer During Catalysis by Human Carbonic Anhydrase II (N62A)

3DV7 の概要

• エントリーDOI: 10.2210/pdb3dv7/pdb
• 関連するPDBエントリー: 3DVB 3DVC 3DVD
• 分子名称: Carbonic anhydrase 2, ZINC ION (3 entities in total)
• 機能のキーワード: hca ii, acetylation, cytoplasm, disease mutation, lyase, metal-binding, polymorphism, zinc
• 由来する生物種: Homo sapiens (human)
• 細胞内の位置: Cytoplasm: P00918
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 29180.25

構造登録者

Avvaru, B.S. (登録日: 2008-07-18, 公開日: 2008-11-11, 最終更新日: 2024-02-21)

主引用文献

Zheng, J.,Avvaru, B.S.,Tu, C.,McKenna, R.,Silverman, D.N.
Role of hydrophilic residues in proton transfer during catalysis by human carbonic anhydrase II.
Biochemistry, 47:12028-12036, 2008

PubMed Abstract: Catalysis by the zinc metalloenzyme human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer between His64 and the zinc-bound solvent molecule. Asn62 extends into the active site cavity of HCA II adjacent to His64 and has been shown to be one of several hydrophilic residues participating in a hydrogen-bonded solvent network within the active site. We compared several site-specific mutants of HCA II with replacements at position 62 (Ala, Val, Leu, Thr, and Asp). The efficiency of catalysis in the hydration of CO 2 for the resulting mutants has been characterized by (18)O exchange, and the structures of the mutants have been determined by X-ray crystallography to 1.5-1.7 A resolution. Each of these mutants maintained the ordered water structure observed by X-ray crystallography in the active site cavity of wild-type HCA II; hence, this water structure was not a variable in comparing with wild type the activities of mutants at residue 62. Crystal structures of wild-type and N62T HCA II showed both an inward and outward orientation of the side chain of His64; however, other mutants in this study showed predominantly inward (N62A, N62V, N62L) or predominantly outward (N62D) orientations of His64. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer between the active site and solution. The site-specific mutant N62D had a mainly outward orientation of His64, yet the difference in p K a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II was 5% that of wild type, showing that His64 mainly in the outward orientation is associated with inefficient proton transfer compared with His64 in wild type which shows both inward and outward orientations. These results emphasize the roles of the residues of the hydrophilic side of the active site cavity in maintaining efficient catalysis by carbonic anhydrase.

PubMed: 18942852
DOI: 10.1021/bi801473w

実験手法

X-RAY DIFFRACTION (1.7 Å)