Atomic Crystal and Molecular Dynamics Simulation Structures of Human Carbonic Anhydrase II: Insights into the Proton Transfer Mechanism

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• 作者：S. Zoë ; Fisher ; C. Mark Maupin ; Monika Budayova-Spano ; Lakshmanan Govindasamy ; Chingkuang Tu ; Mavis Agbandje-McKenna ; David N. Silverman ; Gregory A. Voth ; Robert McKenna
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：March 20, 2007
• 年：2007
• 卷：46
• 期：11
• 页码：2930 - 2937
• 全文大小：324K
• 年卷期：v.46,no.11(March 20, 2007)
• ISSN：1520-4995

文摘

Human carbonic anhydrase II (HCA II) is a zinc-metalloenzyme that catalyzes the reversibleinterconversion of CO₂ and HCO₃^-. The rate-limiting step of this catalysis is the transfer of a protonbetween the Zn-bound solvent molecule and residue His64. In order to fully characterize the active sitestructural features implicated in the proton transfer mechanism, the refined X-ray crystal structure ofuncomplexed wild type HCA II to 1.05 Å resolution with an R_cryst value of 12.0% and an R_free value of15.1% has been elucidated. This structure provides strong clues as to the pathway of the intramolecularproton transfer between the Zn-bound solvent and His64. The structure emphasizes the role of the solventnetwork, the unique positioning of solvent molecule W2, and the significance of the dual conformationof His64 in the active site. The structure is compared with molecular dynamics (MD) simulation calculationsof the Zn-bound hydroxyl/His64⁺ (charged) and the Zn-bound water/His64 (uncharged) HCA II states. Acomparison of the crystallographic anisotropic atomic thermal parameters and MD simulation root-mean-square fluctuation values show excellent agreement in the atomic motion observed between the two methods.It is also interesting that the observed active site solvent positions in the crystal structure are also themost probable positions of the solvent during the MD simulations. On the basis of the comparative studyof the MD simulation results, the HCA II crystal structure observed is most likely in the Zn-bound water/His64 state. This conclusion is based on the following observations: His64 is mainly (80%) orientated inan inward conformation; electron density omit maps infer that His64 is not charged in an either inwardor outward conformation; and the Zn-bound solvent is most likely a water molecule.