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

Abstract

Human carbonic anhydrase II (HCA II) is a zinc-metalloenzyme that catalyzes the reversible interconversion of CO 2 and HCO 3 -. The rate-limiting step of this catalysis is the transfer of a proton between the Zn-bound solvent molecule and residue His64. In order to fully characterize the active site structural features implicated in the proton transfer mechanism, the refined X-ray crystal structure of uncomplexed wild type HCA II to 1.05 Å resolution with an R cryst value of 12.0% and an R free value of 15.1% has been elucidated. This structure provides strong clues as to the pathway of the intramolecular proton transfer between the Zn-bound solvent and His64. The structure emphasizes the role of the solvent network, the unique positioning of solvent molecule W2, and the significance of the dual conformation of His64 in the active site. The structure is compared with molecular dynamics (MD) simulation calculations of the Zn-bound hydroxyl/His64 + (charged) and the Zn-bound water/His64 (uncharged) HCA II states. A comparison of the crystallographic anisotropic atomic thermal parameters and MD simulation root-meansquare 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 the most probable positions of the solvent during the MD simulations. On the basis of the comparative study of 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 in an inward conformation; electron density omit maps infer that His64 is not charged in an either inward or outward conformation; and the Zn-bound solvent is most likely a water molecule.

Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

“…In addition to the electrostatic and related pK a changes that occurred also produced a shorter, unbranched chain of hydrogen-bonded waters that connect ZW to the proton shuttling residues His64. These electrostatic changes and unbranched water network boost the proton transfer activity of HCA II Tyr7Phe mutants (Fisher et al, 2007b). As reported in Table III, the values of k B for TS2 and TS4 are even better than for Tyr7Phe alone at 5.6 and 4.9, respectively.…”

“…Data processing and reduction were done with either d*TREK or the HKL2000 suite of programs (Otwinowski and Minor, 1997;Pflugrath, 1999). The starting model was derived from protein data bank (PDB) accession number 2ili with all the waters and Zn removed, and with the mutated residues changed to Gly (Fisher et al, 2007b). All the structures were refined using PHENIX and manual inspection and model building was done using Coot (Emsley and Cowtan, 2004;Adams et al, 2010).…”

Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II

“…Many previous structural studies of hCAII have shown that His-64 occupies two conformations, termed "in" and "out." Generally it has been seen that the "in" conformation is favored, although there may be pH effects on the ratio of "in" to "out" (10,13). In both the holoCO 2 -bound and the apoCO 2 -bound structures, His-64 was seen to have a preference for the "out" conformation.…”

“…10-l drops of equal amounts of protein and precipitant were equilibrated against precipitant solution (1.3 M sodium citrate, 100 mM Tris-HCl, pH 7.8) by vapor diffusion at room temperature (ϳ20°C) (13). Crystals grew to ϳ0.2 ϫ 0.2 ϫ 0.5 mm in size after ϳ5 days.…”

Characterization of Carbonic Anhydrase Isozyme Specific Inhibition by Sulfamated 2-Ethylestra Compounds

“…Structure Determination-All the crystal structures were determined by molecular replacement methods using wt CA II (PDB code 2ili) [29]. The initial rigid body, individual coordinate and atomic displacement parameter…”