Strong Hydrogen Bond in the Active Site of Human Carbonic Anhydrase II † , ‡

The crystal structure of human carbonic anhydrase II (HCA II) obtained at 0.9 Å resolution reveals that a water molecule, termed deep water, Dw, and bound in a hydrophobic pocket of the active site forms a short, strong hydrogen bond with the zinc-bound solvent molecule, a conclusion based on the observed oxygen-oxygen distance of 2.45 Å. This water structure has similarities with hydrated hydroxide found in crystals of certain inorganic complexes. The energy required to displace Dw contributes in significant part to the weak binding of CO2 in the enzyme-substrate complex, a weak binding that enhances kcat for the conversion of CO2 into bicarbonate. In addition, this short, strong hydrogen bond is expected to contribute to the low pKa of the zinc-bound water and to promote proton transfer in catalysis. The hydration of CO2 to produce bicarbonate and a proton is catalyzed by the carbonic anhydrases (CAs) and plays a significant role in a number of physiological processes, including respiration, fluid secretion, and pHcontrol. There are 14 human gene products classified as CAs, including HCA II which is widespread in tissues and heavily concentrated in red cells. The most efficient of these enzymes, including HCA II, proceed near diffusion control with a kcat/Km for hydration of 10 8 M s (1, 2). Our understanding of the steps in this catalysis is based in significant part on the structure of the active site revealed by X-ray crystallography studies. The first HCA II structures were determined to 2.0 Å resolution and identified the key features of the enzyme mechanism (3), whereas subsequent structures obtained between 2.3 and 1.1 Å resolution have focused on a detailed understanding of the geometry about the zinc, orientations of the proton shuttle residue His64, and solvation of residues of the active site (4-6). Recent structural analysis of HCA II at 0.9 Å resolution reported here allows enhanced interpretation with application to understanding the catalytic mechanism, in particular additional understanding of the role of solvent. The active site cavity of HCA II is conical and 15 Å deep, having one side lined with predominantly hydrophobic residues and the other lined with hydrophilic residues. At the bottom of the cavity is a zinc ion coordinated in a tetrahedral geometry to three histidine residues (His94, -96, and -119) and a solvent ligand (Figure 1). A wide body of spectroscopic and kinetic data are consistent with a pKa near 7 describing the protolysis of the aqueous ligand of themetal forming zinc-bound hydroxide (1, 2). The mechanism of catalysis comprises nucleophilic attack of the zinc-bound hydroxide on CO2, followed by transfer of a proton from zinc-bound water to solution to regenerate the active form (Figure 2). A network of apparently hydrogen bonded water molecules is observed in crystal structures extending from the zinc-bound solvent to the inwardly oriented proton shuttle residue His64 located ∼8 Å from the metal (4, 5). This structure of ordered water molecules is likely closely related to viable pathways of proton transfer during catalysis (7, 8). The final refined 0.9 Å resolution model, 258 residues and 486 water molecules, was refined to an Rcryst of 12.5% and an Rfree of 13.1%. A full description of the structure determination and data collection, and refinement statistics is given in Table S1 (Supporting Information). Of particular interest for this report is the structure of the apparently hydrogen bonded solvent water network that includes the zinc-bound solvent. This network emanates from the deep water (Dw) in the hydrophobic pocket formed in part by the side chains of Val121, Val143, Trp209, and Leu198 to the water molecules labeled W1, W2, W3a, W3b, and W4 shown in Figures 1, 2, and 3. In crystal structures, this chain extends to but is not in hydrogen bond contact with the proton shuttle residue His64. The zinc-bound solvent appears to form a hydrogen bond with the side chain of Thr199, and the deep water molecule (Dw) appears to participate in hydrogen bonds with the backbone amide of Thr199 and with the zinc-bound water molecule. The mechanism of the proton transfer utilizing pathways such as this has been the subject of considerable investigation (2, 7-12). The current high-resolution structure provides a clearer view of the solvation at the active site (Figure 1). The hydrogen bonds in this water network have distances typical of solvent water, with O-O distances near 2.7-2.9 Å. A more detailed picture of distances and bond angles involving active site solvent is provided inFigures S1 and S2 (Supporting Information). However, there is a short hydrogen bond with an O-O distance estimated to be 2.45( 0.03 Å betweenDwand the zinc-bound solvent (Figure 3). The crystallographic occupancy is near 100% for Dw, and both this water molecule and the zinc-bound solvent have B factors that are low (near 10 Å) and close in value to the B factors of the This work was funded by grants from the National Institutes of Health (NIH) (Grant GM25154 D.N.S. and R.M.) and the Thomas Maren Foundation (R.M.), the MacCHESS grant (NIH Grant RR001646), U.S. Department of Energy Grant DE-FG0297ER62443, and CHESS, which is supported by the National Science Foundation (NSF) and the National Institute of General Medical Sciences through NSF Grant DMR-0225180. Coordinates are deposited in the Protein Data Bank with accession code 3KS3. *To whom correspondence should be addressed. D.N. S. : e-mail, silvrmn@ufl.edu; telephone, (352) 392-3556; fax, (352) 392-9696. R.M. : e-mail, rmckenna@ufl.edu; telephone, (352) 392-5695; fax, (352) 392-3422. 250 Biochemistry, Vol. 49, No. 2, 2010 Avvaru et al. surrounding amino acids. Analysis of the anisotropic thermal motions of Dw indicates that the bulk of its movement is perpendicular to the hydrogen bond with the zinc-bound solvent (Table of