Biochemistry 2010, 49, 249–251 249

DOI: 10.1021/bi902007b

A Short, Strong Hydrogen Bond in the Active Site of Human Carbonic Anhydrase II†,‡
Balendu Sankara Avvaru,§ Chae Un Kim,^ Katherine H. Sippel,§ Sol M. Gruner,^,@ Mavis Agbandje-McKenna,§

)
David N. Silverman,*,§, and Robert McKenna*,§

)
§
Department of Biochemistry and Molecular Biology, Department of Pharmacology and Therapeutics, College of Medicine, University
of Florida, Gainesville, Florida 32610, ^Cornell High Energy Synchrotron Source (CHESS), and @Physics Department,
Cornell University, Ithaca, New York 14853
Received November 23, 2009; Revised Manuscript Received December 9, 2009

ABSTRACT: The crystal structure of human carbonic anhy- The active site cavity of HCA II is conical and 15 Å deep,
drase II (HCA II) obtained at 0.9 Å resolution reveals that having one side lined with predominantly hydrophobic residues
a water molecule, termed deep water, Dw, and bound in a and the other lined with hydrophilic residues. At the bottom of
hydrophobic pocket of the active site forms a short, strong the cavity is a zinc ion coordinated in a tetrahedral geometry to
hydrogen bond with the zinc-bound solvent molecule, a three histidine residues (His94, -96, and -119) and a solvent ligand
conclusion based on the observed oxygen-oxygen distance (Figure 1). A wide body of spectroscopic and kinetic data are
of 2.45 Å. This water structure has similarities with consistent with a pKa near 7 describing the protolysis of the
hydrated hydroxide found in crystals of certain inorganic aqueous ligand of the metal forming zinc-bound hydroxide (1, 2).
complexes. The energy required to displace Dw contributes The mechanism of catalysis comprises nucleophilic attack of the
in significant part to the weak binding of CO2 in the zinc-bound hydroxide on CO2, followed by transfer of a proton
enzyme-substrate complex, a weak binding that enhances from zinc-bound water to solution to regenerate the active form
kcat for the conversion of CO2 into bicarbonate. In addi- (Figure 2). A network of apparently hydrogen bonded water
tion, this short, strong hydrogen bond is expected to molecules is observed in crystal structures extending from the
contribute to the low pKa of the zinc-bound water and to zinc-bound solvent to the inwardly oriented proton shuttle
promote proton transfer in catalysis. residue His64 located ∼8 Å from the metal (4, 5). This structure
of ordered water molecules is likely closely related to viable
The hydration of CO2 to produce bicarbonate and a proton is pathways of proton transfer during catalysis (7, 8).
catalyzed by the carbonic anhydrases (CAs) and plays a significant The final refined 0.9 Å resolution model, 258 residues and
role in a number of physiological processes, including respiration, 486 water molecules, was refined to an Rcryst of 12.5% and an
fluid secretion, and pH control. There are 14 human gene products Rfree of 13.1%. A full description of the structure determination
classified as CAs, including HCA II which is widespread in tissues and data collection, and refinement statistics is given in Table S1
and heavily concentrated in red cells. The most efficient of these (Supporting Information).
enzymes, including HCA II, proceed near diffusion control with a Of particular interest for this report is the structure of the
kcat/Km for hydration of 108 M-1 s-1 (1, 2). apparently hydrogen bonded solvent water network that includes
Our understanding of the steps in this catalysis is based in the zinc-bound solvent. This network emanates from the deep
significant part on the structure of the active site revealed by water (Dw) in the hydrophobic pocket formed in part by the side
X-ray crystallography studies. The first HCA II structures were chains of Val121, Val143, Trp209, and Leu198 to the water
determined to 2.0 Å resolution and identified the key features of molecules labeled W1, W2, W3a, W3b, and W4 shown in
the enzyme mechanism (3), whereas subsequent structures ob- Figures 1, 2, and 3. In crystal structures, this chain extends to
tained between 2.3 and 1.1 Å resolution have focused on a but is not in hydrogen bond contact with the proton shuttle
detailed understanding of the geometry about the zinc, orienta- residue His64. The zinc-bound solvent appears to form a hydro-
tions of the proton shuttle residue His64, and solvation of gen bond with the side chain of Thr199, and the deep water
residues of the active site (4-6). Recent structural analysis of molecule (Dw) appears to participate in hydrogen bonds with the
HCA II at 0.9 Å resolution reported here allows enhanced backbone amide of Thr199 and with the zinc-bound water
interpretation with application to understanding the catalytic molecule. The mechanism of the proton transfer utilizing path-
mechanism, in particular additional understanding of the role of ways such as this has been the subject of considerable investiga-
solvent. tion (2, 7-12).
The current high-resolution structure provides a clearer view
†
This work was funded by grants from the National Institutes of of the solvation at the active site (Figure 1). The hydrogen bonds
Health (NIH) (Grant GM25154 D.N.S. and R.M.) and the Thomas in this water network have distances typical of solvent water, with
Maren Foundation (R.M.), the MacCHESS grant (NIH Grant
RR001646), U.S. Department of Energy Grant DE-FG02- O-O distances near 2.7-2.9 Å. A more detailed picture of
97ER62443, and CHESS, which is supported by the National Science distances and bond angles involving active site solvent is provided
Foundation (NSF) and the National Institute of General Medical in Figures S1 and S2 (Supporting Information). However, there is
Sciences through NSF Grant DMR-0225180.
‡
Coordinates are deposited in the Protein Data Bank with accession a short hydrogen bond with an O-O distance estimated to be
code 3KS3. 2.45 ( 0.03 Å between Dw and the zinc-bound solvent (Figure 3).
*To whom correspondence should be addressed. D.N.S.: e-mail, The crystallographic occupancy is near 100% for Dw, and both
silvrmn@ufl.edu; telephone, (352) 392-3556; fax, (352) 392-9696.
R.M.: e-mail, rmckenna@ufl.edu; telephone, (352) 392-5695; fax, this water molecule and the zinc-bound solvent have B factors
(352) 392-3422. that are low (near 10 Å2) and close in value to the B factors of the

r 2009 American Chemical Society Published on Web 12/09/2009 pubs.acs.org/Biochemistry

250 Biochemistry, Vol. 49, No. 2, 2010 Avvaru et al.

FIGURE 1: Stereoview of the active site of HCA II. The zinc is

represented by a gray sphere and the oxygen atoms of water
molecules as smaller red spheres. Dotted lines are presumed hydrogen
bonds. Stick figures are selected amino acids of the active site with
both the inward and outward orientations of His64 shown. The
electron density 2Fo - Fc Fourier map is contoured at 2.0σ. This
figure was created using PyMOL (28).

FIGURE 3: Ordered water network in the active site of HCA II. The
zinc is represented by a gray sphere, and the oxygen atoms of water
molecules are represented as smaller red spheres. Dotted lines are
presumed hydrogen bonds with heavy atom distances given. Stick
figures are selected amino acids of the active site with both the inward
and outward orientations of His64 shown. This figure was created
using PyMOL (28).

Weak hydrogen bonds typical of water molecules in solution

have a favorable enthalpy of formation near 5 kcal/mol; however,
LBHBs can have such enthalpies near 15-25 kcal/mol (13). This
is significant with respect to the catalysis by HCA II since the
binding of CO2 to its catalytically productive binding site
FIGURE 2: Catalytic mechanism: (A) the active site without bound displaces the deep water molecule (Dw) (Figure 2) (16, 17) and
CO2 showing the deep water, (B) the active site showing bound CO2
occupying the site of the deep water, and (C) the binding of thus requires the cleavage of the LBHB contributing to the very
bicarbonate at the active site of T200H HCA II (23). Surface and weak binding of CO2 at this site. A dissociation constant for CO2
stick representation: water molecules depicted as red spheres, the at its catalytic site in HCA II has been estimated to be 100 mM
hydrophobic residues depicted as green shaded regions, and hydro- measured by infrared spectroscopy (18, 19).
philic residues depicted as yellow regions. This figure was created
using PyMOL (28). A tight binding of substrate at the reactive site is a disadvan-
tage for catalysis by HCA II; it adversely affects its physiological
surrounding amino acids. Analysis of the anisotropic thermal function which requires it to enhance catalysis for a maximum
motions of Dw indicates that the bulk of its movement is velocity kcat of 106 s-1 and near diffusion-controlled second-
perpendicular to the hydrogen bond with the zinc-bound solvent order rate constants for hydration. In arguments elucidated by
(Table of Contents Graphic). Fersht (20), the tight binding of substrate (without affecting the
Under specific and well-described conditions, short hydrogen transition state) lowers the energy level of the substrate-enzyme
bonds involving water with O-O distances of ∼2.4 Å have been complex, thereby increasing the activation energy of kcat. When a
observed (13). These are designated low-barrier hydrogen bonds thermodynamic well or pit that accumulates tightly bound
(LBHBs) reflecting the low barrier for hydrogen movement substrate exists, the rate of catalysis is decreased. For an enzyme
between the heteroatoms. Such LBHBs are usually observed in that requires rapid catalysis like carbonic anhydrase, it is
nonprotic solvents and involve closely matched values of pKa for advantageous for substrate binding to be weak and the active
the heteroatoms of the hydrogen bond (13). site to remain largely unbound at physiological levels of substrate
The Dw is bound in a hydrophobic pocket of the active site. CO2. The concentration of CO2 in plasma, for example, is near 1
Moreover, with a solution pKa near 7.0, the zinc-bound solvent in mM; the value of Km for hydration is 10 mM, and the estimated
the crystal structure is probably in large part zinc-bound hydroxide dissociation constant of CO2 is 100 mM. It appears that HCA II
under the conditions of crystallization (pH 7.0) (14). This structure evolved weak substrate binding by having it displace the Dw
has similarities with the identification by crystallography of the which participates in a LBHB.
LBHB of the hydrated hydroxide anion HOHOH- formed in There is likely another role for the LBHB as it may contribute
the hydrophobic region between sheets of phenyl rings in tri- to the low pKa near 7 for the zinc-bound water molecule, the
methylammonium salts of tris(thiobenzohydroximato)chromate- protolysis of which is enhanced using the energy of formation of
(III) (15). In this case, the O-O atom distance is 2.3 Å in a the LBHB. In this respect, the role of the LBHB is analogous with
structure the authors describe as a central proton surrounded by the catalytic mechanism of liver alcohol dehydrogenase in which
two OH- groups. This is probably a good model for the observed removal of a proton from the Zn-coordinated alcohol is pro-
LBHB in HCA II, in which the deep water is in a hydrophobic moted by formation of a LBHB with Ser48 in the reactant
environment and likely involves the zinc-bound hydroxide. state (21). The alkoxide then undergoes hydride transfer to
Rapid Report Biochemistry, Vol. 49, No. 2, 2010 251
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