The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO 2 -pressurized, cryo-cooled crystals to capture the first step of CO 2 hydration catalyzed by the zincmetalloenzyme human carbonic anhydrase II, the binding of substrate CO 2 , for both the holo and the apo (without zinc) enzyme to 1.1 Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO 2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675-3679). These structures provide insight into the long hypothesized binding of CO 2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO 2 . This method may also have a much broader implication for the study of other enzymes for which CO 2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.Since their discovery (2), the carbonic anhydrases (CAs) 3 have been extensively studied because of their important physiological functions in all kingdoms of life (3). This family of enzymes is broadly comprised of three well studied, structurally distinct families (␣, ␤, and ␥) of mostly zinc-metalloenzymes that catalyze the reversible hydration of CO 2 to bicarbonate (3, 4). More recently there have been other more distinct CAs characterized, such as a cadmium ␤ class-mimic CA (5). However, all appear to share the same overall catalytic mechanism composed of two independent stages, shown in Equations 1 and 2, an example of a ping-pong mechanism (6, 7). In the hydration direction, the first stage is the conversion of CO 2 into bicarbonate via a nucleophilic attack on CO 2 by the reactive zinc-bound hydroxide. The resultant bicarbonate is then displaced from the zinc by a water molecule (Reaction 1).The second stage is the transfer of a proton from the zincbound water to bulk solvent to regenerate the zinc-bound hydroxide (Reaction 2). Here B is a proton acceptor in solution or a residue of the enzyme itself.For hCAII (␣ class CA), this reaction is facilitated by a solvent molecule with a pK a near 7 that is directly coordinated to the zinc (6). This centrally located zinc exhibits a tetrahedral configuration with three histidines (His-94, His-96, and His-119) and either a water or a hydroxide molecule. The active site cavity can be loosely described as being conical in shape having a 15 Å diameter entrance that tapers into the center of the enzyme. The cavity is partitioned into two very different environments. On one side of the zinc, deep within the active site, lies a cluster of hydrophobic amino acids (namely , whereas on the other side of the zinc, leading out of the active site to the bulk solvent, the surface is lined with hydrophilic amino acids (namely Tyr-7, Asn-62...

4-Substituted-ureido benzenesulfonamides showing inhibitory activity against carbonic anhydrase (CA, EC 4.2.1.1) II between 3.3-226 nM were crystallized in complex with the enzyme. Hydrophobic interactions between the scaffold of the inhibitors in different hydrophobic pockets of the enzyme were observed, explaining the diverse inhibitory range of these derivatives.

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.

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Recently a convincing body of evidence has accumulated, suggesting that the over-expression of carbonic anhydrase isozyme IX (CA IX) in some cancers contributes to the acidification of the extracellular matrix, which in turn promotes the growth and metastasis of the tumor. These observations have made CA IX an attractive drug target for the selective treatment of certain cancers. Currently there is no available x-ray crystal structure of CA IX and this has hampered the rational design of selective CA IX inhibitors. In light of these observations and based on structural alignment homology, using the crystal structure CA II and the sequence of CA IX, a double mutant of CA II with Ala 65 replaced by Ser and Asn 67 replace by Gln has been constructed to resemble the active site of CA IX. This CA IX mimic has been characterized kinetically using 18 O-exchange and structurally using x-ray crystallography, alone and in complex with five CA sulfonamide based inhibitors; acetazolamide, benzolamide, chlorzolamide, ethoxzolamide, and methazolamide, and compared to CA II. This structural information has been evaluated in relationship to inhibition studies and in vitro cytotoxicity assays and shows a correlated structure-activity relationship. Kinetic and structural studies of CA II and CA IX mimic reveal chlorzolamide to be a more potent inhibitor of CA IX inducing an active site conformational change upon binding. Additionally, chlorzolamide appears to be cytotoxic to prostate cancer cells. This preliminary study demonstrates that the CA IX mimic may provide a useful model to design more isozyme specific CA IX inhibitors which may lead to development of new therapeutic treatments of some cancers.Carbonic anhydrases (CAs) 1 are zinc-metalloenzymes that catalyze the reversible interconversion of CO 2 and HCO 3 -(1). Since their discovery, the CAs have been extensively studied due to their important physiological functions in all kingdoms of life. This family of enzymes † This work was supported by a grant (GM25154 to D.N.S. and R.M.) from the National Institutes of Health and the Maren Foundation (to R.M.). ‡ Coordinates and structure factors have been deposited in the Protein Data Bank as 3DC9.pdb and 3DC9.sf, 3DCS.pdb and 3DCS.sf, 3DCC.pdb and 3DCC.sf, 3DC3.pdb and 3DC3.sf, 3DCW.pdb and 3DCW.sf, 3DBU.pdb and 3DBU.sf, 3DAZ.pdb and 3DAZ.sf, 3D9Z.pdb and 3D9Z.sf, 3DD0.pdb and 3DD0.sf, and 3D8W.pdb and 3D8W.sf.