Balendu Sankara Avvaru

Entrapment of carbon dioxide in the active site of carbonic anhydrase II

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 CO2-pressurized, cryo-cooled crystals to capture the first step of CO2 hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO2, for both the holo and the apo (without zinc) enzyme to 1.1Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO2 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 CO2 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 CO2. This method may also have a much broader implication for the study of other enzymes for which CO2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.

A short, 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 A 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 A. 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 CO(2) in the enzyme-substrate complex, a weak binding that enhances k(cat) for the conversion of CO(2) into bicarbonate. In addition, this short, strong hydrogen bond is expected to contribute to the low pK(a) of the zinc-bound water and to promote proton transfer in catalysis.

Sulfonamides incorporating 1,3,5-triazine moieties selectively and potently inhibit carbonic anhydrase transmembrane isoforms IX, XII and XIV over cytosolic isoforms I and II: Solution and X-ray crystallographic studies.

Reaction of cyanuryl chloride with d,l-amino acids and amino alcohols afforded a new series of triazinyl-substituted benzenesulfonamides incorporating amino acyl/hydroxyalkyl-amino moieties. Inhibition studies of physiologically relevant human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA I, II, IX, XII and XIV with these compounds are reported. They showed moderate-weak inhibition of the cytosolic, offtarget isozymes CA I and II, but many of them were low nanomolar inhibitors of the transmembrane, tumor-associated CA IX and XII (and also of CA XIV). The X-ray crystal structure of two of these compounds in adduct with CA II allowed us to understand the features associated with this strong inhibitory properties and possibly also their selectivity. Two of these compounds were also investigated for the inhibition of other human isoforms, that is, hCA IV, VA, VB, VI, VII and XIII, as well as inhibitors of the fungal pathogenic CAs Nce103 (Candida albicans) and Can2 (Cryptococcus neoformans), showing interesting activity. The 1,3,5-triazinyl-substituted benzenesulfonamides constitute thus a class of compounds with great potential for obtaining inhibitors targeting both α-class mammalian, tumor-associated, and β-class from pathogenic organisms CAs.

Carbonic anhydrase inhibitors. The X-ray crystal structure of human isoform II in adduct with an adamantyl analogue of acetazolamide resides in a less utilized binding pocket than most hydrophobic inhibitors.

We investigated the inhibitory activity of several 1,3,4-thiadiazole-sulfonamides against all catalytically active CA (EC 4.2.1.1), CA I-XV. The tail derivatizing the 5-position in the 1,3,4-thiadiazole-2-sulfonamide scaffold was observed to be critical as an inhibitory determinant of these compounds. The high resolution X-ray crystal structure of hCA II in complex with 5-(1-adamantylcarboxamido)-1,3,4-thiadiazole-2-sulfonamide, showed the adamantyl moiety of the inhibitor residing in a less utilized binding pocket than that of most hydrophobic inhibitors, lined by the amino acid residues Ile91, Val121 and Phe131. This binding site may explain the diverse inhibition profiles of 5-carboxamide- and sufonamide-derivatized 1,3,4-thiadiazole-2-sulfonamides and offers a hot spot for designing isoform selective inhibitors, considering that residues 91 and 131 are highly variable among the 13 catalytically active isoforms.

Coumarinyl-substituted sulfonamides strongly inhibit several human carbonic anhydrase isoforms: solution and crystallographic investigations

We investigated a series of coumarinyl-substituted aromatic sulfonamides as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms with medical applications, the cytosolic hCA I, and II, and the transmembrane, tumor-associated hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4-yl-acetamide-tails and benzenesulfonamide and benzene-1,3-disulfonamide scaffolds showed medium potency inhibition of hCA I (KIs of 73-131 nM), effective hCA II inhibition (KIs of 9.1-36 nM) and less effective hCA IX and XII inhibition (KIs of 55-128 nM). Only one compound, the derivatized 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide with the coumarinyl tail, showed effective inhibition of the transmembrane isoforms, with KIs of 5.9-14.2 nM, although it was less effective as hCA I and II inhibitor (KIs of 36-120 nM). An X-ray crystal structure of hCA II in complex with 4-(7-methoxy-coumarin-4-yl-acetamido)-benzenesulfonamide (KI of 9.1 nM against hCA II) showed the intact inhibitor coordinated to the zinc ion from the enzyme active site by the sulfonamide moiety, and participating in a edge-to-face stacking with Phe131, in addition to other hydrophobic and hydrophilic interactions with water molecules and amino acid residues from the active site. Thus, sulfonamides incorporating coumarin rings have a distinct inhibition mechanism compared to the coumarins, and may lead to compounds with interesting inhibition profiles against various alpha-CAs found in mammals or parasites, such as Plasmodium falciparum.

Design of a carbonic anhydrase IX active-site mimic to screen inhibitors for possible anticancer properties

Recently, a convincing body of evidence has accumulated suggesting that the overexpression 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 lack of availability has hampered the rational design of selective CA IX inhibitors. In light of these observations and on the basis of structural alignment homology, using the crystal structure of carbonic anhydrase II (CA II) and the sequence of CA IX, a double mutant of CA II with Ala65 replaced by Ser and Asn67 replaced 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 by both 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.

Water Networks in Fast Proton Transfer during Catalysis by Human Carbonic Anhydrase II.

Variants of human carbonic anhydrase II (HCA II) with amino acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and (18)O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for transfer of a proton from His64 to the zinc-bound hydroxide during catalysis were 4 and 9 μs(-1) for Y7F and Y7F/N67Q, respectively, compared with a value of 0.8 μs(-1) for wild-type HCA II. These higher values observed for Y7F and Y7F/N67Q HCA II could not be explained by differences in the values of the pK(a) of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by the orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with a reduced level of branching in the networks of hydrogen-bonded water molecules between proton shuttle residue His64 and the zinc-bound solvent molecule as observed in crystal structures at 1.5-1.6 Å resolution. Moreover, Y7F/N67Q HCA II is unique among the variants studied in having a direct, hydrogen-bonded chain of water molecules between the zinc-bound solvent and N(ε) of His64. This study provides the clearest example to date of the relevance of ordered water structure to rate constants for proton transfer in catalysis by carbonic anhydrase.

Role of hydrophilic residues in proton transfer during catalysis by human carbonic anhydrase II.

Catalysis by the zinc metalloenzyme human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer between His64 and the zinc-bound solvent molecule. Asn62 extends into the active site cavity of HCA II adjacent to His64 and has been shown to be one of several hydrophilic residues participating in a hydrogen-bonded solvent network within the active site. We compared several site-specific mutants of HCA II with replacements at position 62 (Ala, Val, Leu, Thr, and Asp). The efficiency of catalysis in the hydration of CO 2 for the resulting mutants has been characterized by (18)O exchange, and the structures of the mutants have been determined by X-ray crystallography to 1.5-1.7 A resolution. Each of these mutants maintained the ordered water structure observed by X-ray crystallography in the active site cavity of wild-type HCA II; hence, this water structure was not a variable in comparing with wild type the activities of mutants at residue 62. Crystal structures of wild-type and N62T HCA II showed both an inward and outward orientation of the side chain of His64; however, other mutants in this study showed predominantly inward (N62A, N62V, N62L) or predominantly outward (N62D) orientations of His64. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer between the active site and solution. The site-specific mutant N62D had a mainly outward orientation of His64, yet the difference in p K a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II was 5% that of wild type, showing that His64 mainly in the outward orientation is associated with inefficient proton transfer compared with His64 in wild type which shows both inward and outward orientations. These results emphasize the roles of the residues of the hydrophilic side of the active site cavity in maintaining efficient catalysis by carbonic anhydrase.

Kinetic and crystallographic studies of the role of tyrosine 7 in the active site of human carbonic anhydrase II

The rate limiting step in catalysis of bicarbonate dehydration by human carbonic anhydrase II (HCA II) is an intramolecular proton transfer from His64 to the zinc-bound hydroxide. We have examined the role of Tyr7 using site-specific mutagenesis and measuring catalysis by the ¹⁸O exchange method using membrane inlet mass spectrometry. The side chain of Tyr7 in HCA II extends into the active-site cavity about 7 Å from the catalytic zinc atom. Replacement of Tyr7 with eight other amino acids had no effect on the interconversion of bicarbonate and CO₂, but in some cases caused enhancements in the rate constant of proton transfer by nearly 10-fold. The variant Y7I HCA II enhanced intramolecular proton transfer approximately twofold; its structure was determined by X-ray crystallography at 1.5 Å resolution. No changes were observed in the ordered solvent structure in the active-site cavity or in the conformation of the side chain of the proton shuttle His64. However, the first 11 residues of the amino-terminal chain in Y7I HCA II assumed an alternate conformation compared with the wild type. Differential scanning calorimetry showed variants at position 7 had a melting temperature approximately 8 °C lower than that of the wild type.

Tracking solvent and protein movement during CO2 release in carbonic anhydrase II crystals

Significance Carbonic anhydrases catalyze the fast interconversion of carbon dioxide and water into bicarbonate and proton. In this study, we use the method of high-pressure cryocooling to capture the gaseous carbon dioxide in crystals of carbonic anhydrase and follow the sequential structure changes as the carbon dioxide is released. These “snapshots” enable us to “slow down” and visualize the water and protein motions that form a “proton wire” as the carbon dioxide exits the enzyme’s active site. This study provides an understanding of the importance of water rearrangements within an enzyme-active site and further suggests that such a method could be generally applied to other protein-mediated reactions that use gaseous molecules. Carbonic anhydrases are mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO2/HCO3−. Previously, the X-ray crystal structures of CO2-bound holo (zinc-bound) and apo (zinc-free) human carbonic anhydrase IIs (hCA IIs) were captured at high resolution. Here, we present sequential timeframe structures of holo- [T = 0 s (CO2-bound), 50 s, 3 min, 10 min, 25 min, and 1 h] and apo-hCA IIs [T = 0 s, 50 s, 3 min, and 10 min] during the “slow” release of CO2. Two active site waters, WDW (deep water) and WDW′ (this study), replace the vacated space created on CO2 release, and another water, WI (intermediate water), is seen to translocate to the proton wire position W1. In addition, on the rim of the active site pocket, a water W2′ (this study), in close proximity to residue His64 and W2, gradually exits the active site, whereas His64 concurrently rotates from pointing away (“out”) to pointing toward (“in”) active site rotameric conformation. This study provides for the first time, to our knowledge, structural “snapshots” of hCA II intermediate states during the formation of the His64-mediated proton wire that is induced as CO2 is released. Comparison of the holo- and apo-hCA II structures shows that the solvent network rearrangements require the presence of the zinc ion.