Structure Of Human Carbonic Anhydrase Research Articles

Crystal growth, morphological, mechanical, spectroscopic studies, optical properties, molecular docking, ADME/T, Hirshfeld surfaces analysis and theoretical calculations of hybrid organic-inorganic phosphate compound

Molecular organic–inorganic single crystals of 4-carboxyanilinium dihydrogenphosphate [4-CADP] have been grown by the slow evaporation technique. The crystal structure was re-investigated by single-crystal X ray diffraction (SCXRD) analysis without signifiant differences from the previous study. 3D Hirshfeld surface and 2D fingerprint plot studies were performed to understand the intermolecular interactions of the 4-CADP crystal in graphical representation. Functional groups identification was studied with FT–IR spectroscopy. High transparency and a wide band gap of 5.44 eV were observed with UV–Vis spectroscopy. Geometrical structure of the 4-CADP compound was performed using the Density Function Theory (DFT) method at the B3LYP/3-21G and PBEPBE/3-21G levels of theory, along with the HF/3-21G basis set. The equilibrium geometry, highest occupied molecular orbitals (HOMO), lowest unoccupied molecular orbitals (LUMO), and molecular electrostatic potential map (MEP) were also calculated and presented. MEP a visual representation of the chemically active sites and comparative reactivity of atoms. Results revealed OH⋯O and NH⋯O contacts have the largest impact the Hirshfeld surface. The mechanical properties of the 4-CADP crystal were studied experimentally Vicker’s microhardness technique, which revealed that the grown crystal belonged to the softer category. 4-CADP crystal void estimation reveals the mechanical strength and porosity of the material.Last, it was tried to compare the biological activities of the studied hybrid molecule against enzymes: the crystal structure of acetylcholinesterase (AChE) (PDB ID: 4M0E), the crystal structure of butyrylcholinesterase (BChE) (PDB ID: 5NN0), crystal structure of alpha-galactosidase (α-Gly) (PDB ID: 1T0O), crystal structure of human carbonic anhydrase I (hCA I) (PDB ID: 2CAB), and the crystal structure of human carbonic anhydrase II (hCA II) (PDB ID: 5AML). Afterwards, ADME/T analysis was performed to predict the effects of molecules on human metabolism.

In-silico Studies of Quinazolinone Analogues to Distinguish their Hypothetical Binding Mode using the X-ray crystal Structure Human carbon Anhydrase II (HCAII) Enzyme Complex with Sugar Sulfamate for Anticonvulsant Activity

The quinazolinone moiety is a significant pharmacophore that depicts various types of pharmacological activities as shown in recent exhaustive ligatures. Quinazolinone exhibit potent central nervous system (CNS) activities like anti-anxiety, analgesic, anti-inflammatory and anticonvulsant. To develop these views and application profiles, attempt have been made to report a drug/ligand or receptor/protein interactions by identifying the suitable active site against X-ray crystal structure of Human Carbonic Anhydrase II (HCA II) enzyme for anticonvulsant activity using Vlife MDS version 4.6 Software because the protein-ligand interaction plays a significant role in structural based drug designing. The interaction was evaluated based on the score comparison between quinazolinone derivatives with sugar sulfamate. The quinazolinone ring forms hydrophobic and hydrogen bond contacts amino acid residues. The ligands 4t and 4s were shown to possess minimum dock score i.e. minimum binding energy in Kcal/mole i.e. these molecules has more affinity for the active site of the receptor. Molecules with low dock score and binding energy show more affinity towards the receptor. The data reported in this article may be helpful for the medicinal chemists who are working in this area.

IN-SILICO DOCKING STUDIES OF CARBONIC ANHYDRASE INHIBITORS IN THE MANAGEMENT OF NEUROPATHIC PAIN

Background: In the present study, the in-silico docking studies of carbonic anhydrase inhibitors with 4RUX i.e. The crystal Structure of Human carbonic Anhydrase II protein was performed in the management of neuropathic pain. Materials and Methods: The crystal structure of protein PDB ID: 4RUX was downloaded from the RCSB PDB database and the ligand molecules of carbonic anhydrase inhibitors were drawn from Marvin Sketch. Docking studies between ligand and protein to predict binding interactions by using AutoDock 4.2 and the receptor-ligand complex interaction viewed by using Biovia Drug Discovery studio. Result: Carbonic anhydrase inhibitors showed binding energy ranging between -5.41 to -8.63. Ganoderic acid A showed better binding energy -8.63 kcal/mol than the standard Acetazolamide -6.22 kcal/mol. Conclusion: The result clearly indicates that among carbonic anhydrase inhibitors, Ganoderic acid A and Morindone show better hydrogen bonding and binding affinity towards carbonic anhydrase II (PDB ID: 4RUX). Thus, conclude that among carbonic anhydrase inhibitors Ganoderic acid A (obtained from Ganoderma lucidium) and Morindone (both obtained from Morinda citrifolia (NONI)} provide the better pharmacological effect.

Directed search for diuretics among 6-substituted pteridine-2,4,7(1H,3H,8H)-triones

Directed search for biologically active compounds among heterocycles still remains a relevant area of medical chemistry. Among the significant number of heterocyclic compounds, pteridines deserve special attention. Among the above-mentioned ones the drugs with antitumor, antimicrobial, antiviral, diuretic and other types of biological action are known. Nevertheless, 6-substituted pteridine-2,4,7(1H,3H,8H)-triones, which are structurally similar to triamterene (6-phenylpteridine-2,4,7-triamine) – a diuretic with potassium-sparing action are interesting objects for search for diuretics. All the more, they are characterized by prototropic tautomerism, able to form hydrogen and donor-acceptor bonds with various ligands, and it is likely that these structural features will provide their diuretic effect. The aim of the study is the directed search for diuretics among 6-substituted pteridine-2,4,7(1H,3H,8H)-triones using in silico and in vivo methodology and elucidation of the probable mechanism of action. 1-methyl-3-R-6- (2-oxo-2-aryl- (hetaryl-) ethyl) pteridine-2,4,7(1H,3H,8H)-triones were selected to study the effect on renal excretory function. and 1-methyl-3-R-6- (2-hydroxy-2-aryl- (hetaryl-) ethyl) pteridine-2,4,7(1H,3H,8H)-triones. Directed search for compounds that affect the excretory function of the kidneys of rats was conducted by the conventional method of E.B. Berkhin with water load. The content of creatinine, sodium, potassium and chlorides in blood and urine plasma was determined by biochemical methods using standard test kits of NPV "Philisit-Diagnostics" (Ukraine) and calculations were performed according to generally accepted methods. Research of the probable mechanism was conducted by flexible molecular docking, as an approach of finding molecules with affinity to a specific biological target. Macromolecular data were downloaded from the Protein Data Bank (PDB) namely, the crystal structures of Human carbonic anhydrase II (PDB ID – 3HS4) and epithelial sodium channel (ENaC) (PDB ID – 4NTX). Studies of the effect of the synthesized compounds on the excretory function of the kidneys of rats showed that 1-methyl-3-R-6- (2-oxo-2-aryl- (hetaryl-) ethyl) pteridine-2,4,7(1H,3H,8H)-triones containing 4-fluorophenyl, 2,4-difluorophenyl, 4-chlorophenyl fragments in the molecule increase diuresis by the second hour by 27.3-70.1% compared with the control group. According to the results of the impact on daily diuresis, it was found that the most active was 1-methyl-6- (2-oxo-2-phenyl) ethyl) pteridine-2,4,7(1H,3H,8H)-triones, which increased daily diuresis by 168.1%, exceeding the effect of Hydrochlorothiazide (41.8%) and Triamterene (49.1%). However, substituted 1-methyl-3-R-6- (2-hydroxy-2-aryl- (hetaryl-) ethyl) pteridine-2,4,7(1H,3H,8H)-triones are inactive compounds. In-depth studies using biological tests and molecular docking have suggested that 1-methyl-6- (2-oxo-2-aryl) ethyl) pteridine-2,4,7(1H,3H,8H)-triones 2.1, 2.5 and 2.6) probable mechanisms of diuretic action are disruption of sodium transport in the distal convoluted tubules, causing sodium excretion and water loss and possibly inhibition of epithelial sodium channels that promote sodium uptake and potassium secretion in the distal convolutions and tubules, which implements potassium-sparing action. A well-founded and developed strategy for the search for diuretics among 6-substituted pteridine-2,4,7(1H,3H,8H)-triones has identified a number of effective compounds that by diuretic effect are superior to the reference drugs "Hydrochlorothiazide" and "Triamterene". Importantly, the results of molecular docking suggested a mechanism of action of the compounds under study, similar to thiazide diuretics. This action may be related to the tautomerism of these compounds and, as a consequence, their ability to form coordination bonds with the zinc cation and the additional interaction of halogens in the active site of CA II. It was possible to detect the presence of potassium-sparing action, probably due to the ability to inhibit epithelial sodium channels (ENaC). The obtained results substantiate the further purposeful search for potential diuretics among this class of compounds.

In the footsteps of an inhibitor unbinding from the active site of human carbonic anhydrase II

The crystal structure of human carbonic anhydrase (HCA) II bound to an inhibitor molecule, 6-hydroxy-2-thioxocoumarin (FC5), shows FC5 to be located in a hydrophobic pocket at the active site. The present work employs classical molecular dynamics (MD) simulation to follow the FC5 molecule for 1 μs as it unbinds from its binding location, adopts the path of substrate/product diffusion (path 1) to leave the active site at around 75 ns. It is then found to undergo repeated binding and unbinding at different locations on the surface of the enzyme in water. Several transient excursions through different regions of the enzyme are also observed prior to its exit from the active site. These transient paths are combined with functionally relevant cavities/channels to enlist five additional pathways (path 2–6). Pathways 1–6 are subsequently explored using steered MD and umbrella sampling simulations. A free energy barrier of 0.969 kcal mol−1 is encountered along path 1, while barriers in the range of 0.57–2.84 kcal mol−1 are obtained along paths 2, 4 and 5. We also analyze in detail the interaction between FC5 and the enzyme along each path as the former leaves the active site of HCA II. Our results indicate path 1 to be the major exit pathway for FC5, although competing contributions may also come from the paths 2, 4 and 5. Communicated by Ramaswamy H. Sarma

Structural Characterization of Carbonic Anhydrase-Arylsulfonamide Complexes Using Ultraviolet Photodissociation Mass Spectrometry.

Numerous mass spectrometry-based strategies ranging from hydrogen-deuterium exchange to ion mobility to native mass spectrometry have been developed to advance biophysical and structural characterization of protein conformations and determination of protein-ligand interactions. In this study, we focus on the use of ultraviolet photodissociation (UVPD) to examine the structure of human carbonic anhydrase II (hCAII) and its interactions with arylsulfonamide inhibitors. Carbonic anhydrase, which catalyzes the conversion of carbon dioxide to bicarbonate, has been the target of countless thermodynamic and kinetic studies owing to its well-characterized active site, binding cavity, and mechanism of inhibition by hundreds of ligands. Here, we showcase the application of UVPD for evaluating structural changes of hCAII upon ligand binding on the basis of variations in fragmentation of hCAII versus hCAII-arylsulfonamide complexes, particularly focusing on the hydrophobic pocket. To extend the coverage in the midregion of the protein sequence, a supercharging agent was added to the solutions to increase the charge states of the complexes. The three arylsulfonamides examined in this study largely shift the fragmentation patterns in similar ways, despite their differences in binding affinities.

Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function.

Post-translational modification of the adeno-associated virus capsids is a poorly understood factor in the development of these viral vectors into pharmaceutical products. Here we report the extensive capsid deamidation of adeno-associated virus serotype 8 and seven other diverse adeno-associated virus serotypes, with supporting evidence from structural, biochemical, and mass spectrometry approaches. The extent of deamidation at each site depended on the vector's age and multiple primary-sequence and three-dimensional structural factors. However, the extent of deamidation was largely independent of the vector recovery and purification conditions. We demonstrate the potential for deamidation to impact transduction activity and, moreover, correlate an early time point loss in vector activity to rapidly progressing spontaneous deamidation at several adeno-associated virus 8 asparagines. We explore mutational strategies that stabilize side-chain amides, improving vector transduction and reducing the lot-to-lot molecular variability that presents a key concern in biologics manufacturing. This study illuminates a previously unknown aspect of adeno-associated virus capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy.

Enhancement of intrinsic fluorescence of human carbonic anhydrase II upon topiramate binding: Some evidence for drug-induced molecular contraction of the protein

In this report, the effect of topiramate (TPM), an anticonvulsant sulfamate drug, on the structure of human carbonic anhydrase II (hCA II) was investigated by spectroscopic techniques. The intrinsic fluorescence experiments indicated that TPM binding causes enhancement of enzyme fluorescence via decreasing the internal quenching and energy transfer efficiency, the result supported by molecular dynamics simulation. Thermodynamic analysis of the binding process suggested that hydrogen bonding and van der Waals interactions are the major forces in the interaction of TPM with hCA II. The far-UV circular dichroism (CD) results showed that TPM caused increment in α-helical and β-sheet content of hCA II whereas, near-UV CD experiments in the presence of the drug showed induction of some compactness in the enzyme tertiary structure. The number of accessible tryptophans and protein surface hydrophobicity index of the enzyme were reduced in the presence of TPM which confirms the enzyme structural compactness upon drug binding. In addition, the enzyme thermal stability was increased in the presence of the drug. It seems that the induction of compactness in the enzyme structure upon drug binding may be responsible for increment of its conformational stability.

Neutron structure of human carbonic anhydrase II in complex with methazolamide: mapping the solvent and hydrogen-bonding patterns of an effective clinical drug.

Carbonic anhydrases (CAs; EC 4.2.1.1) catalyze the interconversion of CO2 and HCO3-, and their inhibitors have long been used as diuretics and as a therapeutic treatment for many disorders such as glaucoma and epilepsy. Acetazolamide (AZM) and methazolamide (MZM, a methyl derivative of AZM) are two of the classical CA inhibitory drugs that have been used clinically for decades. The jointly refined X-ray/neutron structure of MZM in complex with human CA isoform II (hCA II) has been determined to a resolution of 2.2 Å with an Rcryst of ∼16.0%. Presented in this article, along with only the second neutron structure of a clinical drug-bound hCA, is an in-depth structural comparison and analyses of differences in hydrogen-bonding network, water-molecule orientation and solvent displacement that take place upon the binding of AZM and MZM in the active site of hCA II. Even though MZM is slightly more hydrophobic and displaces more waters than AZM, the overall binding affinity (Ki) for both of the drugs against hCA II is similar (∼10 nM). The plausible reasons behind this finding have also been discussed using molecular dynamics and X-ray crystal structures of hCA II-MZM determined at cryotemperature and room temperature. This study not only allows a direct comparison of the hydrogen bonding, protonation states and solvent orientation/displacement of AZM and MZM, but also shows the significant effect that the methyl derivative has on the solvent organization in the hCA II active site.

Structure of a Complex Formed by a Protein and a Helical Aromatic Oligoamide Foldamer at 2.1 Å Resolution

In the search of molecules that could recognize sizeable areas of protein surfaces, a series of ten helical aromatic oligoamide foldamers was synthesized on solid phase. The foldamers comprise three to five monomers carrying various proteinogenic side chains, and exist as racemic mixtures of interconverting right-handed and left-handed helices. Functionalization of the foldamers by a nanomolar ligand of human carbonic anhydrase II (HCA) ensured that they would be held in close proximity to the protein surface. Foldamer-protein interactions were screened by circular dichroism (CD). One foldamer displayed intense CD bands indicating that a preferred helix handedness is induced upon interacting with the protein surface. The crystal structure of the complex between this foldamer and HCA could be resolved at 2.1 Å resolution and revealed a number of unanticipated protein-foldamer, foldamer-foldamer, and protein-protein interactions.

Neutron Structure of Human Carbonic Anhydrase II: A Hydrogen-Bonded Water Network “Switch” Is Observed between pH 7.8 and 10.0

The neutron structure of wild-type human carbonic anhydrase II at pH 7.8 has been determined to 2.0 Å resolution. Detailed analysis and comparison to the previously determined structure at pH 10.0 show important differences in the protonation of key catalytic residues in the active site as well as a rearrangement of the H-bonded water network. For the first time, a completed H-bonded network stretching from the Zn-bound solvent to the proton shuttling residue, His64, has been directly observed.

Emerging from pseudo-symmetry: the redetermination of human carbonic anhydrase II in monoclinicP21with a doubledaaxis

The crystal structure of human carbonic anhydrase II in the monoclinic P2(1) space group with a doubled a axis from that of the usually observed unit cell has recently been reported, with one of the two molecules in the asymmetric unit demonstrating rotational disorder [Robbins et al. (2010), Acta Cryst. D66, 628-634]. The structure has been redetermined, with the coordinates of both pseudo-symmetrically related molecules in the crystallographic asymmetric unit translated by x' = x +/- 1/4, and no rotational disorder is observed. This corresponds to a different choice of how the four molecules in the unit cell should be grouped into pairs that represent a single asymmetric unit.

Structure of a monoclinic polymorph of human carbonic anhydrase II with a doubled a axis.

The crystal structure of human carbonic anhydrase II with a doubled a axis from that of the usually observed monoclinic unit cell has been determined and refined to 1.4 A resolution. The diffraction data with h = 2n + 1 were systematically weaker than those with h = 2n. Consequently, the scaling of the data, structure solution and refinement were challenging. The two molecules comprising the asymmetric unit are related by a noncrystallographic translation of (1/2) along a, but one of the molecules has two alternate positions related by a rotation of approximately 2 degrees. This rotation axis is located near the edge of the central beta-sheet, causing a maximum distance disparity of 1.7 A between equivalent atoms on the diametrically opposite side of the molecule. The crystal-packing contacts are similar to two sequential combined unit cells along a of the previously determined monoclinic unit cell. Abnormally high final R(cryst) and R(free) values (20.2% and 23.7%, respectively) are not unusual for structures containing pseudo-translational symmetry and probably result from poor signal to noise in the weak h-odd data.

Neutron structure of human carbonic anhydrase II: implications for proton transfer.

Human carbonic anhydrase II (HCA II) catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a proton. Despite many high-resolution X-ray crystal structures, mutagenesis, and kinetic data, the structural details of the active site, especially the proton transfer pathway, are unclear. A large HCA II crystal was prepared at pH 9.0 and subjected to vapor H-D exchange to replace labile hydrogens with deuteriums. Neutron diffraction studies were conducted at the Protein Crystallography Station at Los Alamos National Laboratory. The structure to 2.0 A resolution reveals several interesting active site features: (1) the Zn-bound solvent appearing to be predominantly a D(2)O molecule, (2) the orientation and hydrogen bonding pattern of solvent molecules in the active site cavity, (3) the side chain of His64 being unprotonated (neutral) and predominantly in an inward conformation pointing toward the zinc, and (4) the phenolic side chain of Tyr7 appearing to be unprotonated. The implications of these details are discussed, and a proposed mechanism for proton transfer is presented.

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.

High-resolution structure of human carbonic anhydrase II complexed with acetazolamide reveals insights into inhibitor drug design

The crystal structure of human carbonic anhydrase II (CA II) complexed with the inhibitor acetazolamide (AZM) has been determined at 1.1 A resolution and refined to an R(cryst) of 11.2% and an R(free) of 14.7%. As observed in previous CA II-inhibitor complexes, AZM binds directly to the zinc and makes several key interactions with active-site residues. The high-resolution data also showed a glycerol molecule adjacent to the AZM in the active site and two additional AZMs that are adventitiously bound on the surface of the enzyme. The co-binding of AZM and glycerol in the active site demonstrate that given an appropriate ring orientation and substituents, an isozyme-specific CA inhibitor may be developed.

Mapping Proton Wires in Proteins: Carbonic Anhydrase and GFP Chromophore Biosynthesis

We have developed an algorithm for mapping proton wires in proteins and applied it to the X-ray structures of human carbonic anhydrase II (CA-II), the green fluorescent protein (GFP), and some of their mutants. For both proteins, we find more extensive proton wires than typically reported. In CA-II the active site wire exits to the protein surface, and leads to Glu69 and Asp72, located on an electronegative patch on the rim of the active site cavity. One possible interpretation of this observation is that positively charged, protonated buffer molecules dock in that area, from which a proton is delivered to the active site when the enzyme works in the dehydration direction. In GFP we find a new internal proton wire, in addition to the previously reported wire involved in excited state proton transfer. The new wire is located on the other face of the chromophore, and we conjecture that it plays a role in chromophore biosynthesis that occurs following protein folding. In the last step of this process, transient carbanion formation was suggested to occur on the bridge carbon [Pouwels et al. Biochemistry 2008, 47, 10111]. Residues on the new wire (Thr62, His181, Arg96) may participate in proton abstraction from this bridge carbon atom. A possible mechanism involves a rotation of the Thr62 side chain and completion of a short wire by which the proton is transported to His181, while the negative charge is transferred to the imidazolone carbonyl, producing a homoenolate intermediate that is stabilized by Arg96. Finally, comparison of the proton wires in the two proteins reveals common motifs, such as short internalized Ser/Thr-Glu hydrogen-bonded pairs for ultrafast proton abstraction, and threonine side chain rotation functioning as a proton wire switch.

Analyzing the 3D Structure of Human Carbonic Anhydrase II and Its Mutants Using Deep View and the Protein Data Bank

A lab was designed to teach biological chemistry students to critically analyze the 3D structure of a protein. The enzyme studied was human carbonic anhydrase (II) and students also examined mutants and protein bound to a variety of ligands, metals, and inhibitors. The coordinate files for each protein are downloaded by the students from the Protein Data Bank. The structure is viewed and manipulated with the public-domain molecular-viewing software Deep View. Students learn how to interpret Ramachandran plots, study familial homology, make virtual mutations, compare structures, analyze structural details, and produce stunning 3D images. The lab runs on personal computers with Internet access and requires minimal to no supervision. This lab can be used as an independent project or as part of a more comprehensive set of experiments, and it can be adapted to the study of other biomolecules.

Crystal structure of human carbonic anhydrase II at 1.95 A resolution in complex with 667-coumate, a novel anti-cancer agent.

CA (carbonic anhydrase) catalyses the reversible hydration of carbon dioxide into bicarbonate, and at least 14 isoforms have been identified in vertebrates. The role of CA type II in maintaining the fluid and pH balance has made it an attractive drug target for the treatment of glaucoma and cancer. 667-coumate is a potent inhibitor of the novel oncology target steroid sulphatase and is currently in Phase 1 clinical trials for hormone-dependent breast cancer. It also inhibits CA II in vitro. In the present study, CA II was crystallized with 667-coumate and the structure was determined by X-ray crystallography at 1.95 A (1 A=0.1 nm) resolution. The structure reported here is the first for an inhibitor based on a coumarin ring and shows ligation of the sulphamate group to the active-site zinc at 2.15 A through a nitrogen anion. The first two rings of the coumarin moiety are bound within the hydrophobic binding site of CA II. Important residues contributing to binding include Val-121, Phe-131, Val-135, Leu-141, Leu-198 and Pro-202. The third seven-membered ring is more mobile and is located in the channel leading to the surface of the enzyme. Pharmacokinetic studies show enhanced stability of 667-coumate in vivo and this has been ascribed to binding of CA II in erythrocytes. This result provides a structural basis for the stabilization and long half-life of 667-coumate in blood compared with its rapid disappearance in plasma, and suggests that reversible binding of inhibitors to CA may be a general method of delivering this type of labile drug.

Crystal structure of human carbonic anhydrase II complexed with an anti-convulsant sugar sulphamate.

The fructose-based sugar sulphamate RWJ-37497, a potent analogue of the widely used anti-epileptic drug topiramate, possesses anti-convulsant and carbonic anhydrase-inhibitory activities. We have studied the binding interactions of RWJ-37497 in the active site of human carbonic anhydrase II by X-ray crystallography. The atomic positions of the enzyme inhibitor complex were refined at a resolution of 2.1 A (1 A=0.1 nm) to the final crystallographic R and R(free) values of 0.18 and 0.23, respectively. The inhibitor co-ordinates to the active-site zinc ion through its oxygen atom and the ionized nitrogen atom of the sulphamate group by replacing the metal-bound water molecules, although the sulphamoyl oxygen atom provides a rather lengthy co-ordination. The 4,5-cyclic sulphate group is positioned in a hydrophobic pocket of the active site, making contacts with the residues Phe-131, Leu-198, Pro-201 and Pro-202. Since the ligand was found to be intact, concerns about RWJ-37947 irreversibly alkylating the enzyme through its 4,5-cyclic sulphate group were dispelled.