Inhibition and catalysis of carbonic anhydrase. Recent crystallographic analyses.
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Amino Acid Sequence↗
Biomedical subjects
A Liljas
Publications and source records attributed to A Liljas.
X-ray analysis of metal-substituted human carbonic anhydrase II derivatives.
Metal-substituted crystals of human carbonic anhydrase II belonging to space group P2(1) with cell dimensions a = 42.7, b = 41.7, c = 73.0 A and beta = 104.6 degrees were analyzed crystallographically. The resolution limit ranged from 1.82 to 1.92 A with high completeness (86.2-90.7%). Cobalt(II)-substituted carbonic anhydrase has a tetrahedral coordination around the metal both at pH 6 and pH 7.8, similar to the native zinc enzyme. In contrast, the catalytically inactive copper(II), nickel(II) and manganese(II) derivatives showed increased coordination number around the metal ion. Whereas the copper is best described as penta-coordinated, the nickel and manganese are best described as hexa-coordinated. The results are briefly compared with spectroscopic observations and our current view on carbonic anhydrase catalysis.
Journal Article↗
Wild-type and E106Q mutant carbonic anhydrase complexed with acetate.
The molecular structures of the acetate complexes of wild-type human carbonic anhydrase II (HCAII) and of E106Q mutant human carbonic anhydrase II were solved with high completeness (89-91%) to 2.1 and 1.9 A resolution, respectively. Both wild-type and mutant enzyme crystallize in space group P2(1) with cell dimensions a = 42.7, b = 41.7, c = 73.0 A and beta = 104.6 degrees. The altered active-site hydrogen-bond network caused by the mutation results in a different binding of the inhibitor in the two complexes. In the mutant, but not in the wild-type complex, a carboxylate O atom is within hydrogen-bond distance of Thr199 Ogamma1. In the wild-type enzyme ligand hydrogen bonding to this atom is normally only found for hydrogen-bond donors. The importance of this discrimination on catalysis by the enzyme is discussed briefly.
Journal Article↗
Cloning and nucleotide sequence of the cyclic AMP receptor protein-regulated Salmonella typhimurium pepE gene and crystallization of its product, an alpha-aspartyl dipeptidase.
The Salmonella typhimurium pepE gene, encoding an N-terminal-Asp-specific dipeptidase, has been cloned on pBR328 by complementation of the Asp-Pro growth defect conferred by a pepE mutation. Strains carrying the complementing plasmids greatly overproduce peptidase E. The enzyme has been purified from an extract of such a strain, its N-terminal amino acid sequence has been determined, and crystals suitable for X-ray diffraction have been grown. A new assay using L-aspartic acid p-nitroanilide as a substrate has been used to determine the pH optimum (approximately 7.5) and to test the effect of potential inhibitors. Insertions of transposon gamma delta (Tn1000) into one of the plasmids have been used to localize the gene and as sites for priming sequencing reactions. The nucleotide sequence of a 1,088-bp region of one of these plasmids has been determined. This sequence contains an open reading frame that predicts a 24.8-kDa protein with an N-terminal sequence that agrees with that determined for peptidase E. The predicted peptidase E amino acid sequence is not similar to that of any other known protein. The nucleotide sequence of the region upstream from pepE contains a promoter with a cyclic AMP receptor protein (CRP) site, and the effects of growth medium and of a crp mutation on expression of a pepE-lacZ fusion indicate that pepE is a member of the CRP regulon. The unique specificity of peptidase E and its lack of sequence similarity to any other peptidase suggest that this enzyme may be the prototype of a new class of peptidases. Its regulation by CPR and its specificity suggest that the enzyme may play a role in allowing the cell to use peptide aspartate to spare carbon otherwise required for the synthesis of the aspartate family of amino acids.
Amino Acid Sequence↗
Crystal structure of the complex between human carbonic anhydrase II and the aromatic inhibitor 1,2,4-triazole.
The X-ray crystal structure of the complex between human carbonic anhydrase II and the inhibitor 1,2,4-triazole has been refined at 1.9 A resolution to a final R-factor of 0.153. Triazole is an analogue of the competitive inhibitor imidazole, but the crystal structure shows a different type of binding to the enzyme. 1,2,4-Triazole is directly bound to the zinc(II) ion through the nitrogen in position 4, replacing the native water/hydroxyl (Wat263) in a distorted four-co-ordinated complex. The interaction of the inhibitor with the active site is completed by two hydrogen bonds to O gamma of Thr200 and to the amide nitrogen atom of Thr199 through the two adjacent N-1 and N-2 atoms. The binding site of triazole overlaps the proposed binding sites for the substrates, explaining the observed competitive behaviour of the inhibitor towards CO2/HCO3- under equilibrium conditions.
Carbonic Anhydrase Inhibitors↗
The structure of human carbonic anhydrase II in complex with bromide and azide.
The three-dimensional structure of human carbonic anhydrase II complexed with azide and with bromide was investigated crystallographically. Both of these non-protonated inhibitors replace the zinc and the 'deep' water, two catalytically important water molecules in the active site of the molecule. Both the azide and the bromide ions bind in a distorted tetrahedral manner 0.4 and 1.1 A from the zinc water position, respectively, but are in close contact (2.0 and 2.6 A, respectively) with the zinc ion.
Azides↗
Crystallographic analysis of Thr-200-->His human carbonic anhydrase II and its complex with the substrate, HCO3-.
A complex of carbonic anhydrase (CA) with one of its substrates, bicarbonate, has been studied crystallographically. Human isoenzyme II was mutated at position 200 from threonine to histidine, which results in higher affinity for bicarbonate. The HCO3- ion binds in the active site to the zinc ion as a pseudo-bidentate ligand which gives the metal a coordination geometry between tetrahedral and trigonal bipyramide. The water/hydroxide normally bound with tetrahedral coordination to the zinc is probably replaced by the OH group of the bicarbonate ion. The importance of residues Thr-199 and Glu-106 in controlling the binding orientation of HCO3- is discussed as well as the catalytic mechanism. Both the complex as well as the uncomplexed mutant were studied at 1.9 A resolution.
Bicarbonates↗
Metal poison inhibition of carbonic anhydrase.
Carbonic anhydrase is inhibited by the "metal poison" cyanide. Several spectroscopic investigations of carbonic anhydrase where the natural zinc ion has been replaced by cobalt have further strengthened the view that cyanide and cyanate bind directly to the metal. We have determined the structure of human carbonic anhydrase II inhibited by cyanide and cyanate, respectively, by X-ray crystallography. It is shown that the inhibitors replace a molecule of water, which forms a hydrogen bond to the peptide nitrogen of Thr-199 in the native structure. The coordination of the zinc ion is hereby left unaltered compared to the native crystal structure, so that the zinc coordinates three histidines and one molecule of water or hydroxyl ion in a tetrahedral fashion. The binding site of the two inhibitors is identical to what earlier has been suggested to be the position of the substrate (CO2) when attacked by the zinc bound hydroxyl ion. The peptide chain undergoes no significant alterations upon binding of either inhibitor.
Binding Sites↗
Refined structure of bovine carbonic anhydrase III at 2.0 A resolution.
The three-dimensional structure of bovine carbonic anhydrase III (BCA III) from red skeletal muscle cells has been determined by molecular replacement methods. The structure has been refined at 2.0 A resolution by both constrained and restrained structure-factor least squares refinement. The current crystallographic R-value is 19.2% and 121 solvent molecules have so far been found associated with the protein. The structure is highly similar to the refined structure of human carbonic anhydrase II. Some differences in amino acid sequence and structure between the two isoenzymes are discussed. In BCA III, Lys 64 and Arg 91 (His 64 and Ile 91 in HCA II) are both pointing out from the active site cavity forming salt bridges with Glu 4 and Asp 72 (His 4 and Asp 72 in HCA II), respectively. However, Arg 67 and Phe 198 (Asn 67 and Leu 198 in HCA II) are oriented towards the zinc ion and significantly reduce the volume of the active site cavity. Phe 198 particularly reduces the size of the substrate binding region at the "deep water" position at the bottom of the cavity and we suggest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II.
Amino Acid Sequence↗
Structural analysis of the zinc hydroxide-Thr-199-Glu-106 hydrogen-bond network in human carbonic anhydrase II.
The significance of the zinc hydroxide-Thr-199-Glu-106 hydrogen-bond network in the active site of human carbonic anhydrase II has been examined by X-ray crystallographic analyses of site-specific mutants. Mutants with Ala-199 and Ala-106 or Gln-106 have low catalytic activities, while a mutant with Asp-106 has almost full CO2 hydration activity. The structures of these four mutants, as well as that of the bicarbonate complex of the mutant with Ala-199, have been determined at 1.7 to 2.2 A resolution. Removal of the gamma atoms of residue 199 leads to a distorted tetrahedral geometry at the zinc ion, and a catalytically important zinc-bound water molecule has moved towards Glu-106. In the bicarbonate complex of the mutant with Ala-199 one oxygen atom from bicarbonate binds to zinc without displacing this water molecule. Tetrahedral coordination geometries are retained in the mutants at position 106. The mutants with Ala-106 and Gln-106 have a zinc-bound sulfate ion, whereas this sulfate site is only partially occupied in the mutant with Asp-106. The hydrogen-bond network seems to be "reversed" in the mutants with Ala-106 and Gln-106. The network is preserved as in native enzyme in the mutant with Asp-106 but the side chain of Asp-106 is more extended than that of Glu-106 in the native enzyme. These results illustrate the importance of Glu-106 and Thr-199 for controlling the precise coordination geometry of the zinc ion and its ligand preferences which results in an optimal orientation of a zinc-bound hydroxide ion for an attack on the CO2 substrate.
Carbonic Anhydrases↗
Refined structure of the aminobenzolamide complex of human carbonic anhydrase II at 1.9 A and sulphonamide modelling of bovine carbonic anhydrase III.
The binding of aminobenzolamide to human carbonic anhydrase (HCA II) has been investigated by X-ray crystallography. The three dimensional atomic structure of the enzyme inhibitor complex has been refined at 1.9 A resolution. The crystallographic R-factor is 17.8%. All inhibitor atoms are clearly possible to identify from the difference electron density map in the active site of the enzyme. The nitrogen of the sulphonamide group of the inhibitor is bound as a fourth ligand to the zinc ion, the other three are all histidyl residues. The binding conformation of the sulphonamide groups is similar to the previously described sulphonamide inhibitors. One of the oxygens of the outer sulphonamido group of the inhibitor forms a hydrogen bond to the amino group of Gln 92. The higher affinity of the benzolamide inhibitor compared with acetazolamide can be accounted for by the strong aromatic and hydrophobic interactions between the amino benzene ring of the inhibitor and the residues Phe 131 and Leu 198. In modelling studies of bovine carbonic anhydrase III (BCA III) it was evident that Phe 198 prevents an optimal interaction with sulphonamides.
Animals↗
Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes.
In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.
Anions↗
Ribosomal proteins from Thermus thermophilus for structural investigations.
In parallel with crystallographic studies of ribosomes from Thermus thermophilus, a long-term program on the crystallization and structural investigations of ribosomal proteins from the same microorganism has been started at the Institute of Protein Research (Pushchino, Russia). At present, more than half of the individual ribosomal proteins from T thermophilus have been purified without denaturating agents on a preparative scale and some of them have been obtained in the crystalline form. X-ray structural analysis of two ribosomal proteins, L1 and S6, is being carried out jointly with the Institute of Molecular Biology (Moscow, Russia) and laboratory of professor A Liljas (Lund University, Sweden). L1 is the large protein of the large ribosomal subunit. It can bind not only to a specific site on the 23S rRNA, but also to the mRNA that codes for L1 and L11, thereby acting as a translational repressor for the synthesis of these proteins. The crystals of L1 are orthorhombic and diffract to about 2 A resolution. Native data and data for several heavy atom derivatives have been collected. S6 is a small acidic protein from the small ribosomal subunit. The crystals of S6 are orthorhombic and diffract to 2 A resolution. Native data and derivatives' data have been collected.
Amino Acid Sequence↗
Crystals of protein S6 from the 30 S ribosomal subunit of Thermus thermophilus.
Crystals of protein S6 from the small ribosomal subunit of an extreme thermophile, Thermus thermophilus, have been obtained by the hanging-drop/vapor diffusion technique using methane pentanediol as a precipitant in the presence of potassium fluoride. The crystals belong to the space group C222 with cell parameters a = 106.7, b = 52.8, c = 41.0 A. They diffract to 2.0 A resolution.
Amino Acid Sequence↗
Crystallization and preliminary X-ray investigation of a recombinant form of rat catechol O-methyltransferase.
Rat catechol O-methyltransferase cDNA was introduced into an E. coli expression vector pKEX14, which utilizes the inducible T7 promoter. Active and soluble recombinant catechol O-methyltransferase was produced in bacteria and purified to electrophoretic homogeneity by chromatographic procedures. The purified enzyme has been crystallized by the method of vapor diffusion using polyethylene glycol as precipitant. The space group is P3(1)21 or P3(2)21 with a = b = 51.3 A and c = 168.5 A and one molecule in the asymmetric unit. The crystals diffract beyond 3.2 A and are suitable for three-dimensional X-ray structure determination.
Animals↗
Comparative biochemistry and biophysics of ribosomal proteins.
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Amino Acid Sequence↗
Characterization of the binding sites of protein L11 and the L10.(L12)4 pentameric complex in the GTPase domain of 23 S ribosomal RNA from Escherichia coli.
Ribonuclease and chemical probes were used to investigate the binding sites of ribosomal protein L11 and the pentameric complex L10.(L12)4 on Escherichia coli 23 S RNA. Protein complexes were formed with an RNA fragment constituting most of domains I and II or with 23 S RNA and they were investigated by an end-labelling method and a reverse transcriptase procedure, respectively. The results demonstrate that the two protein moieties bind at adjacent sites within a small RNA region. The L11 binding region overlaps with those of the modified peptide antibiotics thiostrepton and micrococcin and is constrained structurally by a three-helix junction while the L10.(L12)4 site is centred on an adjacent internal loop. The secondary structure of the whole region was determined in detail by the phylogenetic sequence comparison method, and the results for the L11 binding region, together with the experimental data, were used in a computer graphics approach to build a partial RNA tertiary structural model. The model provides insight into the topography of the L11 binding site. It also provides a structural rationale for the mutually co-operative binding of protein L11 with the antibiotics thiostrepton and micrococcin, and with the L10.(L12)4 protein complex.
Base Sequence↗
Refined structure of the acetazolamide complex of human carbonic anhydrase II at 1.9 A.
The binding of acetazolamide to human carbonic anhydrase II (HCA II) has been investigated by X-ray crystallography. The atomic positions of the enzyme inhibitor complex have been refined at 1.9 A resolution using the least squares refinement program package PROLSQ. The crystallographic R-factor is 17.6%. The bound inhibitor is clearly resolved in the active site of the enzyme. The acetazolamide amine group is bound as a fourth ligand to the zinc ion, the other three are all histidine residues. In addition to van der Waals' interactions and the previously described binding of the sulphonamide group, the inhibitor forms a hydrogen bond from the carbonyl oxygen of the acetylamido group to the amino group of Gln 92.
Acetazolamide↗