Forms Of Human Carbonic Anhydrase Research Articles

Characterization of the Copper(II) Binding Sites in Human Carbonic Anhydrase II.

Human carbonic anhydrase (CA) is a well-studied, robust, mononuclear Zn-containing metalloprotein that serves as an excellent biological ligand system to study the thermodynamics associated with metal ion coordination chemistry in aqueous solution. The apo form of human carbonic anhydrase II (CA) binds 2 equiv of copper(II) with high affinity. The Cu(2+) ions bind independently forming two noncoupled type II copper centers in CA (CuA and CuB). However, the location and coordination mode of the CuA site in solution is unclear, compared to the CuB site that has been well-characterized. Using paramagnetic NMR techniques and X-ray absorption spectroscopy we identified an N-terminal Cu(2+) binding location and collected information on the coordination mode of the CuA site in CA, which is consistent with a four- to five-coordinate N-terminal Cu(2+) binding site reminiscent to a number of N-terminal copper(II) binding sites including the copper(II)-amino terminal Cu(2+) and Ni(2+) and copper(II)-β-amyloid complexes. Additionally, we report a more detailed analysis of the thermodynamics associated with copper(II) binding to CA. Although we are still unable to fully deconvolute Cu(2+) binding data to the high-affinity CuA site, we derived pH- and buffer-independent values for the thermodynamics parameters K and ΔH associated with Cu(2+) binding to the CuB site of CA to be 2 × 10(9) and -17.4 kcal/mol, respectively.

Location of Binding Sites in Small Molecule Rescue of Human Carbonic Anhydrase II

Small molecule rescue of mutant forms of human carbonic anhydrase II (HCA II) occurs by participation of exogenous donors/acceptors in the proton transfer pathway between the zinc-bound water and solution. To examine more thoroughly the energetics of this activation, we have constructed a mutant, H64W HCA II, which we have shown is activated by 4-methylimidazole (4-MI) by a mechanism involving the binding of 4-MI to the side chain of Trp-64 ∼8 Å from the zinc. A series of experiments are consistent with the activation of H64W HCA II by the interaction of imidazole and pyridine derivatives as exogenous proton donors with the indole ring of Trp-64; these experiments include pH profiles and H/D solvent isotope effects consistent with proton transfer, observation of approximately fourfold greater activation with the mutant containing Trp-64 compared with Gly-64, and the observation by x-ray crystallography of the binding of 4-MI associated with the indole side chain of Trp-64 in W5A-H64W HCA II. Proton donors bound at the less flexible side chain of Trp-64 in W5A-H64W HCA II do not show activation, but such donors bound at the more flexible Trp-64 of H64W HCA II do show activation, supporting suggestions that conformational mobility of the binding site is associated with more efficient proton transfer. Evaluation using Marcus theory showed that the activation of H64W HCA II by these proton donors was reflected in the work functions w r and w p rather than in the intrinsic Marcus barrier itself, consistent with the role of solvent reorganization in catalysis.

Molecular Basis for the Origin of Differential Spectral and Binding Profiles of Dansylamide with Human Carbonic Anhydrase I and II

Sulfonamide derivatives serve as potent inhibitors of carbonic anhydrases (CAs), and a few such inhibitors have been currently used as drugs for the treatment of different pathogenic conditions in humans. In pursuit of designing the isozyme-specific inhibitors of human CAs, we observed that the fluorescence spectral properties and binding profiles of a fluorogenic sulfonamide derivative, 5-(dimethylamino)-1-naphthalenesulfonamide (dansylamide, DNSA), were markedly different between the recombinant forms of human carbonic anhydrase I (hCA I) and II (hCA II). The kinetic evaluation of the overall microscopic pathways for the binding of DNSA to hCA I versus hCA II revealed that the protein isomerization step served as a major determinant of the above discrepancy. Arguments are presented that the detailed structural-functional investigations of enzyme-ligand interactions may provide insights into designing the isozyme-specific inhibitors of CAs.

Changing the efficiency and specificity of the esterase activity of human carbonic anhydrase II by site-specific mutagenesis.

Rates of hydrolysis of 4-, 3-, and 2-nitrophenyl acetate and 4-nitrophenyl propionate catalyzed by wild-type and mutant forms of human carbonic anhydrase II have been measured. The results show that the mutations Tyr7-->Phe and Ala65-->Leu lead to activity enhancements with all the investigated substrates, but there is no significant effect on the specificity. In contrast, some mutations at sequence position 200 have large effects on specificity. For example, while the mutation Thr200-->Gly results in a threefold increase of the rate of hydrolysis of 4-nitrophenyl acetate, the activity is enhanced 10 times with the meta-substituted substrate and 380 times with the ortho-substituted substrate. These results are interpreted in terms of the removal in the mutant of a steric interference between the 2-NO2 group, in particular, and the side chain of Thr200. Mutants involving residues lining a hydrophobic pocket near the catalytically essential zinc ion have also been investigated. The most pronounced effect on specificity was found for the Val143-->Gly mutant. This mutation leads to a sixfold decrease of the rate of hydrolysis of 4-nitrophenyl acetate but a 20-fold increase of the activity with the propionyl ester as substrate. These results suggest that the side chain of Val143 interferes sterically with the acyl moiety of 4-nitrophenyl propionate. Based on these results, we have constructed a hypothetical model of the location of these ester substrates in the enzymic active site.

Models of the active sites of zinc-containing enzymes: the structure of acetato[hydrotris(3-tert-butyl-5-methylpyrazol-1-yl)borato]zinc(II)

The zinc complex acetato[hydrotris(3-tert-butyl-5-methylpyrazol-1-yl)borato]zinc(II) has been prepared and structurally characterised as a model of the active sites of the zinc-containing enzymes carbonic anhydrase and dihydroorotase. Crystals are monoclinic, space group P21/n, a 15.995(2), b 19.861(4), c 10.530(2)A, β 90.00(2)°, Z 4 and the structure has been refined to a residual of 0.048 based on 4096 reflections. The complex deviates from C3 symmetry as a consequence of interactions between the tripod ligand and the fourth ligand bound to the Zn atom. The Zn–N (tripod) bond lengths are affected by the degree of steric crowding but bonds to the fourth ligand are not. Comparison with the I–, OH– and HCO3– forms of human carbonic anhydrase I confirms that the complexes with tripodal ligands are excellent structural models for the active site of this enzyme.

Insights into the function of the zinc hydroxide-Thr199-Glu106 hydrogen bonding network in carbonic anhydrases

The exact functional role of the zinc hydroxide (water)-Thr199-Glu106 hydrogen bond network in the carbonic anhydrases is unknown. However, from the results of molecular dynamics simulations (MD) we are able to better define its function. From computer graphics analysis and MD simulations on the zinc hydroxide form of human carbonic anhydrase II we find that this interaction forces the hydroxide hydrogen atom to be in a “down” position relative to the deep water-binding pocket. From previous work we have found that this pocket is a high-affinity binding site for CO 2. We also note that during the timescale of our simulation (126 ps) the hydrogen bonds between the hydroxide hydrogen atom and Thr199 and the one between Thr199 and Glu106 are not fluxional. We propose that the role of the zinc hydroxide (water)-Thr199-Glu106 hydrogen bond network is to lock the hydrogen atom in the down position in order to expose the CO 2 molecule bound in the deep water pocket to a lone pair of the hydroxide oxygen atom. This would allow for the rapid reaction of the CO 2 molecule around the zinc ion. Furthermore, if the hydroxide hydrogen atom were not locked in the down position the binding of CO 2 to the deep water pocket could be interfered with by the unrestrained hydroxide hydrogen atom (e.g. the NZnOH torsion could undergo rotational transitions that would partially block the deep water pocket). In summary, the roles we ascribe to this hydrogen bonding network are (1) to allow for facile access of CO 2 to the deep water pocket and (2) to allow for maximal exposure of a hydroxide oxygen lone pair to the CO 2 carbon atom.

Dynamic 13C NMR investigations of substrate interaction and catalysis by cobalt(II) human carbonic anhydrase I.

Using 13C NMR spectroscopy, we have further investigated the binding of HCO3- in the active site of an artificial form of human carbonic anhydrase I in which the native zinc is replaced by Co(II). The Co(II) enzyme, unlike all other metal-substituted derivatives, has functional properties closely similar to those of the native zinc enzyme. By measuring the spin-lattice relaxation rate and the line width for both the CO2 and HCO3- at two field strengths, we have determined both the paramagnetic effects that reflect substrate binding and the exchange effects due to catalysis at chemical equilibrium. The following are the results at 14 degrees C and pH 6.3 (1) HCO3- is bound in the active site of the catalytically competent enzyme with the 13C of the HCO3- located 3.22 +/- 0.02 A from the Co(II); (2) the apparent equilibrium dissociation constant for the bound HCO3- is 7.6 +/- 1.5 mM, determined by using the paramagnetic effects on the line widths, and 10 +/- 2 mM, determined by using the exchange effects; (3) the lifetime of HCO3- bound to the metal is (4.4 +/- 0.4) X 10(-5) s; (4) the overall catalyzed CO2 in equilibrium HCO3- exchange rate constant of the Co(II) enzyme is (9.6 +/- 0.4) X 10(3) s-1; (5) the electron spin relaxation time of the Co(II), determined by using paramagnetic effects on the bound HCO3-, is (1.1 +/- 0.1) X 10(-11) s. The data did not provide any direct information on the binding of CO2.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of human carbonic anhydrase III from skeletal muscle.

A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to beta-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.

Catalysed hydrolysis of S-p-nitrophenyl thioacetate by human carbonic anhydrase

Sigmoidal kenz–pH profiles are observed for catalysis of the hydrolysis of S-p-nitrophenyl thioacetate by native, cobalt(II), and copper(II) forms of human carbonic anhydrase B and by native C.

Human renal carbonic anhydrase. Purification and properties.

Carbonic ahyndrase was isolated from fresh human donor kidneys which had been thoroughly perfused free from blood. The isolation procedure involved biospecific affinity chromatography on a sulfanilamide-agarose column and yielded one soluble form of the enzyme, which was homogenous with respect to sedimentation in the ultracentrifuge, electrophoresis, isoelectric focusing and immunodiffusion. The renal enzyme had an amino acid composition and beahved chromatographically, electrophoretically, and immunochemically like the erythrocyte form human carbonic anhydrase C, isolated by the same technique. The kinetic behaviour of the renal enzyme was similar to that of human carbonic anhydrase C when compared by the stopped-flow pH-indicator technique. The results therefore suggest that the cytoplasmic carbonic anhydrase of the human kidney is very similar, if not identical, to the high-activity erythrocyte form of human carbonic anhydrase C.

Human Carbonic Anhydrases

Abstract A polymorphic form of human carbonic anhydrase C has been isolated in crystalline form from the erythrocytes of Negroes who were either homozygous or heterozygous for this enzyme. It differs from carbonic anhydrase C in having an aspartic acid rather than an asparagine at a position 9 residues removed from the COOH-terminal end of the molecule.

Purification and properties of carbonic anhydrase from human erythrocytes

1. 1. In fractionation experiments several forms of human erythrocyte carbonic anhydrase were obtained. Purification methods, involving ion-exchange chromatography and zone electrophoresis, are described for the two forms which account for the greatest part of the enzyme activity in ethanol-chloroform extracts of the erythrocytes. 2. 2. The two purified forms of the enzyme show a high degree of homogeneity in electrophoresis and in the ultracentrifuge. Both have a molecular weight of 34 000, but, in contrast to other cases of microheterogeneity, they differ in their specific activities. The enzyme contains 0.36% S and 0.20% Zn, corresponding to 4 atoms of sulfur and 1 atom of zinc per protein molecule. 3. 3. The possible existence of preparation artefacts is discussed and comparisons of the physical and chemical properties of different forms of the human carbonic anhydrase and bovine enzyme are made.