Inhibition of the hydration of CO2 catalyzed by carbonic anhydrase III from cat muscle

Using stopped flow methods, we have measured the steady state rate constants and the inhibition by N3- and I- of the hydration of CO2 catalyzed by carbonic anhydrase III from cat muscle. Also, using fluorescence quenching of the enzyme at 330 nm, we have measured the binding of the sulfonamide chlorzolamide to cat carbonic anhydrase III. Inhibition by the anions was uncompetitive at pH 6.0 and was mixed at higher values of pH. The inhibition constant of azide was independent of pH between 6.0 and 7.5 with a value of KIintercept = 2 X 10(-5) M; the binding constant of chlorzolamide to cat carbonic anhydrase III was also independent of pH in the range of 6.0 to 7.5 with a value Kdiss = 2 X 10(-6) M. Both of these values increased as pH increased above 8. There was a competition between chlorzolamide and the anions N-3 and OCN- for binding sites on cat carbonic anhydrase III. The pH profiles for the kinetic constants and the uncompetitive inhibition at pH 6.0 can be explained by an activity-controlling group in cat carbonic anhydrase III with a pKa less than 6. Moreover, the data suggest that like isozyme II, cat isozyme III is limited in rate by a step occurring outside the actual interconversion of CO2 and HCO3- and involving a change in bonding to hydrogen exchangeable with solvent water.     

Purification and properties of cyclostome carbonic anhydrase from erythrocytes of hagfish

1. Carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been purified from erythrocytes of hagfish (Myxine glutinosa). A single form with low specific CO2 hydration activity was isolated. The purified carbonic anhydrase appeared homogeneous judging from polyacrylamide gel electrophoresis and gel filtration experiments. The protein has a molecular weight of about 29 000, corresponding to about 260 amino acid residues. This molecular weight is in accordance with other vertebrate carbonic anhydrases with the exception of the elasmobranch enzymes, which have Mr 36 000--39 000. 2. The molecular weight obtained for hagfish carbonic anhydrase indicates that a carbonic anhydrase with Mr approx. 29 000 is the ancestral type of the vertebrate enzyme rather than, as in sharks, a heavier carbonic anhydrase molecule. 3. The circular dichroism spectrum may indicate a somewhat different structural arrangement of aromatic amino acid residues in this enzyme than in the mammalian carbonic anhydrases. 4. The enzyme is strongly inhibited by acetazolamide and also to a lesser extent by monovalent anions. 5. Zn2+, which is essential for activity, appears, contrary to other characterized carbonic anhydrases, less strongly bound in the active site of the enzyme.     

Studies on the binding of copper to dopamine beta-monooxygenase and other proteins using the Cu2+ ion-selective electrode

The binding of Cu2+ to native and copper-free dopamine beta-monooxygenase has been investigated by potentiometric titrations using a Cu2+-selective electrode. Stoichiometric formation constants have been determined from regression analysis of the resulting titration curves. The results establish a stoichiometry of four high-affinity binding sites for Cu/+ (log Kf approximately 11) per enzyme tetramer, and more binding sites of lower affinity (log Kf approximately 5-7). The data for binding of the first four Cu2+ to the enzyme tetramer indicate interactions in the binding to the sites. Bovine serum albumin, metal-free carbonic anhydrase, and ovotransferrin have also been titrated with Cu2+, and the formation constants of both high-affinity binding sites and other sites have been determined. The stoichiometry of one high-affinity binding site of Cu2+ for carbonic anhydrase (log Kf approximately 10-12) and two sites for ovotransferrin (log Kf approximately 11) agree with the reported metal binding properties of these proteins. The number of high-affinity binding sites for bovine serum albumin was pH dependent.     

Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O

We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme.     

Catalytic enhancement of human carbonic anhydrase III by replacement of phenylalanine-198 with leucine

Carbonic anhydrase III, a cytosolic enzyme found predominantly in skeletal muscle, has a turnover rate for CO2 hydration 500-fold lower and a KI for inhibition by acetazolamide 700-fold higher (at pH 7.2) than those of red cell carbonic anhydrase II. Mutants of human carbonic anhydrase III were made by replacing three residues near the active site with amino acids known to be at the corresponding positions in isozyme II (Lys-64----His, Arg-67----Asn, and Phe-198----Leu). Catalytic properties were measured by stopped-flow spectrophotometry and 18O exchange between CO2 and water using mass spectrometry. The triple mutant of isozyme III had a turnover rate for CO2 hydration 500-fold higher than wild-type carbonic anhydrase III. The binding constants, KI, for sulfonamide inhibitors of the mutants containing Leu-198 were comparable to those of carbonic anhydrase II. The mutations at residues 64, 67, and 198 were catalytically independent; the lowered energy barrier for the triple mutant was the sum of the energy changes for each of the single mutants. Moreover, the triple mutant of isozyme III catalyzed the hydrolysis of 4-nitrophenyl acetate with a specific activity and pH dependence similar to those of isozyme II. Phe-198 is thus a major contributor to the low CO2 hydration activity, the weak binding of acetazolamide, and the low pKa of the zinc-bound water in carbonic anhydrase III. Intramolecular proton transfer involving His-64 was necessary for maximal turnover.     

A closer look at the active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila

The prototype of the gamma-class of carbonic anhydrase has been characterized from the methanogenic archaeon Methanosarcina thermophila. Previously reported kinetic studies of the gamma-class carbonic anhydrase are consistent with this enzyme having a reaction mechanism similar to that of the mammalian alpha-class carbonic anhydrase. However, the overall folds of these two enzymes are dissimilar, and apart from the zinc-coordinating histidines, the active site residues bear little resemblance to one another. The crystal structures of zinc-containing and cobalt-substituted gamma-class carbonic anhydrases from M. thermophila are reported here between 1.46 and 1.95 A resolution in the unbound form and cocrystallized with either SO(4)(2)(-) or HCO(3)(-). Relative to the tetrahedral coordination geometry seen at the active site in the alpha-class of carbonic anhydrases, the active site of the gamma-class enzyme contains additional metal-bound water ligands, so the overall coordination geometry is trigonal bipyramidal for the zinc-containing enzyme and octahedral for the cobalt-substituted enzyme. Ligands bound to the active site all make contacts with the side chain of Glu 62 in manners that suggest the side chain is likely protonated. In the uncomplexed zinc-containing enzyme, the side chains of Glu 62 and Glu 84 appear to share a proton; additionally, Glu 84 exhibits multiple conformations. This suggests that Glu 84 may act as a proton shuttle, which is an important aspect of the reaction mechanism of alpha-class carbonic anhydrases. A hydrophobic pocket on the surface of the enzyme may participate in the trapping of CO(2) at the active site. On the basis of the coordination geometry at the active site, ligand binding modes, the behavior of the side chains of Glu 62 and Glu 84, and analogies to the well-characterized alpha-class of carbonic anhydrases, a more-defined reaction mechanism is proposed for the gamma-class of carbonic anhydrases.     

Novel carbonic anhydrase isozymes I, II and IV activators incorporating sulfonyl-histamino moieties.

Sulfonylamido(ureido) derivatives of histamine were synthesized by an original procedure in order to obtain tight-binding activators of the zinc enzyme carbonic anhydrase (CA), exploiting the binding energy of the alkyl/arylsulfonyl moieties with amino acid residues at the entrance of the active site. In contrast to the lead molecule, histamine, the new derivatives possessed higher affinity for three different CA isozymes, as evidenced by compairing the affinity constants of these compounds for isozyme CA II.     

Thermodynamic parameters of the interaction between Co(II) bovine carbonic anhydrase and anionic inhibitors.

The pH dependence of the apparent affinity constants of perchlorate for cobalt(II)bovine carbonic anhydrase II has been measured by electronic absorption spectroscopy. The obtained data have been analyzed in terms of the ionization of two acidic groups of CoBCAII, and the affinity of perchlorate for the two water-containing species of the enzyme have been estimated. Furthermore, the affinity constants of nitrate, perchlorate, and azide for CoBCAII in the temperature range 5 degrees C-30 degrees C have been determined by spectrophotometric titrations at pH 7. The affinity constants for these ligands decrease with increasing temperatures. The temperature dependence of binding was used to estimate the enthalpy and entropy parameters for the formation of the corresponding 1:1 adducts. The obtained results indicate that binding of these anions to the cobalt enzyme is an enthalpy driven process which is opposed by a moderate entropy change.     

Pt(CN)2-4 and Au(CN)-2: potential general probes for anion-binding sites of proteins. 35Cl and 81Br nuclear-magnetic-resonance studies.

Nuclear magnetic quadrupole relaxation appears to be a general method for studying the binding of anions to proteins. This is shown by the increase in transverse quadrupole relaxation rate of 35Cl- and 81Br- in the presence of horse liver alcohol dehydrogenase, lysozyme, trypsin, alpha-chymotrypsin, human carbonic anhydrase, fructose-1,6-bisphosphate aldolase and human serum albumin. Of the many possible binding sites at the surface of a protein (e.g. positively charged amino acid side-chains) only a few account for the main part of the relaxation enhancement. This is shown by the decrease in 35Cl- and 81Br- relaxation rate on addition of functional ligands. Large, kinetically inert, complex anions like Pt(CN)2-4 and Au(CN)-2 are found to act as strong competitors towards halogen ions for the high-affinity anion binding sites of a number of proteins. Titrations with complex anions following the 35Cl- or 81Br- relaxation rates are found to be helpful in attempts to elucidate binding mechanisms. Especially, the complex anions may be useful probes for the discrimination between general and metallic anion binding sites in proteins and they also permit correlation of information from X-ray investigations of crystals with that from physical measurements in solution. From the change in halide ion quadrupole relaxation rate on addition of strongly binding ligands the quadrupole coupling constants of the high affinity Cl- and Br- binding sites are estimated using certain assumptions. It is found that for several proteins, comprising the metal-free proteins but also alcohol dehydrogenase and Escherichia coli alkaline phosphatase, the 35Cl quadrupole coupling constants have approximately the same values. For some other metallo-proteins like carbonic anhydrase and a zinc - serum-albumin complex considerably greater quadrupole coupling constants were obtained. The estimated quadrupole coupling constants are used as a basis for a discussion of the interactions involved in anion-protein interactions.     

Interaction of amide inhibitors with the active site of carbonic anhydrase: metal-induced deprotonation of the bound amide group is indicated by slow binding kinetics, by visible spectra of complexes with cobalt enzyme, and by pH effects on binding affinity

Most carbonic anhydrase (CA) inhibitors bind at the active site metal and either are anions or are capable of deprotonation to yield anions. Much less is known about the interaction of CA with inhibitors that have hitherto been considered to bind as neutral species. We report a study of the reversible amide inhibition of Co(II)-substituted CA by iodoacetamide and ethyl carbamate (urethane), as well as the ambivalent oxamate, the monoamide of oxalate. Visible cobalt spectral changes indicate coordination of all these inhibitors to the metal. The pH dependence of the affinity of carbonic anhydrase isozyme I (CA I) for ethyl carbamate and iodoacetamide is formally consistent with their binding either as anionic species to the acid form of the enzyme or as neutral species to the basic form of the enzyme. The former view is in better accord with the spectral data. Most strikingly, reversible binding of iodoacetamide and ethyl carbamate leads to uniquely slow kinetics of ligand association and dissociation that could be followed by simple mixing. The slow association kinetics suggest the involvement of energetically unfavorable deprotonation of the amide group preceding final coordination. The complex pH profile for inhibition of CA I by the ambivalent oxamate is consistent with coordination through the carboxylate group at low pH and through the deprotonated amide group at high pH. The visible spectrum of the complex of Co(II)CA I with oxamate shows a parallel dependence on pH, reflecting this dual coordination mode. Similarly, oxamate dissociation kinetics were biphasic and could be correlated with the pH-dependent spectral changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitive Inhibitory Effects of Acetazolamide upon Interactions with Bovine Carbonic Anhydrase II

Sulfonamide drugs mediate their main therapeutic effects through modulation of the activity of membrane and cytosolic carbonic anhydrases. How interactions of sulfonamide drugs impact structural properties and activity of carbonic anhydrases requires further study. Here the effect of acetazolamide on the structure and function of bovine carbonic anhydrase II (cytosolic form of the enzyme) was evaluated. The Far-UV CD studies indicated that carbonic anhydrase, for the most part, retains its secondary structure in the presence of acetazolamide. Fluorescence measurements using iodide ions and ANS, along with ASA calculations, revealed that in the presence of acetazolamide minimal conformational changes occurred in the carbonic anhydrase structure. These structural changes, which may involve spatial reorientation of Trp 4 and Trp 190 or some other related aminoacyl residues near the active site, considerably reduced the catalytic activity of the enzyme while its thermal stability was slightly increased. Our binding results indicated that binding of acetazolamide to the protein could occur with a 1:1 ratio, one mole of acetazolamide per one mole of the protein. However, the obtained kinetic results supported the existence of two acetazolamide binding sites on the protein structure. The occupation of each of these binding sites by acetazolamide completely inactivates the enzyme. Advanced analysis of the kinetic results revealed that there are two substrate (p-NPA) binding sites whose simultaneous occupation is required for full enzyme activity. Thus, these studies suggest that the two isoforms of CA II should exist in the medium, each of which contains one substrate binding site (catalytic site) and one acetazolamide binding site. The acetazolamide binding site is equivalent to the catalytic site, thus, inhibiting enzyme activity by a competitive mechanism.     

Syntheses and structures of M(L)(X)BPh4 complexes {M=Co(II), Zn(II); L=cis-1,3,5-tris[3-(2-furyl)prop-2-enylideneamino]cyclohexane, X=OAc, NO3}: structural models of the active site of carbonic anhydrase

The metal coordination geometries in the structures of the zinc(II) and cobalt(II) complexes of the ligand cis-1,3,5-tris[3-(2-furyl)prop-2-enylideneamino]cyclohexane (fr-protach) and with the anions nitrate and acetate are structural models for the active site of carbonic anhydrase. The acetate structures show a striking structural correlation with the metal coordination environments in the known bicarbonate forms of the enzyme. Such structures provide a basis for understanding the marked effect of different metal substitution on the catalytic rate of the enzyme.     

13C and 15N NMR and time-resolved fluorescence depolarization study of bovine carbonic anhydrase--4-methylbenzenesulfonamide complex

The influence of the binding of the high-affinity inhibitor, 4-methylbenzenesulfonamide, to the active site of bovine carbonic anhydrase B was studied by 15N- and 13C-NMR spectroscopy. The rotational correlation time dependence on temperature and concentration of the complex was determined by time-resolved fluorescence depolarization measurements. Our experiment provides evidence that the stoichiometry of the interaction of 4-methylbenzenesulfonamide with carbonic anhydrase B is 1:1 and the inhibitor is bound in anionic form. The 15N-NMR relaxation parameters confirm our previous conclusions about the presence of librational motions in the active site of carbonic anhydrase and indicate that the internal motion in the enzyme-inhibitor complex is more restricted than the backbone motion in the uncomplexed native enzyme.     

A fast screening method for histochemical localization of carbonic anhydrase. Application to kidney, skeletal muscle, and thrombocytes

A simple method for histochemical localization of carbonic anhydrase using 5-dimethyl-amino-naphthalene-1-sulfonamide (DNSA) is described. Cryosections of tissues, or cell smears, are incubated in 3 to 10 X 10(-5) M DNSA and viewed in a fluorescence microscope. Upon excitation with ultraviolet light, sites of carbonic anhydrase localization can be identified by an intense blue fluorescence, which is due to the emission of blue light (lambda max = 470 nm) by carbonic anhydrase-DNSA complexes. This fluorescence can be largely suppressed by simultaneous incubation with 1 X 10(-4) to 2 X 10(-3) M concentrations of nonfluorescent carbonic anhydrase inhibitors, displacing DNSA from its binding site on the enzyme. Application of the method to kidney, skeletal muscle, and thrombocytes yields patterns of carbonic anhydrase localization that are in good agreement with results that have been obtained with a variety of other techniques.     

Carbonic anhydrase activity in Madin Darby canine kidney cells. Evidence for intercalated cell properties

Madin Darby canine kidney (MDCK) renal epithelial cell cultures have been investigated with respect to their potency to express carbonic anhydrase activity using histochemical methods. Acetazolamide inhibitable carbonic anhydrase activity could be detected in the cytoplasmic compartment as well as in the apical membrane of cells when grown on solid culture supports. Cells forming domes in MDCK monolayers exhibit the highest histochemically detectable enzyme activity. The attempt to subculture clonal cell lines from MDCK monolayer cultures resulted in the establishment of 5 clones, slightly different with respect to size and shape of cells and their potency to form domes. Scanning electron microscopy ensured the identification of one clone (1A4), which distinctly differed from the others with respect to the apical membrane architecture. Co-localization of peanut agglutinin and carbonic anhydrase activity at the plasma membrane always revealed a combined occurrence of enzyme reactivity and lectin binding in the apical membrane domain. Both, lectin binding and carbonic anhydrase activity were distinctly more intense in plasma membrane regions equipped with microvilli. From the results it is concluded that MDCK cells in tissue culture retained properties of intercalated cells of the nephron collecting duct segment.     

Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.     

Role of hemoglobin in proton transfer to the active site of carbonic anhydrase.

The binding of bovine oxyhemoglobin to bovine carbonic anhydrase with a dissociation constant between 10(-5) and 10(-7) M has been determined by countercurrent distribution using aqueous, biphasic polymer systems. This result provides an explanation for the very efficient proton transfer between hemoglobin and carbonic anhydrase, a transfer which enhances the catalytic activity of carbonic anhydrase as measured by 18O exchange between bicarbonate and water at chemical equilibrium (Silverman, D. N., Tu, C. K., and Wynns, G. C. (1978) J. Biol. Chem, 253, 2563-2567). Two rate constants describing 18O exchange activity of carbonic anhydrase at pH 7.5 show saturation behavior when plotted against hemoglobin concentration consistent with a dissociation constant of 2.5 X 10(-6) M between bovine hemoglobin and carbonic anhydrase. Interpretation of these rate constants in terms of a two-step model for 18O exchange indicates that hemoglobin enhances the rate of exchange from carbonic anhydrase of water containing the oxygen abstracted from bicarbonate, but does not affect the catalytic interconversion of CO2 and HCO3- at chemical equilibrium.     

Kinetic and magnetic properties of cobalt(III) ion in the active site of carbonic anhydrase.

Cobalt(III)bovine carbonic anhydrase B was prepared by the oxidation of the cobalt(II) enzyme with hydrogen peroxide and was purified by affinity chromatography. The oxidation reaction is inhibited by specific inhibitors of carbonic anhydrase. The inhibition is explained by the fact that the Co(II)-enzyme . inhibitor complex cannot be directly oxidized by hydrogen peroxide, but has to dissociate to give free Co(II) enzyme which is then oxidized. The Co(III) ion in Co(III) carbonic anhydrase cannot be directly substituted by zinc ions. It can be reduced by either dithionite or BH-4 ions to give, first, their complexes with the Co(II) enzyme, and upon their removal, a fully active Co(II) enzyme. Cyanide and azide bind to cobalt(III) carbonic anhydrase with similar rate constants of 0.060 +/- 0.005 and 0.070 +/- 0.007 M-1 S-1 respectively. These rates are faster than those found for Co(III) inorganic complexes. The Co(III) ion in both Co(III) carbonic anhydrase and Co(III) carboxypeptidase A was found to be diamagnetic, indicating a near octahedral symmetry.     

Complete amino acid sequence of ovine salivary carbonic anhydrase

The primary structure of the secreted carbonic anhydrase from ovine salivary glands has been determined by automated Edman sequence analysis of peptides generated by cyanogen bromide and tryptic cleavage of the protein and Staphylococcus aureus V8 protease, trypsin, and alpha-chymotrypsin subdigests of the large cyanogen bromide peptides. The enzyme is a single polypeptide chain comprising 307 amino acids and contains two apparent sites of carbohydrate attachment at Asn-50 and Asn-239. The protein contains two half-cystine residues at 25 and 207 which appear to form an intramolecular disulfide bond. Salivary carbonic anhydrase shows 33% sequence identity with the ovine cytoplasmic carbonic anhydrase II enzyme, with residues involved in the active site highly conserved. Compared to the cytoplasmic carbonic anhydrases, the secreted enzyme has a carboxyl-terminal extension of 45 amino acids. This is the first report of the complete amino acid sequence of a secreted carbonic anhydrase (CA VI).     

Hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle

We report three experiments which show that the hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle occurs at a site on the enzyme different than the active site for CO2 hydration. This is in contrast with isozymes I and II of carbonic anhydrase for which the sites of 4-nitrophenyl acetate hydrolysis and CO2 hydration are the same. The pH profile of kcat/Km for hydrolysis of 4-nitrophenyl acetate was roughly described by the ionization of a group with pKa 6.5, whereas kcat/Km for CO2 hydration catalyzed by isozyme III was independent of pH in the range of pH 6.0-8.5. The apoenzyme of carbonic anhydrase III, which is inactive in the catalytic hydration of CO2, was found to be as active in the hydrolysis of 4-nitrophenyl acetate as native isozyme III. Concentrations of N-3 and OCN- and the sulfonamides methazolamide and chlorzolamide which inhibited CO2 hydration did not affect catalytic hydrolysis of 4-nitrophenyl acetate by carbonic anhydrase III.