Activation of mammalian skeletal-muscle carbonic anhydrase III by arginine modification

Purified carbonic anhydrase isozymes I, II, and III (CA I, CA II, CA III) from various sources were treated with 2,3-butanedione and their bicarbonate dehydration reactions followed. The specific activities of human and bovine CA I and CA II and chicken CA III were not affected by the butanedione treatment, whereas the activities of human, gorilla, and bovine CA III were rapidly activated. These findings suggest that one, or both, of the two arginyl residues which appear to be unique to the active sites of the mammalian CA III isozymes are modified by butanedione.     

Crystallization of and preliminary X-ray data for bovine carbonic anhydrase III

Crystals of bovine carbonic anhydrase III have been grown in a solution of polyethylene glycol. The crystals are monoclinic, space group P2(1), with the unit cell parameters a = 50.6 A, b = 44.7 A, c = 56.9 A, and beta = 90.3 degrees. The asymmetric unit contains 1 molecule. The diffraction pattern extends beyond 2.0-A resolution.     

The inhibition of bovine carbonic anhydrase by saccharin and 2- and 4-carbobenzoxybenzene sulfonamide

The present work demonstrates that the high-activity zinc metalloenzyme, carbonic anhydrase (CA II) from bovine erythrocytes is inhibited by the cyclic sulfimide, saccharin, and 2- and 4-carbobenzoxybenzene sulfonamide. A spectrophotometric method was employed to monitor the enzymatically catalyzed hydrolysis of p-nitrophenyl acetate by following the increase in absorbance at 410 nm which accompanies p-nitrophenoxide/p-nitrophenol formation. The more rapid enzymatic hydration of CO2 was monitored by using a stopped-flow spectrophotometer as well as by a modified colorimetric method of Wilbur and Anderson. The studies show that, at a given molar ratio of inhibitor to enzyme, the degree of inhibition of the enzymaic hydration of CO2 and hydrolysis of p-nitrophenyl acetate by the inhibitory compounds is essentially the same. Kinetic analyses were made at 25.0 degrees at pH 6.5 (MES buffers), pH 6.9 (HEPES buffers) and pH 7.9 (HEPES buffers) with ionic strength regulated by the addition of appropriate quantities of sodium sulfate. Lineweaver-Burk plots were used to evaluate apparent inhibition constants for each of the three inhibitors. For all the inhibitors studied, inhibition appears to be mixed (competitive/noncompetitive). For saccharin in the presence of sodium sulfate, the extent of inhibition is considerably decreased. It was found for the three inhibitors that the inhibitory potency decreases with increasing pH, and that the inhibitory potency is extremely sensitive to the shape of these rather closely related molecules. For example, apparent inhibition constants for the enzymatic hydrolysis of p-nitrophenyl acetate at pH 6.9 were Ki (saccharin) = 0.20 mM, Ki (2-carbobenzoxybenzene sulfonamide) = 0.54 mM and Ki (4-carbobenzoxybenzene sulfonamide) = 1.6 microM. For the enzymatic hydration of CO2 at pH 6.9, 0.10 mM saccharin caused 50% inhibition while 7.0 nM 4-carbobenzoxybenzene sulfonamide resulted in 50% inhibition. The results suggest that sulfonamide inhibition is caused by formation of a monodentate ligand at the zinc ion of the enzyme active site and that the more linear 4-carbobenzoxybenzene sulfonamide is better able to enter a conical enzyme active site than is 2-carbobenzoxybenzene sulfonamide or saccharin.     

Formation of amyloid fibrils by bovine carbonic anhydrase

Amyloids are typically characterized by extensive aggregation of proteins where the participating polypeptides are involved in formation of intermolecular cross beta-sheet structures. Alternate structure attainment and amyloid formation has been hypothesized to be a generic property of a polypeptide, the propensities of which vary widely depending on the polypeptide involved and the physicochemical conditions it encounters. Many proteins that exist in the normal form in-vivo have been shown to form amyloid when incubated in partially denaturing conditions. The protein bovine carbonic anhydrase II (BCA II) when incubated in mildly denaturing conditions showed that the partially unfolded conformers assemble together and form ordered amyloid aggregates. The properties of these aggregates were tested using the traditional Congo-Red (CR) and Thioflavin-T (ThT) assays along with fluorescence microscopy, transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy. The aggregates were found to possess most of the characteristics ascribed to amyloid fibers. Thus, we report here that the single-domain globular protein, BCA II, is capable of forming amyloid fibrils. The primary sequence of BCA II was also analyzed using recurrence quantification analysis in order to suggest the probable residues responsible for amyloid formation.     

Study of bovine carbonic anhydrase by 13C-NMR-spectroscopy

The study of internal mobility in enzymes is of considerable importance for the understanding of their catalytic function, which cannot be adequately described as a property of a rigid protein. [13C]NMR spectroscopy permits simultaneous and selective observation of spectral lines from carbon atoms in many different residues in the enzyme with the chemical shift and relaxation parameters sensitive to structure, conformation and local motion. The changes in internal mobility in bovine carbonic anhydrase B (carbonate hydrolase, EC 4.2.1.1) in the native form and at various stages of denaturation are studied. Measurements of the relaxation parameters (T1, T1 rho) and of the NOE of 13C nuclei in the native protein showed that the extensive beta-sheet together with groups in the active center has a considerable internal librational mobility with tau G about 10(-11) s. This librational mobility is fairly uniform for all the alpha-carbons in the native enzyme. The use of a semiempirical modification of the motional theory proposed by Woessner allows to use simultaneously all the relaxation parameters measured in order to determine reliable values of the various correlation times.     

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.     

Expression of carbonic anhydrase isozymes II and III in developing bovine parotid gland

Two cytosolic carbonic anhydrase isozymes (CA-II and CA-III) were studied by immunohistochemistry in bovine parotid glands during fetal development. In a 3-month-old fetus of crown-rump length (CRL) 17 cm, the expression of CA-II in undifferentiated epithelial cells was observed, whereas immunostaining for CA-III remained negative. At 26 cm CRL (4–5 months old), weak expression of CA-III in large ductal epithelial cells was noted. The accumulation of secreted granules in primary acinar cells was initially observed at this stage. In a newborn calf, anti-CA-II reactivity almost disappeared from most duct segments. The time-dependent expression and distribution of the isozymes in parotid glands may reflect different biological functions of these structurally closely related isozymes. Bovine parotid acinar cells of fetuses would thus appear to possess all the cellular structures and immunohistochemical properties at 4 and 5 months of gestation. CA-II subsequently disappeared from duct segments and nearly all acinar cells in adults were present at or just after birth.     

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. © 1993 Wiley-Liss, Inc.     

Refined structure of bovine carbonic anhydrase III at 2.0 Å 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 Å 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 sugest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II. © 1993 Wiley-Liss, Inc.     

Molecular weight determination of bovine carbonic anhydrase

The molecular weight of bovine carbonic anhydrase was determined by osmometric and sedimentation equilibrium methods. The solvents used were 0.15 M KCl and 6.0 M guanidinium chloride. The value found was 28300 ± 300 which is lower than the values found by other investigators.As a part of the studies the intrinsic viscosities of the enzyme in 4.5 M guanidinium thiocyanate and 6.0 M guanidinium chloride were also ascertained. The values found, 25.4 ml/g and 24.7 ml/g, respectively, are smaller than expected on the basis of the molecular weight. This finding, however, is in agreement with the low value. 0.72 × 10^?3 cm³ mol/g² of the second virial coefficient in 6.0 M guanidinium chloride.     

The inhibition by anion binding of reactions of inorganic radical anions with bovine carbonic anhydrase B

Reactions of the inorganic radical anions, Br(2) and (SCN)2, with bovine carbonic anhydrase (carbonate hydrolyase, EC 4.2.1.1) have been studied by pulse radiolysis. Reaction is almost completely inhibited by the binding of Br-, SCN- and ClO4- to an electrophilic site at the active centre of the enzyme. Dissociation constants for anion binding calculated from the reduction in free radical reactivity agree well with inhibition constants for these anions. The anions OCN- and CN-, although potent inhibitors of carbonic anhydrase activity, have relatively little effect on the reactivity of radical anions with the enzyme. Reaction of radical anions occurs mainly with tryptophan and tyrosine residues in the hydrophobic core of the enzyme, through a channel at the active site. This channel is closed by the anions in accord with their position in the lyotropic series.     

Relaxation of solvent protons by cobalt bovine carbonic anhydrase.

In a recent report, Bertini et al. (Biochem. Biophys. Res. Comm.78, 158–160 (1977)) argued that the low-pH form of Co²⁺-substituted bovine carbonic anhydrase contains a rapidly exchanging water molecule at the cobalt site. The basis for this was the observation of a pH-independent contribution to the solvent water proton relaxation rate; it was suggested that the result was unobserved by previous workers because of the presence of sulfate in the sample buffer. We have repeated the experiments of Bertini et al. and find that the results can be attributed to an ionic strength-induced shift of the pK of the group responsible for the relaxation enhancement. The amount of high-pH form of the enzyme present (determined spectrophotometrically) at every pH correlates with the relaxation rate, whereas the fraction of high-pH form present at a given pH depends on ionic strength. These results are in agreement with earlier data indicating that the low-pH form of the enzyme does not contribute to solvent water proton relaxation.     

Membrane-associated carbonic anhydrase purified from bovine lung

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.     

Activation of carbonic anhydrase II by active-site incorporation of histidine analogs

The hydration of CO2 catalyzed by human carbonic anhydrase II (HCA II) is accompanied by proton transfer from the zinc-bound water of the enzyme to solution. We have replaced the proton shuttling residue His 64 with Ala and placed cysteine residues within the active-site cavity by mutating sites Trp 5, Asn 62, Ile 91, and Phe 131. These mutants were modified at the single inserted cysteine with imidazole analogs to introduce new potential shuttle groups. Catalysis by these modified mutants was determined by stopped-flow and 18O-exchange methods. Specificity in proton transfer was demonstrated; only modifications of the Cys 131-containing mutant showed enhancement in the proton transfer step of catalysis compared with unmodified Cys 131-containing mutant. Modifications at other sites resulted in up to 3-fold enhancement in rates of CO2 hydration, with apparent second-order rate constants near 350 microM(-1) s(-1). These are among the largest values of kcat/Km observed for a carbonic anhydrase.     

Trigger factor-assisted folding of bovine carbonic anhydrase II

Spontaneous refolding of GdnHCl denatured bovine carbonic anhydrase II (BCA II) shows at least three phases: a burst phase, a fast phase, and a slow phase. The fast and slow phases are both controlled by proline isomerization. However, we find that in trigger factor (TF)-assisted BCA II folding, only the fast phase is catalyzed by wild-type TF, suggesting that certain proline residues are accessible in folding intermediates. The refolding yields of BCA II assisted by wild-type TF and TF mutants which lack PPIase activity are about the same, which provides further experimental evidence that the PPIase and chaperone activities of TF are independent. The binding of TF to folding intermediates during BCA II refolding was characterized by chemical crosslinking and Western blotting. A scheme for TF-assisted BCA II folding is proposed and the possible role of the TF dimer as a "binding" chaperone in vivo is discussed.     

Thermal inactivation and conformational lock of bovine carbonic anhydrase

The kinetics of thermal inactivation of bovine carbonic anhydrase (BCA) was studied in a 50 mM Tris-HCl buffer, pH 7.8 using p-nitrophenyl acetate as substrate in absorbance of 400 nm by UV-VIS spectrophotometry. The number of conformational locks and inter-subunit amino acid residues of BCA were obtained by thermal inactivation analysis. The cleavage bonds between dimers of BCA during thermal dissociation and type of interactions between specific amino acid residues were also detected. The thermal inactivation curves were plotted in temperatures ranging between 40-70°C. It was shown several phases for inactivation of BCA at 65°C. Analyses of the curves were done by the conformational lock theory. The subunits are dissociated and several intermediates appear during inactivation through increasing the temperature in comparison with native state. Dynamic light scattering measurements was done to study the changes in hydrodynamic radius during thermal inactivation. Three distinct zones were shown in DLS data. Biochemical computation using ligplot is performed to find the inter-subunit amino acid residues for BCA.     

Immunohistochemical detection of bovine ruminal carbonic anhydrase isoenzyme

Summary Rabbits immunized with low-activity ruminal carbonic anhydrase (RCA) isoenzyme, extracted from ruminal epithelial cells isolated by digestion with trupsin, yielded anti-RCA sera which reacted specifically with bovine RCA in double agar gel diffusion and immunoelectrophoretic tests, but failed to cross-react with bovine erythrocyte CA. The localization of RCA was identified in histological sections and isolated ruminal epithelial cell preparations by indirect immunofluorescence and immunoperoxidase tests as the basal, spinosum and granulosum layers of ruminal mucous epithelium.