Esterase Activity Of Carbonic Anhydrase Research Articles

Gas Chromatography with Modified pH-Sensitive Pellets in Evaluating Esterase Activity of Carbonic Anhydrase III Enzyme: Drug Discovery Approach

Gas Chromatography with Modified pH-Sensitive Pellets in Evaluating Esterase Activity of Carbonic Anhydrase III Enzyme: Drug Discovery Approach

Benazepril and Moexipril Inhibit the Esterase Activity of Carbonic Anhydrase I and II at Micromolar Concentration, an in Vitro Investigation

Benazepril and Moexipril Inhibit the Esterase Activity of Carbonic Anhydrase I and II at Micromolar Concentration, an in Vitro Investigation

Captopril/enalapril inhibit promiscuous esterase activity of carbonic anhydrase at micromolar concentrations: An in vitro study

The inhibitory activity of captopril, a thiol-containing competitive inhibitor of the angiotensin-converting enzyme, ACE, against esterase activity of carbonic anhydrase, CA was investigated. This small molecule, as well as enalapril, was selected in order to represents both thiol and carboxylate, as two well-known metal binding functional groups of metalloprotein inhibitors. Since captopril, has also been observed to inhibit other metalloenzymes such as tyrosinase and metallo-beta lactamase through binding to the catalytic metal ions and regarding CA as a zinc-containing metallo-enzyme, in the current study, we set out to determine whether captopril/enalapril inhibit CA esterase activity of the purified human CA II or not? Then, we revealed the inhibitors' potencies (IC50, Ki and Kdiss values) and also mode of inhibition. Our results also showed that enalapril is more potent CA inhibitor than captopril. Since enalapril represents no sulfhydryl moiety, thus carboxylate group may have a determinant role in inhibiting of CA esterase activity, the conclusion confirmed by molecular docking studies. Additionally, since CA inhibitory potencies of captopril/enalapril were much lower than those of classic sulfonamide drugs, the findings of the current study may explain why these drugs exhibit no effective CA inhibition at the concentrations reached in vivo and also may shed light on the way of generating new class of inhibitors that will discriminately inhibit various CA isoforms.

Degradation products of the artificial azo dye, Allura red, inhibit esterase activity of carbonic anhydrase II: A basic in vitro study on the food safety of the colorant in terms of enzyme inhibition

Allura red is a widely used food colorant, but there is debate on its potential security risk. In the present study, we found that degradation products of the dye were more potent agents with higher carbonic anhydrase inhibitory action than the parent dye. The mechanism by which the compounds inhibit the enzyme activity has been determined as competitive mode. In addition, the enzyme binding properties of the compounds were investigated employing different spectroscopic techniques and molecular docking. The analyses of fluorescence quenching data revealed the existence of the same binding site for the compounds on the enzyme molecule. The thermodynamic parameters of ligand binding were not similar, which indicates that different interactions are responsible in binding of the parent dye and degradation products to the enzyme. It appears that enzyme inhibition should be considered, more seriously, as a new opened dimension in food safety.

<i>Mytilus galloprovincialis</i> Carbonic Anhydrase II: Activity and cDNA Sequence Analysis

The esterase activity of carbonic anhydrase (CA) was investigated in mussels sampled at 24 locations along the Croatian coast of Adriatic Sea. The gills were the target tissue because the respiratory, ionic transport and pH regulatory enzyme function of CA and its potential usage as biomarkers of environmental pollution was the main topic. Total esterase activity was measured in cytosolic fraction by colorimetric end-point reaction using p-nitrophenyl acetate as enzyme substrate. CA activity was estimated by the same enzymatic reaction using acetazolamide as a specific CA inhibitor. The results of total esterase activities in winter (March; all sites average value 0.137±0.057) were lower than determined for summer season (August; 0.153±0.036) at almost all investigated locations. CA activities determined in gills of mussel sampled in winter ranged from 1.75% to 24.65% of total esterase activities and in summer samples were lower (0.83 to 13.45%). Although recent research showed potential application of CA activity in bioassay and biomarker in pollution studies, further research is needed. Here we report a short-term simple colorimetric microplate method which can be applied for analyses of large numbers of samples. Furthermore in this study we characterized full length coding sequence (cDNA) of M. galloprovincialis carbonic anhydrase II (CAII). The CAII cDNA (with the 5’ and 3’ untranslated regions) is 1317 bp long. The putative open reading frame encodes a polypeptide of 256 amino acids, with a theoretical pI/Mw 5.87/28.416 kDa and conserved domains (active site and zinc binding site).

Esterase activity of carbonic anhydrases serves as surrogate for selecting antibodies blocking hydratase activity

Carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) were proposed as potential targets for cancer therapy more than 20 years ago. However, to date, there are only very few antibodies that have been described to specifically target CA9 and CA12 and also block the enzymatic activity of their targets. One of the early stage bottlenecks in identifying CA9- and CA12-inhibiting antibodies has been the lack of a high-throughput screening system that would allow for rapid assessment of inhibition of the targeted carbon dioxide hydratase activity of carbonic anhydrases. In this study, we show that measuring the esterase activity of carbonic anhydrase offers a robust and inexpensive screening method for identifying antibody candidates that block both hydratase and esterase activities of carbonic anhydrase’s. To our knowledge, this is the first implementation of a facile surrogate-screening assay to identify potential therapeutic antibodies that block the clinically relevant hydratase activity of carbonic anhydrases.

Allura red, the artificial azo dye, inhibits esterase activity of carbonic anhydrase II: A preliminary study on the food safety in term of enzyme inhibition

Allura red, the artificial azo dye, inhibits esterase activity of carbonic anhydrase II: A preliminary study on the food safety in term of enzyme inhibition

Directed Evolution of the Promiscuous Esterase Activity of Carbonic Anhydrase II

A promiscuous activity of an existing enzyme can confer an evolutionary advantage by providing an immediate response to a new selection pressure and a starting point for the divergence of a new enzyme. This work seeks to examine how this process might take place. Human carbonic anhydrase II (hCAII) is an enzyme that evolved to catalyze the reversible hydration of CO(2) and performs this task at a remarkable rate (k(cat) approximately 10(6) s(-)(1)). hCAII also exhibits promiscuous activity toward highly activated esters such as 4-nitrophenyl acetate. We describe a much weaker esterase activity of hCAII toward the bulkier and much less activated ester substrate 2-naphthyl acetate (2NA). Directed evolution of hCAII produced a variant with 40-fold higher rates toward 2NA, owing to two mutations: one within the active site (Ala65Val) and one at its mouth (Thr200Ala). Structure-activity studies suggest that these mutations led to adaptation of the active site for bulkier substrates and for the catalysis of nonactivated esters. The mutations did not, however, significantly alter the native activity of hCAII. Our results support the notion that the evolution of a new function can be driven by mutations that increase a promiscuous function (which serves as the starting point for the evolutionary process) but do not harm the native function.

Peroxynitrite Inactivates Carbonic Anhydrase II by Releasing Active Site Zinc Ion

Peroxynitrite enters erythrocytes through band 3 anion exchanger and oxidizes cytosolic proteins therein. As a protein associated with band 3, carbonic anhydrase II may suffer from peroxynitrite-induced oxidative damages. Esterase activity of carbonic anhydrase II decreased as the concentration of peroxynitrite increased. Neither hydrogen peroxide nor hypochlorite affected the enzyme activity. Inactivation of the enzyme was in parallel with the release of zinc ion, which is a component of the enzyme's active site. SDS-PAGE of peroxynitrite-treated samples showed no indication of fragmentation but non-denaturing PAGE exhibited new bands with lower positive charges. Western analysis demonstrated that nitration of tyrosine residues increased with the peroxynitrite concentration but the sites of nitration could not be determined. Instead MALDI-TOF analysis identified tryptophan-245 as a site of nitration. Such modification of tryptophan residues is responsible for the decrease in tryptophan fluorescence. These results demonstrate that peroxynitrite nitrates tyrosine and tryptophan residues of carbonic anhydrase II without causing fragmentation or dimerization. The peroxynitrite-induced inactivation of the enzyme is primarily due to the release of zinc ion in the enzyme's active site.

Semi-automated enzymatic measurement of serum zinc concentration

Objective: To measure serum zinc concentration by means of carbonic anhydrase reactivation using an automated analyzer. Methods: The zinc content of carbonic anhydrase (CA), whose cofactor is zinc, was removed by dialysis against pyridine 2 to 6 dicarboxylic acid and a pure apoenzyme was obtained. Serum proteins were precipitated with trichloroacetic acid (TCA) solution and the supernatant fraction of the sample was used to determine the zinc concentration. The negative effects of the precipitant on CA activity in the assay were completely removed, reaction conditions for maximal CA activity were provided and the color of the product was enhanced and stabilized. P-nitrophenyl acetate was used as the substrate and the change of absorbance of p-nitrophenol which was produced was followed at 400 nm. The initial rate of the esterase activity of CA was measured by using an automated analyzer. Analytical performance characteristics of the assay were determined. The zinc concentrations in serum samples of healthy subjects and patients were measured. Results: The enzymatic assay is accurate, sensitive, specific and is not affected by other metals. There was excellent agreement with the results obtained using an atomic absorption spectrophotometer (AAS) (y = 0.98X + 0.18, r = 0.99). Serum zinc concentrations were found to be higher in patients with vivax malaria, and lower in patients with cutaneous leishmaniasis than in healthy subjects. Conclusion: The enzymatic method is suitable for semiautomated measurement of serum zinc concentration.

Inhibition of bovine carbonic anhydrase by new sulfonamide compounds.

Inhibitory effects of three new derivatives of 2-acetylamino-1,3,4-thiadiazole-5-sulfonamide on bovine carbonic anhydrase have been investigated. The new compounds are 2-(3-chloropropionylamino)-1,3,4-thiadiazole-5-sulfonamide, 2-(2,2-dichloroacetylamino)-1,3,4-thiadiazole-5-sulfonamide, and 2-(3-phenylpropionylamino)-1,3,4-thiadiazole-5-sulfonamide. The new compounds inhibit the esterase activity of carbonic anhydrase noncompetitively and have inhibition constants and I(50) values very similar to those for 2-acetylamino-1,3,4-thiadiazole-5-sulfonamide, the latter being clinically used in the treatment of glaucoma.

The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO 2 hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K m of 2.8 m m. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate ( K i = 1.22 × 10 −3M), arsenate ( K i = 1.17 × 10 −3M), and molybdate ( K i = 1.34 × 10 −7M), with these inhibitors having no effect on the CO 2 hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO 2 hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO 2 hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.

Combination of Bovine Carbonic Anhydrase with a Fluorescent Sulfonamide

Abstract Bovine erythrocyte carbonic anhydrase forms a highly complex with 5-dimethylaminonaphthalene-1-sulfonamide (DNSA). The binding, studied either by enhancement of ligand fluorescence or by the quenching of protein ultraviolet fluorescence, shows that only 1 mole of DNSA is bound per mole of protein; the dissociation constant at pH 7.4 is 2.5 x 10-7 m. The fluorescence of free DNSA in water has peak emission at 580 mµ and a quantum yield of only 0.055, but bound DNSA has an emission maximum at 468 mµ and a yield of 0.84. Arguments are presented to explain the large emission blue shift on the basis that the binding site is extremely hydrophobic and that the —SO2NH2 group of the ligand loses a proton upon binding to the enzyme. The binding appears specifically to involve the sulfonamide site known to exist in carbonic anhydrase; several other fluorescent probe compounds showed no evidence of binding to the enzyme. Calculation of the energy transfer efficiency gave the surprising result that 85% of the photons absorbed by the 7 tryptophan residues are transferred to the single bound DNSA molecule. The transfer efficiency is much higher than hitherto observed for a protein having only one 5-dimethylaminonaphthalene-1-sulfonyl group. Although the diameter of the protein is roughly 51 A, the bound DNSA group is probably within the critical transfer distance R0 (- 21.3 A) of all the tryptophans. The effective average distance between DNSA and tryptophan was found to be 16 A. The fluorescence properties of the complex were quite different from those of a conjugate prepared by reaction of 5-dimethylaminonaphthalene-1-sulfonyl chloride with carbonic anhydrase. Various considerations lead to the conclusion that the sulfonamide-binding site and the tryptophan residues are in the interior of the protein. The tryptophan fluorescence of the protein was 73% quenched by the binding of 1 DNSA molecule. Although large, this degree of quenching was less than the over-all efficiency of energy transfer of photons absorbed by the protein. This result indicates that the fluorescence efficiencies of the 7 tryptophans are different, and that DNSA is bound in such a way that energy transfer occurs with greater probability from those tryptophan residues which are relatively less fluorescent. DNSA inhibits the esterase activity of carbonic anhydrase as tested with the substrate, p-nitrophenyl acetate. Direct measurements of fluorescence decay times permitted calculation of the rotational relaxation time of carbonic anhydrase from depolarization of fluorescence data. The value of about 30 nsec for the relaxation time is consistent with a low degree of molecular asymmetry.