Activity Of Human Carbonic Anhydrase Research Articles

Sulfonamides stimulate ROS formation upon glycation of human carbonic anhydrase II

Advanced glycation end products are the most important species of glycation pathway, and cause disorders such as oxidative stress and diabetes. Sulfonamide compounds, which are generally known as widespread inhibitors, are potential agents used in different drug products, which can readily enter biological matrices. In the present work, the structure and activity of human carbonic anhydrase II studied in the presence of glucose as well as four sulfonamide agents from different views. These included enzyme kinetics, free lysine content, fluorescence spectroscopy, circular dichroism, and ROS measurement. Our results indicated that upon glycation, the structure of HCA II collapses and 8 to 13 lysine residues will be more available based on ligand incubation. Secondary and tertiary structural changes were also observed in the presence and absence of sulfonamide agents using fluorescence and circular dichroism methods, respectively. These spectroscopic data also showed a remarkable increase in hydrophobicity and decrease in α-helix contents during glycation, especially after 35 days of incubation. ROS assay was studied in the presence of glucose and sulfonamide compounds, that demonstrated the role of sulfonamide compounds in ROS formation in the presence of glucose in a synergistic manner. Overall, our data indicated that sulfonamides act as a stimulant factor upon prolonged interaction with HCA II and may intensify the complications of some disorders, such as diabetes and other conformational diseases.

Energetics and dynamics of the proton shuttle of carbonic anhydrase II

Human carbonic anhydrase II catalyzes the reversible reaction of carbon dioxide and water to form bicarbonate and a proton. His64-mediated proton shuttling between the active site and the bulk solvent is rate limiting. Here we investigate the protonation behavior of His64 as well as its structural and dynamic features in a pH dependent way. We derive two pKa values for His64, 6.25 and 7.60, that we were able to assign to its inward and outward conformation. Furthermore, we show that His64 exists in both conformations equally, independent of pH. Both conformations display an equal distribution of their two neutral tautomeric states. The life time of each conformation is short and both states display high flexibility within their orientation. Therefore, His64 is never static, but rather poised to change conformation. These findings support an energetic, dynamic and solution ensemble-based framework for the high enzymatic activity of human carbonic anhydrase II.

The Effect of Nanoparticles on the Structure and Enzymatic Activity of Human Carbonic Anhydrase I and II.

Human carbonic anhydrases (hCAs) belong to a well characterized group of metalloenzymes that catalyze the conversion of carbonic dioxide into bicarbonate. There are currently 15 known human isoforms of carbonic anhydrase with different functions and distribution in the body. This links to the relevance of hCA variants to several diseases such as glaucoma, epilepsy, mountain sickness, ulcers, osteoporosis, obesity and cancer. This review will focus on two of the human isoforms, hCA I and hCA II. Both are cytosolic enzymes with similar topology and 60% sequence homology but different catalytic efficiency and stability. Proteins in general adsorb on surfaces and this is also the case for hCA I and hCA II. The adsorption process can lead to alteration of the original function of the protein. However, if the function is preserved interesting biotechnological applications can be developed. This review will cover the knowledge about the interaction between hCAs and nanomaterials. We will highlight how the interaction may lead to conformational changes that render the enzyme inactive. Moreover, the importance of different factors on the final effect on hCAs, such as protein stability, protein hydrophobic or charged patches and chemistry of the nanoparticle surface will be discussed.

Mechanistic Explanation of the Weak Carbonic Anhydrase's Esterase Activity.

In order to elucidate the elementary mechanism of the promiscuous esterase activity of human carbonic anhydrase (h-CA), we present an accurate theoretical investigation on the hydrolysis of fully-acetylated d-glucose functionalized as sulfamate. This h-CA’s inhibitor is of potential relevance in cancer therapy. The study has been performed within the framework of three-layer ONIOM (QM-high:QM’-medium:MM-low) hybrid approach. The computations revealed that the hydrolysis process is not energetically favored, in agreement with the observed weak carbonic anhydrase’s esterase activity.

Synthesis of 3-chloro-1-substituted aryl pyrrolidine-2,5-dione derivatives: discovery of potent human carbonic anhydrase inhibitors

Carbonic anhydrase isoenzymes are important metalloenzymes that are involved in many physiologic processes, in which they catalyze the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3 –) and protons (H+) via a metal hydroxide nucleophilic mechanism. Because of their known biological activities and potential as carbonic anhydrase inhibitors, the present study focused on developing of a convenient route to synthesize 3-chloro-1-aryl pyrrolidine-2,5-diones (2b). This synthetic route started with (Z)-4-oxo-4-(arylamino)but-2-enoic acid (3b) and sulfurous dichloride (SOCl2) and resulted in a ring closing reaction that produced a series of 3-chloro-N-aryl maleimide derivatives (20–29) in good yields. This is the first report of the syntheses of 3-chloro-1-aryl pyyrolidine-2,5-diones 20–23 and 27–29 by ring-closing reactions of (Z)-4-oxo-4-(arylamino)but-2-enoic acid. The structures of all of the products were determined by 1H-NMR, 13C-NMR and infrared spectroscopy. Their biological activities were studied against human carbonic anhydrase I, and II. The 3-chloro-1-aryl pyrrolidine-2,5-diones strongly inhibited the activity of human carbonic anhydrase I and II, with K i values in the low nanomolar range of 23.27–36.83 nmol/L against human carbonic anhydrase I and 10.64–31.86 nmol/L against human carbonic anhydrase II.

Azobenzene-based inhibitors of human carbonic anhydrase II.

Aryl sulfonamides are a widely used drug class for the inhibition of carbonic anhydrases. In the context of our program of photochromic pharmacophores we were interested in the exploration of azobenzene-containing sulfonamides to block the catalytic activity of human carbonic anhydrase II (hCAII). Herein, we report the synthesis and in vitro evaluation of a small library of nine photochromic sulfonamides towards hCAII. All molecules are azobenzene-4-sulfonamides, which are substituted by different functional groups in the 4´-position and were characterized by X-ray crystallography. We aimed to investigate the influence of electron-donating or electron-withdrawing substituents on the inhibitory constant Ki. With the aid of an hCAII crystal structure bound to one of the synthesized azobenzenes, we found that the electronic structure does not strongly affect inhibition. Taken together, all compounds are strong blockers of hCAII with Ki = 25–65 nM that are potentially photochromic and thus combine studies from chemical synthesis, crystallography and enzyme kinetics.

Inactivation and adsorption of human carbonic anhydrase II by nanoparticles.

The enzymatic activity of human carbonic anhydrase II (HCAII) was studied in the presence of nanoparticles of different nature and charge. Negatively charged nanoparticles inhibit HCAII whereas no effect is seen for positively charged particles. The kinetic effects were correlated with the strength of binding of the enzyme to the particle surface as measured by ITC and adsorption assays. Moreover, conformational changes upon adsorption were observed by circular dichroism. The main initial driving force for the adsorption of HCAII to nanoparticles is of electrostatic nature whereas the hydrophobic effect is not strong enough to drive the initial binding. This is corroborated by the fact that HCAII do not adsorb on positively charged hydrophobic polystyrene nanoparticles. Furthermore, the dehydration of the particle and protein surface seems to play an important role in the inactivation of HCAII by carboxyl-modified polystyrene nanoparticles. On the other hand, the inactivation by unmodified polystyrene nanoparticles is mainly driven by intramolecular interactions established between the protein and the nanoparticle surface upon conformational changes in the protein.

Insight into the promiscuous activity of human carbonic anhydrase against the cyanic acid substrate from a combined QM and QM/MM investigation

The promiscuous activity of human carbonic anhydrase (hCAII) against a non-physiological cyanic acid substrate has been investigated by using a combined QM and QM/MM level of theory. Results show that the hCAII is able to hydrate the cyanic acid by a reaction mechanism similar to that of the CO2 native substrate. The energy barrier for the nucleophilic attack is found to be 15.6 and 4.3 kcal mol(-1) at QM and QM/MM levels, respectively. This result underlines the importance of taking into account the surrounding residues around the active site in the presence of the substrate having small molecular sizes. The carbamate is strongly stabilized with respect to the bicarbonate of the native substrate indicating a more difficult release of the reaction product.

Human Carbonic Anhydrase III: Structural and Kinetic Study of Catalysis and Proton Transfer,

The residue phenylalanine 198 (Phe 198) is a prominent cause of the lower activity of human carbonic anhydrase III (HCA III) compared with HCA II and other isozymes which have leucine at this site. We report the crystal structures of HCA III and the site-directed mutant F198L HCA III, both at 2.1 A resolution, and the enhancement of catalytic activity by exogenous proton donors containing imidazole rings. Both enzymes had a hexahistidine extension at the carboxy-terminal end, used to aid in purification, that was ordered in the crystal structures bound in the active site cavity of an adjacent symmetry-related enzyme. This observation allowed us to comment on a number of possible binding sites for imidazole and derivatives as exogenous proton donors/acceptors in catalysis by HCA III. Kinetic and structural evidence indicates that the phenyl side chain of Phe 198 in HCA III, about 5 A from the zinc, is a steric constriction in the active site, may cause altered interactions at the zinc-bound solvent, and is a binding site for the activation of catalysis by histidylhistidine. This suggests that sites of activation of the proton-transfer pathway in carbonic anhydrase are closer to the zinc than considered in previous studies.

COX-2 inhibitors and carbonic anhydrase activity

Purpose To compare the carbonic anhydrase inhibition (CAI) profile of selective and non-selective COX-2 inhibitors with the prototypic CAI acetazolamide (ACZ). Methods: The CO2 hydration activity of human carbonic anhydrase II (hCAII) was determined by a colorimetric method. The enzyme concentration was 5nM. Stock solutions of the inhibitors were dissolved in either ethanol or DMSO. The fraction of inhibitory activity was calculated by comparing CO2 hydration activity of the enzyme in the absence and presence of the test compounds (tested blind to the study hypothesis). Duplicate determinations were performed. Commercially available compounds purchased at the pharmacy and excipient free compounds were used. Results: The IC50 (nM) values (from the most potent CAI to least) were: ACZ, 7.5; celecoxib, 410; valdecoxib, 24800; rofecoxib > 3X10 5 and diclofenac > 3X10 5. The structural basis underlying the differences in potency is in all likelihood the pharmacophore SO2NH2, the common molecular moiety of acetazolamide, celecoxib and valdecoxib. The sulfonamide motif is absent from rofecoxib and diclofenac. Discussion: We have demonstrated an unusual inhibition binding profile for the COX-2 inhibitor celecoxib against hCAII, an isozyme with numerous physiological roles. The nanomolar CAI is linked to the pharmacophore SO2 NH2. We suggest that celecoxib may mediate some of its actions, e.g. anti-cancer effects, HCO3- modulation, by the COX-2 independent mechanism of CAI. Clinical Pharmacology & Therapeutics (2004) 75, P49–P49; doi: 10.1016/j.clpt.2003.11.185

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.

Interactions of active-site residues and catalytic activity of human carbonic anhydrase III.

To elucidate the interactions between residues found in the active-site cavity of human carbonic anhydrase III, we have prepared a series of single and double mutants with Lys-64, Arg-67, and Phe-198 replaced with Ala, Asp, Glu, His, and Leu. Rates of catalysis were determined using 18O exchange between CO2 and water measured by mass spectrometry and initial velocity measured by stopped-flow spectrophotometry. Replacement of these residues resulted in increases in kcat/Km for CO2 hydration as much as 200-fold and increases in the pKa of the zinc-bound water by as much as 3.5 units. We conclude that the effect of replacements made at positions 64, 67, and 198 were in general additive for kcat/Km for CO2 hydration, indicating that there is no interaction between these sites that affects the catalytic interconversion of CO2 and HCO3-. One notable exception is the antagonism exhibited by the double mutant of human carbonic anhydrase III containing Glu-64 and Leu-198. The data also show that one source of the large enhancement of kcat/Km for the mutant containing Asp-198 in human carbonic anhydrase III is the presence of both Asp-198 and Lys-64; when Lys-64 was replaced with Ala, a reduction of catalytic activity was observed. These results provide an additional view of the independent interactions of amino acids that affect the catalytic pathway of isozyme III, the least active of the known carbonic anhydrase isozymes.

Histidine 64 is not required for high CO 2 hydration activity of human carbonic anhydrase II

To test the hypothesis that histidine 64 in carbonic anhydrase II has a crucial role as a ‘proton shuttle group’ during catalysis of CO 2-HCO − 3 interconversion, this residue was replaced by lysine, glutamine, glutamic acid and alanine by site-directed mutagenesis. All these variants turned out to have high CO 2 hydration activities. The k cat values at pH 8.8 and 25°C were only reduced by 1.5–3.5-fold compared to the unmodified enzyme. These results show that intramolecular proton transfer via His 64 is not a dominating pathway in the catalytic reaction. The variants also catalyze the hydrolysis of 4-nitrophenyl acetate. The p K a values for the activity-controlling group are between 6.8 and 7.0 for all studied forms of the enzyme except the Glu 64 variant which shows a complex pH dependence with the major p K a shifted to 8.4

Effects of high concentrations of salt on the esterase activity of human carbonic anhydrase

Effects of high concentrations of salt on the esterase activity of human carbonic anhydrase

The Dinitrophenylation of Human Carbonic Anhydrase B

Abstract The CO2 hydration activity of human carbonic anhydrase B is rapidly inactivated by 1-fluoro-2,4-dinitrobenzene at neutral pH. The inactivation reaction is second order and is retarded by the presence of various enzyme inhibitors. The second order rate constant for inactivation is about 100 times the second order rate constant for the reaction of 1-fluoro-2,4-dinitrobenzene and α-N-acetylhistidine. The inactivated enzyme was purified and was found to contain one 2,4-dinitrophenyl group per molecule, at the 3' position of a histidine residue. While such a reacted enzyme has lost more than 95% of its CO2 hydration activity, it retains over half its esterase activity. The dinitrophenyl group can be quantitatively removed by thiolysis, regenerating a fully active native carbonic anhydrase. Dinitrophenyl carbonic anhydrase binds iodoacetate with somewhat greater affinity than the unmodified enzyme. However, iodoacetate does not react covalently with the dinitrophenyl enzyme, as it does with the native enzyme. Analysis of the tryptic peptides of dinitrophenyl carbonic anhydrase shows that reaction takes place with histidine residue 204, the same residue modified by iodoacetate. It is suggested that histidine 204, while clearly in the active site, may not play any direct role in enzyme catalysis. Modifications at this position may disrupt the normal substrate binding site.

The activity of concentrated solutions of carbonic anhydrase.

A rapidly recording stopped-flow apparatus has been used to measure the rate of reactions catalysed by carbonic anhydrase. The method allows high concentrations of enzyme to be used so that the contributions to the observed rate by the non-catalysed and buffer-catalysed rates become unimportant. The rates observed were proportional to the enzyme concentration up to the highest concentrations studied, about one tenth of that found in the red blood cell. It was shown that under physiological conditions the enzyme is largely in the form uncombined with substrate. The activity of human carbonic anhydrase at physiological temperature, pH, ionic strength and substrate concentration was measured directly. From this it can be estimated that the enzyme in the red cell increases the rate of CO 2 output from bicarbonate by about 13 000 fold. This figure may be compared with the 700-fold increase necessary for CO 2 evolution during the transit of the blood through the lung capillaries.

The activity of concentrated solutions of carbonic anhydrase

Abstract A rapidly recording stopped-flow apparatus has been used to measure the rate of reactions catalysed by carbonic anhydrase. The method allows high concentrations of enzyme to be used so that the contributions to the observed rate by the non-catalysed and buffer-catalysed rates become unimportant. The rates observed were proportional to the enzyme concentration up to the highest concentrations studied, about one tenth of that found in the red blood cell. It was shown that under physiological conditions the enzyme is largely in the form uncombined with substrate. The activity of human carbonic anhydrase at physiological temperature, pH, ionic strength and substrate concentration was measured directly. From this it can be estimated that the enzyme in the red cell increases the rate of CO 2 output from bicarbonate by about 13 000 fold. This figure may be compared with the 700-fold increase necessary for CO 2 evolution during the transit of the blood through the lung capillaries.