Carbonic anhydrase II (CAII) is a metalloenzyme that catalyzes the reversible hydration/dehydration of CO2/HCO3-. In addition, CAII is attributed to other catalytic reactions, including esterase activity. Aspirin (acetyl-salicylic acid), an everyday over-the-counter drug, has both ester and carboxylic acid moieties. Recently, compounds with a carboxylic acid group have been shown to inhibit CAII. Hence, we hypothesized that Aspirin could act as a substrate for esterase activity, and the product salicylic acid (SA), an inhibitor of CAII. Here, we present the crystal structure of CAII in complex with SA, a product of CAII crystals pre-soaked with Aspirin, to 1.35Å resolution. In addition, we provide kinetic data to support the observation that CAII converts Aspirin to its deacetylated form, SA. This data may also explain the short half-life of Aspirin, with CAII so abundant in blood, and that Aspirin could act as a suicide inhibitor of CAII.

Human carbonic anhydrase II (hCAII) is a metalloenzyme essential to critical physiological processes in the body. hCA inhibitors are used clinically for the treatment of indications ranging from glaucoma to epilepsy. Targeted protein degraders have emerged as a promising means of inducing the degradation of disease-implicated proteins by using the endogenous quality control mechanisms of a cell. Here, a series of heterobifunctional degrader candidates targeting hCAII were developed from a simple aryl sulfonamide fragment. Degrader candidates were functionalized to produce either cereblon E3 ubiquitin ligase (CRBN) recruiting proteolysis targeting chimeras (PROTACs) or adamantyl-based hydrophobic tags (HyTs). Screens in HEK293 cells identified two PROTAC small-molecule degraders of hCA. Optimization of linker length and composition yielded a degrader with sub-nanomolar potency and sustained depletion of hCAII over prolonged treatments. Mechanistic studies suggest that this optimized degrader depletes hCAII through the same mechanism as previously reported CRBN-recruiting heterobifunctional degraders.

The role of protein dynamics in enzyme catalysis is one of the most highly debated topics in enzymology. The main controversy centers around what may be defined as functionally significant conformational fluctuations and how, if at all, these fluctuations couple to enzyme catalyzed events. To shed light on this debate, the conformational dynamics along the transition path surmounting the highest free energy barrier have been herein investigated for the rate limiting proton transport event in human carbonic anhydrase (HCA) II. Special attention has been placed on whether the motion of an excess proton is correlated with fluctuations in the surrounding protein and solvent matrix, which may be rare on the picosecond and subpicosecond time scales of molecular motions. It is found that several active site residues, which do not directly participate in the proton transport event, have a significant impact on the dynamics of the excess proton. These secondary participants are shown to strongly influence the active site environment, resulting in the creation of water clusters that are conducive to fast, moderately slow, or slow proton transport events. The identification and characterization of these secondary participants illuminates the role of protein dynamics in the catalytic efficiency of HCA II.

The effect of some sulfonamide ligands on the structure and function of human carbonic anhydrase II (HCA II) was investigated using different spectroscopic techniques including UV-Vis, fluorescence, circular dichroism and molecular dynamics simulation tools. Kinetic measurements were performed in 50 mM Tris-HCl, pH 7.4 at 27 °C. Kinetic data revealed that sulfonamide ligands inhibit the HCA II esterase activity in a linear competitive manner with Ki in the nanomolar range. Fluorescence measurements illustrated that ligands act as the enzyme quenchers. Stern-Volmer analysis of the quenching data at different temperatures demonstrated that the quenching of the HCA II intrinsic fluorescence occurred through static and dynamic quenching mechanisms. Analysis of the binding thermodynamic parameters showed that hydrogen bonding and hydrophobic interactions play an important role in the stabilization of enzyme-drug complex. Job plot confirmed the 1:1 stoichiometry of ligand-protein complex, and therefore, the existence of one binding site for the ligand. Molecular simulations confirmed that acetazolamide induced impressive conformational changes in two domains adjacent to the active site, including amino acids 19-25 and 61-67. RMSF studies showed sharp changes in three distinct regions near the active site including amino acids 15-25, 160-180 and 190-210 upon drug binding.

Carbonic anhydrase II (CAII) is expressed along the nephron where it interacts with a number of transport proteins augmenting their activity. Aquaporin-1 (AQP1) interacts with CAII to increase water flux through the water channel. Both CAII and aquaporin-1 are expressed in the thin descending limb (TDL); however, the physiological role of a CAII-AQP1 interaction in this nephron segment is not known. To determine if CAII was required for urinary concentration, we studied water handling in CAII-deficient mice. CAII-deficient mice demonstrate polyuria and polydipsia as well as an alkaline urine and bicarbonaturia, consistent with a type III renal tubular acidosis. Natriuresis and hypercalciuria cause polyuria, however, CAII-deficient mice did not have increased urinary sodium nor calcium excretion. Further examination revealed dilute urine in the CAII-deficient mice. Urinary concentration remained reduced in CAII-deficient mice relative to wild-type animals even after water deprivation. The renal expression and localization by light microscopy of NKCC2 and aquaporin-2 was not altered. However, CAII-deficient mice had increased renal AQP1 expression. CAII associates with and increases water flux through aquaporin-1. Water flux through aquaporin-1 in the TDL of the loop of Henle is essential to the concentration of urine, as this is required to generate a concentrated medullary interstitium. We therefore measured cortical and medullary interstitial concentration in wild-type and CAII-deficient mice. Mice lacking CAII had equivalent cortical interstitial osmolarity to wild-type mice: however, they had reduced medullary interstitial osmolarity. We propose therefore that reduced water flux through aquaporin-1 in the TDL in the absence of CAII prevents the generation of a maximally concentrated medullary interstitium. This, in turn, limits urinary concentration in CAII deficient mice.

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.

Nitric oxide (NO) promotes vasodilation through the activation of guanylate cyclase, resulting in the relaxation of the smooth muscle vasculature and a subsequent decrease in blood pressure. Therefore, its regulation is of interest for the treatment and prevention of heart disease. An example is pulmonary hypertension which is treated by targeting this NO/vasodilation pathway. In bacteria, plants and fungi, nitrite (NO2 -) is utilized as a source of NO through enzymes known as nitrite reductases. These enzymes reduce NO2 - to NO through a catalytic metal ion, often copper. Recently, several studies have shown nitrite reductase activity of mammalian carbonic anhydrase II (CAII), yet the molecular basis for this activity is unknown. Here we report the crystal structure of copper-bound human CAII (Cu-CAII) in complex with NO2 - at 1.2 Å resolution. The structure exhibits Type 1 (T-1) and 2 (T-2) copper centers, analogous to bacterial nitrite reductases, both required for catalysis. The copper-substituted CAII active site is penta-coordinated with a 'side-on' bound NO2 -, resembling a T-2 center. At the N terminus, several residues that are normally disordered form a porphyrin ring-like configuration surrounding a second copper, acting as a T-1 center. A structural comparison with both apo- (without metal) and zinc-bound CAII (Zn-CAII) provides a mechanistic picture of how, in the presence of copper, CAII, with minimal conformational changes, can function as a nitrite reductase.

By using a proteomic approach, we found increased levels of carbonic anhydrase II (CA II) in the brain of Ts65Dn mice, a mouse model for Down syndrome (DS). Further immunoblot analyses showed that the levels of CA II are increased not only in the brain of adult Ts65Dn mice but also in the brain of infants and young children with DS. Cellular localization of the enzyme in human brain, predominantly in the oligodendroglia and primitive vessels in fetal brain and in the oligodendroglia and some GABAergic neurons postnatally, was similar in DS subjects and controls. Given the role of CA II in regulation of electrolyte and water balance and pH homeostasis, up-regulation of CA II may reflect a compensatory mechanism mobilized in response to structural/functional abnormalities in the developing DS brain. However, this up-regulation may also have an unfavorable effect by increasing susceptibility to seizures of children with DS.

Here, we report the synthesis, carbonic anhydrase-II (CA-II) inhibition and structure-activity relationship studies of cinnamaldehyde-clubbed thiosemicarbazones derivatives. The derivatives showed potent activities in the range of 10.3 ± 0.62-46.6 ± 0.62 µM. Among all the synthesized derivatives, compound 3n (IC50 = 10.3 ± 0.62 µM), 3g (IC50 = 12.1 ± 1.01 µM), and 3h (IC50 = 13.4 ± 0.52 µM) showed higher inhibitory activity as compared to the standard inhibitor, acetazolamide. Furthermore, molecular docking of all the active compounds was carried out to predict their behavior of molecular binding. The docking results indicate that the most active hit (3n) specifically mediate ionic interaction with the Zn ion in the active site of CA-II. Furthermore, the The199 and Thr200 support the binding of thiosemicarbazide moiety of 3n, while Gln 92 supports the interactions of all the compounds by hydrogen bonding. In addition to Gln92, few other residues including Asn62, Asn67, The199, and Thr200 play important role in the stabilization of these molecules in the active site by specifically providing H-bonds to the thiosemicarbazide moiety of compounds. The docking score of active hits are found in range of - 6.75 to - 4.42 kcal/mol, which indicates that the computational prediction correlates well with the in vitro results.

Carbonic anhydrase inhibitors are used to treat glaucoma and cancers. Carbonic anhydrases perform a crucial role in the conversion of carbon dioxide and water into bicarbonate and protons. However, there is little information about carbonic anhydrase isoforms during the process of ageing. Mitochondrial dysfunction is implicit in ageing brain and muscle. We have interrogated isolated mitochondrial fractions from young adult and middle aged mouse brain and skeletal muscle. We find an increase of tissue specific carbonic anhydrases in mitochondria from middle-aged brain and skeletal muscle. Mitochondrial carbonic anhydrase II was measured in the Purkinje cell degeneration (pcd5J) mouse model. In pcd5J we find mitochondrial carbonic anhydrase II is also elevated in brain from young adults undergoing a process of neurodegeneration. We show C.elegans exposed to carbonic anhydrase II have a dose related shorter lifespan suggesting that high CAII levels are in themselves life limiting. We show for the first time that the mitochondrial content of brain and skeletal tissue are exposed to significantly higher levels of active carbonic anhydrases as early as in middle-age. Carbonic anhydrases associated with mitochondria could be targeted to specifically modulate age related impairments and disease.

Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins.

Carbonic anhydrase II, belonging to one of the most important enzyme groups of the human body, is a well-studied isozyme from the family of the carbonic anhydrases. Since it is involved in several physiological processes, it has been a pharmaceutical target for many years. In this study we synthesized a number of sulfamates derived from pentacyclic methyl triterpenoates, and we demonstrate their potential as carbonic anhydrase II inhibitors using the well-established photometric 4-nitrophenyl acetate assay. Inhibition constants, as an indicator of their inhibition strength, were in the micromolar range; one compound (10, methyl (3β) 3-(aminosulfonyloxy)-oleanoate) showed a Ki value as low as 0.3 μM. This Ki value is comparable to that of acetazolamide which is a potent carbonic anhydrase inhibitor and a drug for the treatment of glaucoma.

Although it has been reported that bovine carbonic anhydrase CAII is capable of generating NO from nitrite, the function and mechanism of CAII in nitrite-dependent NO formation and vascular responses remain controversial. We tested the hypothesis that CAII catalyses NO formation from nitrite and contributes to nitrite-dependent inhibition of platelet activation and vasodilation.

Cancer, one of the principal causes of death, is a global social health problem. Autoantibodies developed against the organism's self-antigens are detected in the sera of subjects with cancer. In recent years carbonic anhydrase (CA) I and II autoantibodies have been shown in some autoimmune diseases and carcinomas, but the mechanisms underlying this immune response have not yet been explained. The aim of this study was to evaluate CA I and II autoantibodies in patients with acute myeloid leukemia (AML) and to provide a novel perspective regarding the autoimmune basis of the disease.

Knowledge of folding/unfolding pathway is fundamental basis to study protein structure and stability. Human carbonic anhydrase II (HCAII) is a ∼29kDa, β-sheet dominated monomeric protein of 259 amino acid residues. In the present study, the urea-induced denaturation of HCAII was carried out which was a tri-phasic process, i.e., N (native) ↔ XI ↔ XII ↔ D (denatured) with stable intermediates XI and XII populated around 2 and 4M urea, respectively. The far-UV CD was used to characterize the intermediate states (XI and XII) for secondary structural content, near-UV CD for tertiary structure, dynamic light scattering for hydrodynamic radius and ANS fluorescence spectroscopy for the presence of exposed hydrophobic patches. Based on these experiments, we concluded that urea-induced XI state has characteristics of molten globule state while XII state bears characteristics features of pre-molten globule state. Characterization of the intermediates on the folding pathway will contribute to a deeper understanding of the structure-function relationship of HCAII. Furthermore, this system may provide an excellent model to study urea stress and the strategies adopted by the organisms to combat such a stress.

Early detection and intervention are key strategies to reduce mortality, increase long-term survival, and improve the therapeutic effects of hepatocellular carcinoma (HCC) patients. Herein, the isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic strategy was used to study the secretomes in conditioned media from HCC cancerous tissues, surrounding noncancerous tissues, and distal noncancerous tissues to identify diagnostic and prognostic biomarkers for HCC. In total, 22 and 49 dysregulated secretory proteins were identified in the cancerous and surrounding noncancerous tissues, respectively, compared with the distal noncancerous tissues. Among these proteins, carbonic anhydrase II (CA2) was identified to be significantly upregulated in the secretome of cancerous tissues; correspondingly, the serum concentrations of CA2 were remarkably increased in HCC patients compared with that in normal populations. Interestingly, a significant increase of serum CA2 in recurrent HCC patients after radical resection was also confirmed compared with HCC patients without recurrence, and the serum level of CA2 could act as an independent prognostic factor for time to recurrence and overall survival. Regarding the mechanism, the secreted CA2 enhances the migration and invasion of HCC cells by activating the epithelial mesenchymal transition pathway. Taken together, this study identified a novel biomarker for HCC diagnosis and prognosis, and provided a valuable resource of HCC secretome for investigating serological biomarkers.

Two acetazolamide (AAZ) complexes with ruthenium(II) η6-p-cymene chloride were synthesised, characterised and tested for their inhibitory effects on several carbonic anhydrase (CA, EC 4.2.1.1) isoforms with pharmacological applications. Against human (h) isoform hCA I, the two complexes showed inhibition constants in the range of 8.5-23.4 nM (AAZ has a KI of 250 nM), against hCA II of 0.48-4.2 nM, whereas against hCA IX of 0.63-3.8 nM and against hCA XII of 0.04-0.52 nM, respectively. These highly effective ruthenium acetazolamide derivatives against the tumour-associated CA isoforms IX and XII warrant further in vivo studies, in hypoxic tumours overexpressing these enzymes.

Four human (h) carbonic anhydrase isoforms (CA, EC 4.2.1.1), hCA I, II, IV, and VII, were investigated for their activation profile with piperazines belonging to various classes, such as N-aryl-, N-alkyl-, N-acyl-piperazines as well as 2,4-disubstituted derivatives. As the activation mechanism involves participation of the activator in the proton shuttling between the zinc-coordinated water molecule and the external milieu, these derivatives possessing diverse basicity and different scaffolds were appropriate for being investigated as CA activators (CAAs). Most of these derivatives showed CA activating properties against hCA I, II, and VII (cytosolic isoforms) but were devoid of activity against the membrane-associated hCA IV. For hCA I, the KAs were in the range of 32.6-131 µM; for hCA II of 16.2-116 µM, and for hCA VII of 17.1-131 µM. The structure-activity relationship was intricate and not easy to rationalize, but the most effective activators were 1-(2-piperidinyl)-piperazine (KA of 16.2 µM for hCA II), 2-benzyl-piperazine (KA of 17.1 µM for hCA VII), and 1-(3-benzylpiperazin-1-yl)propan-1-one (KA of 32.6 µM for hCA I). As CAAs may have interesting pharmacologic applications in cognition and for artificial tissue engineering, investigation of new classes of activators may be crucial for this relatively new research field.

Cancer is the second most important cause of mortality, and millions of people either have or have had the disease. Leukaemia is one of the most common forms of cancer. Autoantibodies that have developed against the organism's self-antigens are detected in the sera of subjects with cancer. In recent years carbonic anhydrase (CA) autoantibodies have been determined in some autoimmune diseases and carcinomas, but the mechanisms underlying this immune response have not yet been fully explained. The purpose of this study was to determine CA I and II autoantibodies in subjects with chronic lymphocytic leukaemia (CLL) and to provide a novel perspective regarding the autoimmune basis of the disease. Autoantibody levels were investigated using enzyme-linked immunosorbent assay (ELISA) in serum samples from 37 patients with CLL and 37 healthy peers. Anti-CA I titres in the CLL group were significantly higher compared with the control group (p = 0.0001). However, there was no significant difference between CLL and control groups in terms of anti-CA II titres (p = 0.278). The prevalences of CA I and II autoantibodies in patients with CLL in this study were 27% and 24.3%, respectively. Our results suggest that these autoantibodies may be involved in the pathogenesis of CLL. More extensive studies are now needed to reveal the entire mechanism.

Some Brucella isolates are known to require an increased concentration of CO2 for growth, especially in the case of primary cultures obtained directly from infected animals. Moreover, the different Brucella species and biovars show a characteristic pattern of CO2 requirement, and this trait has been included among the routine typing tests used for species and biovar differentiation. By comparing the differences in gene content among different CO2-dependent and CO2-independent Brucella strains, we have confirmed that carbonic anhydrase (CA) II is the enzyme responsible for this phenotype in all the Brucella strains tested. Brucella species contain two CAs of the β family, CA I and CA II; genetic polymorphisms exist for both of them in different isolates, but only those putatively affecting the activity of CA II correlate with the CO2 requirement of the corresponding isolate. Analysis of these polymorphisms does not allow the determination of CA I functionality, while the polymorphisms in CA II consist of small deletions that cause a frameshift that changes the C-terminus of the protein, probably affecting its dimerization status, essential for the activity. CO2-independent mutants arise easily in vitro, although with a low frequency ranging from 10-6 to 10-10 depending on the strain. These mutants carry compensatory mutations that produce a full-length CA II. At the same time, no change was observed in the sequence coding for CA I. A competitive index assay designed to evaluate the fitness of a CO2-dependent strain compared to its corresponding CO2-independent strain revealed that while there is no significant difference when the bacteria are grown in culture plates, growth in vivo in a mouse model of infection provides a significant advantage to the CO2-dependent strain. This could explain why some Brucella isolates are CO2 dependent in primary isolation. The polymorphism described here also allows the in silico determination of the CO2 requirement status of any Brucella strain.