Carbonic anhydrase inhibitors. Inhibition studies of the human secretory isoform VI with anions

The unique secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified. The kinetic parameters for the CO(2) hydration reaction proved hCA VI to possess a k(cat) of 3.4x10(5)s(-1) and k(cat)/K(M) of 4.9x10(7)M(-1)s(-1) (at pH 7.5 and 20 degrees C). hCA VI has a significant catalytic activity for the physiological reaction, of the same order of magnitude as isoforms CA I or CA IX. A series of anions (such as bicarbonate, chloride, nitrate, etc.) were shown to inhibit the activity of the enzyme, with inhibition constants typically in the range of 0.60-0.90mM. The best hCA VI inhibitors were cyanide, azide, sulfamide, and sulfamate, with inhibition constants in the range of 70-90microM.     

Carbonic anhydrase inhibitors: inhibition of the transmembrane isozyme XIV with sulfonamides

The inhibition of the last human carbonic anhydrase (CA, EC 4.2.1.1) isozyme (hCA XIV) discovered has been investigated with a series of sulfonamides, including some clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and zonisamide), as well as the sulfamate antiepileptic drug topiramate. The full-length hCA XIV is an enzyme showing a medium-low catalytic activity, quite similar to that of hCA XII, with the following kinetic parameters at 20 degrees C and pH 7.5, for the CO2 hydration reaction: k(cat) = 3.12 x 10(5) s(-1) and k(cat)/K(M) = 3.9 x 10(7) M(-1) s(-1). All types of activities have been detected for the investigated compounds, with several micromolar inhibitors, including zonisamide, topiramate, and simple sulfanilamide derivatives (K(I)-s in the range of 1.46-6.50 microM). In addition, topiramate and zonisamide were observed to behave as weak hCA XII inhibitors, while zonisamide was an effective hCA IX inhibitor (K(I) of 5.1 nM). Some benzene-1,3-disulfonamide derivatives or simple five-membered heteroaromatic sulfonamides showed K(I)-s in the range of 180-680 nM against hCA XIV, whereas the most effective of such inhibitors, including 3-chloro-/bromo-sulfanilamide, benzolamide-like, ethoxzolamide-like, and acetazolamide/methazolamide-like derivatives, showed inhibition constant in the range of 13-48 nM. The best hCA XIV inhibitor was aminobenzolamide (K(I) of 13 nM), but no CA XIV-selective derivatives were evidenced. There are important differences of affinity of these sulfonamides/sulfamates for the three transmembrane CA isozymes, with CA XII showing the highest affinity, followed by CA IX, whereas CA XIV usually showed the lowest affinity for these inhibitors.     

Carbonic anhydrase inhibitors: cloning, characterization, and inhibition studies of the cytosolic isozyme III with sulfonamides

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(-1) and k(cat)/K(M) of 2.5 x 10(5) M(-1) s(-1), being a slower catalyst for the physiological reaction as compared to the genetically related cytosolic isoforms hCA I and II. An inhibition study with a library of sulfonamides and one sulfamate, some which are clinically used compounds, is reported. hCA III is less prone to be inhibited by these compounds as compared to hCA I and II for which many low nanomolar inhibitors were detected earlier. The best hCA III inhibitors were prontosil, sulpiride, indisulam, benzolamide, aminobenzolamide, and 4-amino-6-chloro-benzene-1,3-disulfonamide which showed K(I)s in the range of 2.3-18.1 microM. Clinically used compounds such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, topiramate, zonisamide, celecoxib, and valdecoxib were less effective hCA III inhibitors, with affinities in the range of 154-2200 microM. This is the first study in which low micromolar hCA III inhibitors are reported.     

Carbonic anhydrase inhibitors. Inhibition of the human cytosolic isozyme VII with aromatic and heterocyclic sulfonamides

The inhibition of a newly cloned human carbonic anhydrase (CA, EC 4.2.1.1), isozyme VII (hCA VII), has been investigated with a series of aromatic and heterocyclic sulfonamides, including some of the clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide and benzolamide), as well as the sulfamate antiepileptic drug topiramate. Inhibition data for the the other physiologically relevant cytosolic isoforms hCA I, hCA II and mCA XIII are also provided for comparison. hCA VII shows a high catalytic activity for the CO(2) hydration reaction, with a k(cat) of 9.5 x 10(5)s(-1) and k(cat)/K(m) of 8.3 x 10(7)M(-1)s(-1) at pH7.5 and 20 degrees C. A very interesting inhibition profile against hCA VII with this series of 32 sulfonamides/sulfamates was observed. hCA VII shows high affinity for all the investigated compounds, with inhibition constants in the range of 0.45-210 nM. Topiramate, ethoxzolamide and benzolamide showed subnanomolar hCA VII inhibitory activity, whereas acetazolamide, methazolamide, dorzolamide and brinzolamide showed K(I)-s in the range of 2.1-3.5 nM. Dichlorophenamide was slightly less active (K(I) of 26.5 nM). A number of heterocyclic or bicyclic aromatic sulfonamides also showed excellent hCA VII inhibitory properties (K(I)-s in the range of 4.3-7.0 nM) whereas many monosubstituted or disubstituted benzenesulfonamides were less active (K(I)-s in the range of 45-89 nM). The least active hCA VII inhibitors were some substituted benzene-1,3-disulfonamides as well as some halogenated sulfanilamides (K(I)-s in the range of 100-210 nM). The inhibition profile of hCA VII is rather different of that of the other cytosolic isozymes, providing thus a possibility for the design of more selective, hCA VII-specific inhibitors. In addition, these data furnish further evidence that hCA VII is the isozyme responsible for the anticonvulsant/antiepileptic activity of sulfonamides and sulfamates.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 μM against hCA I, of 0.381-470 μM against hCA II and of 0.578-1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Carbonic anhydrase inhibitors. Cloning, characterization and inhibition studies of the cytosolic isozyme III with anions

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(- 1) and k(cat)/K(M) of 2.5.10(5) M(- 1) s(- 1). The first detailed inhibition study of this enzyme with anions is reported. Inhibition data of the cytosolic isozymes hCA I - hCA III with a large number of anions (halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, sulfate, sulfamic acid, sulfamide, etc.), were determined and these values are comparatively discussed for these three cytosolic isoforms. Fluoride, nitrate, nitrite, phenylboronic acid and phenylarsonic acid (as anions) were weak hCA III inhibitors (K(I)s of 21-78.5 mM), whereas bicarbonate, chloride, bromide, sulfate and several other simple anions showed K(I)s around 1 mM. The best hCA III inhibitors were carbonate, cyanide, thiocyanate, azide and hydrogensulfide, which showed K(I)s in the range of 10-90 microM. It is difficult to explain the inhibitory activity of carbonate (K(I) of 10 microM) against hCA III, also considering the fact that this ion has an affinity of 15-73 mM for hCA I and II and is in equilibrium with one of the substrates of this enzyme, i.e., bicarbonate, which is a much weaker inhibitor (K(I) of 0.74 mM against hCA III, of 12 mM against hCA I and of 85 mM against hCA II).     

Carbonic anhydrase inhibitors: purification and inhibition studies of pigeon (Columba livia var. domestica) red blood cell carbonic anhydrase with sulfonamides

A carbonic anhydrase (CA, EC 4.2.1.1) from red blood cells of pigeons (Columba livia var. domestica), clCA, was purified to homogeneity. Its kinetic parameters for the CO(2) hydration reaction were measured. With a k(cat)/K(m) of 1.1?×?10(8) M(-1) s(-1), and a k(cat) of 1.3?×?10(6) s(-1), clCA has a high activity, similar to that of the human isoform hCA II. A group of 25 aromatic/heterocyclic sulfonamides incorporating the sulfanilamide, homosulfanilamide, benzene-1,3-disulfonamide, and acetazolamide scaffolds showed variable inhibitory activity against the pigeon enzyme, with K(I)s in the range of 1.9-3460?nM. Red blood cells of pigeons, like those of ostriches, contain thus just one CA isoform, unlike the blood of mammals, which normally contain two isoforms, one of low (CA I-like) and one of very high activity (CA II-like). However, from the sulfonamide inhibition viewpoint, the pigeon enzyme was more similar to hCA II than to the ostrich enzyme.     

Carbonic anhydrase inhibitors: 2-substituted-1,3,4-thiadiazole-5-sulfamides act as powerful and selective inhibitors of the mitochondrial isozymes VA and VB over the cytosolic and membrane-associated carbonic anhydrases I, II and IV

A series of 2-substituted-1,3,4-thiadiazole-5-sulfamides was prepared and assayed as inhibitors of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, the membrane-associated CA IV and the mitochondrial CA VA and VB. The new compounds showed weak inhibitory activity against hCA I (K(I)s of 102 nM-7.42 microM), hCA II (K(I)s of 0.54-7.42 microM) and hCA IV (K(I)s of 4.32-10.05 microM) but were low nanomolar inhibitors of hCA VA and hCA VB, with inhibition constants in the range of 4.2-32 nM and 1.3-74 nM, respectively. Furthermore, the selectivity ratios for inhibiting the mitochondrial enzymes over CA II were in the range of 67.5-415, making these sulfamides the first selective CA VA/VB inhibitors.     

Purification and inhibition studies with anions and sulfonamides of an α-carbonic anhydrase from the Antarctic seal Leptonychotes weddellii

A high activity α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified from various tissues of the Antarctic seal Leptonychotes weddellii. The new enzyme, denominated lwCA, has a catalytic activity for the physiologic CO(2) hydration to bicarbonate reaction, similar to that of the high activity human isoform hCA II, with a k(cat) of 1.1×10(6) s(-1), and a k(cat)/K(m) of 1.4×10(8) M(-1) s(-1). The enzyme was highly inhibited by cyanate, thiocyanate, cyanide, bicarbonate, carbonate, as well as sulfamide, sulfamate, phenylboronic/phenylarsonic acids (K(I)s in the range of 46-100 μM). Many clinically used sulfonamides, such as acetazolamide, methazolamide, dorzolamide, brinzolamide and benzolamide were low nanomolar inhibitors, with K(I)s in the range of 5.7-67 nM. Dichlorophenamide, zonisamide, saccharin and hydrochlorothiazide were weaker inhibitors, with K(I)s in the range of 513-5390 nM. The inhibition profile with anions and sulfonamides of the seal enzyme was rather different from those of the human isoforms hCA I and II. The high sensitivity to bicarbonate inhibition of lwCA, unlike that of the human enzymes, may reflect an evolutionary adaptation to the deep water, high CO(2) partial pressure and hypoxic conditions in which Weddell seals spend much of their life.     

Carbonic anhydrase inhibitors: cloning and sulfonamide inhibition studies of a carboxyterminal truncated alpha-carbonic anhydrase from Helicobacter pylori

A library of sulfonamides/sulfamates has been investigated for the inhibition of the carboxyterminal truncated form of the alpha-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the gastric pathogen Helicobacter pylori (hpCA). This enzyme, incorporating 202 amino acid residues, showed a catalytic activity similar to that of the full length hpCA, with k(cat) of 2.35 x 10(5)s(-1) and k(cat)/K(M) of 1.56 x 10(7)M(-1)s(-1) at 25 degrees C and pH of 8.9, for the CO(2) hydration reaction. All types of activity for inhibition of the bacterial enzyme have been detected. Dorzolamide and simple 4-substituted benzenesulfonamides were weak hpCA inhibitors (inhibition constants, K(I)s, in the range of 830-4310 nM). Sulfanilamide, orthanilamide, some of their derivatives, and indisulam showed better activity (K(I)s in the range of 310-562 nM), whereas most of the clinically used CA inhibitors, such as methazolamide, ethoxzolamide, dichlorophenamide, brinzolamide, topiramate, zonisamide, etc., acted as medium potency hpCA inhibitors (K(I)s in the range of 124-287 nM). Some potent hpCA inhibitors were detected too (K(I)s in the range of 20-96 nM) such as acetazolamide, 4-amino-6-chloro-1,3-benzenedisulfonamide, 4-sulfanilyl-aminoethyl-benzenesulfonamide, and 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. Most of the investigated derivatives acted as better inhibitors of the human isoform hCA II than as hpCA inhibitors. Since hpCA is essential for the survival of the pathogen in acid, its inhibition by compounds such as those investigated here might be used as a new pharmacologic tool in the management of drug resistant H. pylori.     

Carbonic anhydrase activators: activation of the human isoforms VII (cytosolic) and XIV (transmembrane) with amino acids and amines

An activation study of the human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes VII and XIV using a small library of natural/non-natural amino acids and aromatic/heterocyclic amines is reported. hCA VII was efficiently activated by L-/D-His, dopamine and serotonin (K(A)s of 0.71-0.93 microM). The best hCA XIV activators were histamine (K(A) of 10 nM), L-Phe, L-/D-His and 4-amino-L-Phe (K(A)s of 0.24-2.90 microM). In view of the significant expression levels of CA VII and CA XIV in the brain, selective activation of these isoforms may be useful when developing pharmacologic agents for the management of major disorders such as epilepsy and Alzheimer's disease.     

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with organic phosphates and phosphonates

The interaction of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, that is, hCA I, II, IV, V, and IX with a small library of phosphonic acids/organic phosphates, including methylphosphonic acid, MPA; phenylphosphonic acid, PPA; N-(phosphonoacetyl)-L-aspartic acid, PALA, methylene diphosphonic acid MDPA, the O-phosphates of serine (Ser-OP) and threonine (Thr-OP) as well as the antiviral phosphonate foscarnet has been studied. hCA I was activated by all these compounds, with the best activators being MPA and PPA (K(A)s of 0.10-1.20 microM). MPA and PPA were on the other hand nanomolar inhibitors of hCA II (K(I)s of 98-99 nM). PALA showed an affinity of 7.8 microM, whereas the other compounds were weak, millimolar inhibitors of this isozyme. The best hCA IV inhibitors were PALA (79 nM) and PPA (5.4 microM), whereas the other compounds showed K(I)s in the range of 0.31-5.34 mM. The mitochondrial isozyme was weakly inhibited by all these compounds (K(I)s in the range of 0.09-41.7 mM), similarly to the transmembrane, tumor-associated isozyme (K(I)s in the range of 0.86-2.25 mM). Thus, phosphonates may lead to CA inhibitors with selectivity against two physiologically relevant isozymes, the cytosolic hCA II or the membrane-bound hCA IV.     

Carbonic anhydrase activators: activation of the human tumor-associated isozymes IX and XII with amino acids and amines

The first activation study of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms associated to tumors, hCA IX and XII, with a small library of natural and non-natural amino acids as well as aromatic/heterocyclic amines is reported. hCA IX was activated efficiently by dopamine, adrenaline and heterocyclic amines possessing aminoethyl-/aminomethyl-moieties (K(A)s of 9 nM-1.07 microM), whereas the best hCA XII activators were serotonin, L-adrenaline, 4-(2-aminoethyl)-morpholine and d-Phe (K(A) of 0.24-0.41 microM). Precise steric and electronic requirements are needed to be present in the molecules of effective hCA IX/hCA XII activators, in order to assure an adequate fit within the enzyme active site cavity for the formation of the enzyme-activator complex, and for an efficient proton transfer process within this complex, leading to the release of a proton and formation of the catalytically active, zinc-hydroxide species of the enzyme. Selective activation of these CA isoforms might be useful to develop pharmacologic tools or to understand whether some of these biogenic amines/amino acids may influence the progression of tumors overexpressing CA IX and/or CA XII.     

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with carboxylates

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with carboxylates including aliphatic (formate, acetate), dicarboxylic (oxalate, malonate), hydroxy/keto acids (l-lactate, l-malate, pyruvate), tricarboxylic (citrate), or aromatic (benzoate, tetrafluorobenzoate) representatives, some of which are important intermediates in the Krebs cycle, is presented. The cytosolic isozyme hCA I was strongly activated by acetate, oxalate, pyruvate, l-lactate, and citrate (K(A) around 0.1 microM), whereas formate, malonate, malate, and benzoate were weaker activators (K(A) in the range 0.1-1mM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with inhibition constants in the range of 0.03-24 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by carboxylates, showing a K(I) of 99 nM for citrate and oxalate, of 2.8 microM for malonate and of 14.5 microM for pyruvate among others. The mitochondrial isozyme hCA V was weakly inhibited by all these carboxylates (K(I)s in the range of 1.67-25.9 mM), with the best inhibitor being citrate (K(I) of 1.67 mM), whereas this is the most resistant CA isozyme to pyruvate inhibition (K(I) of 5.5mM), which may be another proof that CA V is the isozyme involved in the transfer of acetyl groups from the mitochondrion to the cytosol for the provision of substrate(s) for de novo lipogenesis. Furthermore, the relative resistance of CA V to inhibition by pyruvate may be an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of this anion within this organelle. The transmembrane, tumor-associated isozyme hCA IX was similar to isozyme II in its slight inhibition by all these anions (K(I) in the range of 1.12-7.42 mM), except acetate, lactate, and benzoate, which showed a K(I)>150 mM. The lactate insensitivity of CA IX also represents an interesting finding, since it is presumed that this isozyme evolved in such a way as to show a high catalytic activity in hypoxic tumors rich in lactate, and suggests a possible metabolon in which CA IX participates together with the monocarboxylate/H(+) co-transporter in dealing with the high amounts of lactate/H(+) present in tumors.     

Carbonic anhydrase inhibitors: inhibition of the beta-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with simple anions

The catalytic activity and inhibition of the beta-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Candida albicans (Nce103) and Cryptococcus neoformans (Can2) with inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate were investigated. The two enzymes showed appreciable CO(2) hydrase activity (k(cat) in the range of (3.9-8.0)x10(5)s(-1), and k(cat)/K(m) in the range of (4.3-9.7)x10(7)M(-1)s(-1)). Can2 was weakly inhibited by cyanide and sulfamic acid (K(I)s of 8.22-13.56 mM), while all other anions displayed more potent inhibition. Nce103 was strongly inhibited by cyanide and carbonate (K(I)s of 10-11 microM), and weakly inhibited by sulfate, phenylboronic, and phenyl arsonic acid (K(I)s of 14.15-30.85 mM). These data demonstrate that pathogenic, fungal beta-CAs may be targets for the development of antifungals that have a novel mechanism of action.     

Catalytic reaction pathway for the mitogen-activated protein kinase ERK2

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (/=56 s(-1)), from the ERK2 active site.     

Anomalous pH dependence of the reactions of carbenicillin and sulbenicillin with Bacillus cereus beta-lactamase I. Influence of the alpha-substituent charge on the kinetic parameters

The pH dependence of k(cat) for the Bacillus cereus beta-lactamase I catalyzed hydrolysis of carbenicillin(VI), which differs from benzylpenicillin (I) in having a carboxylic moiety alpha to the phenyl ring, exhibits a profile consistent with a model in which the alpha-COOH and alpha-COO forms of the ES complex turn over with respective rate constants of 2152 s(-1) and 384 s(-1). The pK(a)(app) for the alpha-COOH is shifted from 3.2 in solution to 6.1 in the ES complex. The normalized k(cat)/K(m) vs. pH profile for VI is not superimposable on that of I, indicating that both the neutral and anionic forms of the carboxyl moiety of VI combine with the enzyme to give the first irreversibly formed complex, presumably the acyl-enzyme. Quantitative accord with the kinetic data is achieved only through fitting to a model where kinetically significant proton transfer in the ES complex is permitted. The second-order rate constants for the reaction of the enzyme with the alpha-COOH and alpha-COO forms of VI are 2.2 x 10(8) M(-1) s(-1) and 3.8 x 10(6) M(-1) s(-1), respectively. The high value for the alpha-COOH form suggests that this reaction may be in part diffusion controlled. This conjecture is borne out by the observation that the sensitivity of k(cat)/K(m) to eta(rel) decreases with increasing pH for VI, whereas this sensitivity is pH independent for I. These conclusions are further supported by the results of a kinetic investigation of the pH dependence of sulbenicillin (VII) where an alpha-SO3H replaces the alpha-COOH of VI. The strongly acidic sulfonic acid moiety of VII is fully ionized throughout nearly the entire pH range of interest, and its kinetics, as a function of pH, are very similar to those observed and calculated for the alpha-COO form of VI. Solvent deuterium kinetic isotope effects are reported for k(cat) and k(cat)/K(m) for both VI and VII.     

Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the yeast Saccharomyces cerevisiae with anions

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a beta-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically, and investigated for its inhibition with a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate, and some of its isosteric species. The enzyme showed high CO(2) hydrase activity, with a k(cat) of 9.4x10(5) s(-1) and k(cat)/K(m) of 9.8x10(7) M(-1) s(-1). scCA was weakly inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K(I)s of 16.8-55.6 mM) and strongly inhibited by bromide, iodide, and sulfamide (K(I)s of 8.7-10.8 microM). The other investigated anions showed inhibition constants in the low millimolar range.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I,II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K_i values of the molecules 3–6 were in the range of 41.12–363 μM against hCA I, of 0.381–470 μM against hCA II and of 0.578–1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K_A values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

QSAR studies on the activation of the human carbonic anhydrase cytosolic isoforms I and II and secretory isozyme VI with amino acids and amines

The first QSAR study on the activation of the human secretory isoform of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), CA VI, with a series of amines and amino acids is reported. A large set of topological indices have been used to obtain several tri-/tetra-parametric models. We compared the CA VI activating QSAR models with those calculated for activation of the cytosolic human isozymes hCA I and hCA II. In addition, the effect of D- and L-amino acids as activators of hCA I, hCA II and of hCA VI as compared to those of structurally related biogenic amines was investigated for obtaining statistically significant and predictive QSAR equations. The obtained models are discussed using a variety of statistical parameters. The best models were obtained for hCA II activation, followed by hCA I, whereas the QSAR models for the activation of hCA VI were statistically weaker.