Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the beta- and gamma-class

Catalysis of the dehydration of HCO(3)(-) by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide. Carbonic anhydrases in each of the alpha, beta, and gamma classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the alpha class. We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (beta class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (gamma class). A series of structurally homologous imidazole and pyridine buffers were used as proton acceptors in the activation of CO(2) hydration at steady state and as proton donors of the exchange of (18)O between CO(2) and water at chemical equilibrium. Free energy plots of the rate constants for this intermolecular proton transfer as a function of the difference in pK(a) of donor and acceptor showed extensive curvature, indicating a small intrinsic kinetic barrier for the proton transfers. Application of Marcus rate theory allowed quantitative estimates of the intrinsic kinetic barrier which were near 0.3 kcal/mol with work functions in the range of 7-11 kcal/mol for mutants in the beta and gamma class, similar to results obtained for mutants of carbonic anhydrase in the alpha class. The low values of the intrinsic kinetic barrier for all three classes of carbonic anhydrase reflect proton transfer processes that are consistent with a model of very rapid proton transfer through a flexible matrix of hydrogen-bonded solvent structures sequestered within the active sites of the carbonic anhydrases.     

Acipimox inhibits human carbonic anhydrases

Acipimox, a nicotinic acid derivative in clinical use for the treatment of hyperlipidaemia, incorporates a free carboxylic acid and an N-oxide moiety, functionalities known to interact with the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and inhibit its activity. Herein we report that acipimox acts as a low micromolar CA inhibitor (CAI) against most human (h) isoforms possessing catalytic activity, hCA I – XIV. By using computational techniques (docking and molecular dynamics simulations), we propose that acipimox coordinates through its carboxylate group to the zinc ion from the enzyme active site cavity, whereas the N-oxide group is hydrogen-bonded to the proton shuttle His residue in some isoforms (hCA I) or to active site Thr or Gln residues in other isoforms (hCA II, III, IV, VII, etc). As some CA isoforms are involved in lipogenesis, these data may be useful for the design of more effective CAIs with antiobesity activity.     

Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.     

Proton transfer from exogenous donors in catalysis by human carbonic anhydrase II

In the site-specific mutant of human carbonic anhydrase in which the proton shuttle His64 is replaced with alanine, H64A HCA II, catalysis can be activated in a saturable manner by the proton donor 4-methylimidazole (4-MI). From 1H NMR relaxivities, we found 4-MI bound as a second-shell ligand of the tetrahedrally coordinated cobalt in Co(II)-substituted H64A HCA II, with 4-MI located about 4.5 A from the metal. Binding constants of 4-MI to H64A HCA II were estimated from: (1) NMR relaxation of the protons of 4-MI by Co(II)-H64A HCA II, (2) the visible absorption spectrum of Co(II)-H64A HCA II in the presence of 4-MI, (3) the inhibition by 4-MI of the catalytic hydration of CO2, and (4) from the catalyzed exchange of 18O between CO2 and water. These experiments along with previously reported crystallographic and catalytic data help identify a range of distances at which proton transfer is efficient in carbonic anhydrase II.     

Cross-reactions among carbonic anhydrases I,II, and III studied by binding tests and with monoclonal antibodies

Cross-reactions among carbonic anhydrases (CAs) I, II, and III were studied using a variety of antisera: (1) a rabbit antiserum to bovine CA III, (2) mouse antisera to human CA I, CA II, and CA III; and (3) five monoclonal antibodies prepared by the hybridoma technique using splenocytes from a mouse immunized with human CAs I and II and bovine CA III. Cross-reactions between CAs were readily found by binding assays using these antisera. Human CA I, but not human CA II, inhibited the reaction of the rabbit anti-CA III with its homologous antigen. Mouse antisera to CA I or CA II bound the homologous I or II with nearly as great efficiency as the autologous isozyme and sometimes weakly bound CA III. Mouse antisera to CA III frequently bound CA I or II. These cross-reactions were confirmed by the first use of hybridoma-prepared, monoclonal antibodies to CAs. The mouse monoclonal antibodies to CA isozymes varied in the amount of cross-reactivity among I, II, and III: at one extreme, one monoclonal was highly specific for the autologous CA III; at the other extreme, one monoclonal weakly reacted with some examples of CAs I, II, and III.This work was supported by NIH Grant GM-24681 and a grant from the National Foundation-March of Dimes.     

Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n = 24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n = 16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.     

Large-scale preparation of the human carbonic anhydrases

A procedure for the large-scale preparation of human carbonic anhydrases B and C is described. The procedure has been adopted for routine use in this laboratory for preparing the large amounts of protein required for primary structural studies on both enzymes.     

Herbicide oryzalin inhibits human carbonic anhydrases in vitro

Herbicides of the dinitroaniline chemical class, widely used oryzalin and trifluralin, and also nitralin were tested as inhibitors of recombinant human carbonic anhydrases (CAs). Oryzalin bound and inhibited 11 out of 12 catalytically active CA isoforms present in the human body with the affinities in the same range as clinically used CA drugs, while no effect was detected for the other two compounds. Binding of all three herbicides was examined by fluorescence‐based thermal shift assay, isothermal titration calorimetry, and the inhibition of carbon dioxide hydratase activity. During the last decade, dinitroaniline compound‐based therapies against protozoan diseases are being developed. Therefore, it is important to investigate their potential off‐target effects, including human CAs.     

Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n?=?24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n?=?16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.     

Inhibition of carbonic anhydrases I and II by N-unsubstituted carbamate esters.

We previously showed that the zinc metalloenzyme carbonic anhydrases (CA I and II isozymes) bind "neutral" amides and related compounds as anions through coordination of their deprotonated amide nitrogen to the active site zinc (Rogers, J. I., Mukherjee, J., and Khalifah, R. G. (1987) Biochemistry 26, 5672-5679). Urethan, the ethyl carbamate ester, was among such compounds. The present study was designed to test whether other N-unsubstituted carbamate esters of pharmacological interest (as sedatives, hypnotics, anxiolytics, and skeletal muscle relaxants) were capable of binding to CA in the same manner. We studied the interaction of human CA I and II with urethan, phenyl carbamate, ethinamate, meprobamate, and methocarbamol. Phenyl carbamate studies were greatly complicated by its uncatalyzed hydrolysis via an elimination mechanism to form cyanate, a powerful CA inhibitor. In general, the compounds display: 1) slow on-off inhibition binding kinetics in the seconds range, 2) maximal inhibitor affinity at alkaline pH, and 3) characteristic three-band visible spectra of their complexes with cobalt-substituted CA I. These properties are shared with the previously studied amide inhibitors and are taken as evidence that the deprotonated carbamate nitrogen coordinates to the active site metal ion. CA I appeared to bind carbamate esters more tightly than CA II, an unusual 1000-fold selectivity being seen in the case of methocarbamol. The inhibition by these drugs is not sufficiently strong to implicate CA I and II in their mechanism of action. However, it does suggest the possible existence of previously unsuspected similarities between binding to CA and to their physiological receptors or targets, particularly the involvement of zinc.     

Natural product coumarins that inhibit human carbonic anhydrases

Natural products (NPs) have proven to be an invaluable source of new chemotherapies yet very few have been explored to source small molecule carbonic anhydrase (CA) inhibitors. CA enzymes underpin physiological pH and are critical to the progression of several diseases including cancer. The present study is the first to more widely investigate NP coumarins for CA inhibition following the recent discovery of a NP coumarin CA inhibitor. We assembled a NP library comprising 24 plant coumarins (compounds 4–27) and three ascidian coumarins (compounds 28–30) that together provide a diverse collection of structures containing the coumarin pharmacophore. This library was then evaluated for inhibition of six human CA isozymes (CAs I, II, VII, IX, XII and XIII) and a broad range of inhibition and isozyme selectivity profiles were evident. Our findings provide a platform to support further evaluation of NPs for the discovery of new chemotypes that inhibit disease relevant CA enzymes.     

Role of hydrophilic residues in proton transfer during catalysis by human carbonic anhydrase II

Catalysis by the zinc metalloenzyme human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer between His64 and the zinc-bound solvent molecule. Asn62 extends into the active site cavity of HCA II adjacent to His64 and has been shown to be one of several hydrophilic residues participating in a hydrogen-bonded solvent network within the active site. We compared several site-specific mutants of HCA II with replacements at position 62 (Ala, Val, Leu, Thr, and Asp). The efficiency of catalysis in the hydration of CO 2 for the resulting mutants has been characterized by (18)O exchange, and the structures of the mutants have been determined by X-ray crystallography to 1.5-1.7 A resolution. Each of these mutants maintained the ordered water structure observed by X-ray crystallography in the active site cavity of wild-type HCA II; hence, this water structure was not a variable in comparing with wild type the activities of mutants at residue 62. Crystal structures of wild-type and N62T HCA II showed both an inward and outward orientation of the side chain of His64; however, other mutants in this study showed predominantly inward (N62A, N62V, N62L) or predominantly outward (N62D) orientations of His64. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer between the active site and solution. The site-specific mutant N62D had a mainly outward orientation of His64, yet the difference in p K a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II was 5% that of wild type, showing that His64 mainly in the outward orientation is associated with inefficient proton transfer compared with His64 in wild type which shows both inward and outward orientations. These results emphasize the roles of the residues of the hydrophilic side of the active site cavity in maintaining efficient catalysis by carbonic anhydrase.     

Thiazole-substituted benzenesulfonamides as inhibitors of 12 human carbonic anhydrases

Four series of para or meta - substituted thiazolylbenzenesulfonamides bearing Cl substituents were designed, synthesized, and evaluated as inhibitors of all 12 catalytically active recombinant human carbonic anhydrase (CA) isoforms. Observed affinities were determined by the fluorescent thermal shift assay and the intrinsic affinities were calculated based on the fractions of binding-ready deprotonated sulfonamide and CA bearing protonated hydroxide bound to the catalytic Zn(II) in the active site. Several compounds exhibited selectivity towards CA IX, an anticancer target. Intrinsic affinities reached 30 pM, while the observed affinities - 70 nM. The structure-intrinsic affinity relationship map of the compounds showed the energetic contributions of the thiazole ring and its substituents.     

Gamma carbonic anhydrases in plant mitochondria

Three genes from Arabidopsis thaliana with high sequence similarity to gamma carbonic anhydrase (γCA), a Zn containing enzyme from Methanosarcina thermophila(CAM), were identified and characterized. Evolutionary and structural analyses predict that these genes code for active forms of γCA. Phylogenetic analyses reveal that these Arabidopsis gene products cluster together with CAM and related sequences from α and γ proteobacteria, organisms proposed as the mitochondrial endosymbiont ancestor. Indeed, in vitro and in vivo experiments indicate that these gene products are transported into the mitochondria as occurs with several mitochondrial protein genes transferred, during evolution, from the endosymbiotic bacteria to the host genome. Moreover, putative CAM orthologous genes are detected in other plants and green algae and were predicted to be imported to mitochondria. Structural modeling and sequence analysis performed in more than a hundred homologous sequences show a high conservation of functionally important active site residues. Thus, the three histidine residues involved in Zn coordination (His 81, 117 and 122), Arg 59, Asp 61, Gin 75, and Asp 76 of CAM are conserved and properly arranged in the active site cavity of the models. Two other functionally important residues (Glu 62 and Glu 84 of CAM) are lacking, but alternative amino acids that might serve to their roles are postulated. Accordingly, we propose that photosynthetic eukaryotic organisms (green algae and plants) contain γCAs and that these enzymes codified by nuclear genes are imported into mitochondria to accomplish their biological function.