Complex Of Carbonic Anhydrase Research Articles

Enhanced CO2 fixation and lipid production of Chlorella vulgaris through the carbonic anhydrase complex

Photosynthesis of C. vulgaris shows slow growth and low lipid production due to the low solubility of CO2, and it is thus necessary to increase the dissolved inorganic carbon source to solve this problem. In this study, carbonic anhydrase (CA) was fused with dockerin to form a CA complex by cohesion-dockerin interaction. The CA complex was displayed on the surface of C. vulgaris by a cellulose binding module. The CA complex increased activity and stability compared to those of a single enzyme. Additionally, C. vulgaris showed an average of 1.6-fold rapid growth during log phase through the influence of the CA complex. The bicarbonate produced by the CA complex increased the lipid production about 1.7-fold (23.3%), compared to 13.6% for the control group. The present results suggest that the CA complex successfully enhances the CO2 fixation, which should be an essential study for 4th generation biofuels.

Carbon Dioxide Sequestration by Using a Model Carbonic Anhydrase Complex in Tertiary Amine Medium.

Globally, the elevation of carbon dioxide (CO2 ) levels due to the anthropogenic effect poses a serious threat to the ecosystem. Hence, it is important to control and/or mitigate the level of CO2 in the atmosphere, which necessitates novel tools. Herein, it is proposed to improve CO2 sequestration by using model complexes based on the enzyme carbonic anhydrase (CA) in aqueous tertiary amine medium. The effect of substituents on the model CA model complexes on CO2 absorption and desorption was determined by using a stopped-flow spectrophotometer to follow pH changes through coupling to pH indicator and a continuous stirred-tank reactor (CSTR). The CO2 hydration rate constants were determined under basic conditions and compound 6, which contained a hydrophilic group, showed the highest absorption or hydration levels of CO2 (2.860×10(3) L mol(-1) s(-1) ). In addition, CSTR results for the absorption and desorption of CO2 suggest that simple model CA complexes could be used in post-combustion processing.

Ligand-induced protein mobility in complexes of carbonic anhydrase II and benzenesulfonamides with oligoglycine chains.

This paper describes a biophysical investigation of residual mobility in complexes of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamide ligands with chains of 1–5 glycine subunits, and explains the previously observed increase in entropy of binding with chain length. The reported results represent the first experimental demonstration that BCA is not the rigid, static globulin that has been typically assumed, but experiences structural fluctuations upon binding ligands. NMR studies with 15N-labeled ligands demonstrated that the first glycine subunit of the chain binds without stabilization or destabilization by the more distal subunits, and suggested that the other glycine subunits of the chain behave similarly. These data suggest that a model based on ligand mobility in the complex cannot explain the thermodynamic data. Hydrogen/deuterium exchange studies provided a global estimate of protein mobility and revealed that the number of exchanged hydrogens of BCA was higher when the protein was bound to a ligand with five glycine subunits than when bound to a ligand with only one subunit, and suggested a trend of increasing number of exchanged hydrogens with increasing chain length of the BCA-bound ligand, across the series. These data support the idea that the glycine chain destabilizes the structure of BCA in a length-dependent manner, causing an increase in BCA mobility. This study highlights the need to consider ligand-induced mobility of even “static” proteins in studies of protein-ligand binding, including rational ligand design approaches.

Atomic resolution studies of carbonic anhydrase II

Carbonic anhydrase has been well studied structurally and functionally owing to its importance in respiration. A large number of X-ray crystallographic structures of carbonic anhydrase and its inhibitor complexes have been determined, some at atomic resolution. Structure determination of a sulfonamide-containing inhibitor complex has been carried out and the structure was refined at 0.9 A resolution with anisotropic atomic displacement parameters to an R value of 0.141. The structure is similar to those of other carbonic anhydrase complexes, with the inhibitor providing a fourth nonprotein ligand to the active-site zinc. Comparison of this structure with 13 other atomic resolution (higher than 1.25 A) isomorphous carbonic anhydrase structures provides a view of the structural similarity and variability in a series of crystal structures. At the center of the protein the structures superpose very well. The metal complexes superpose (with only two exceptions) with standard deviations of 0.01 A in some zinc-protein and zinc-ligand bond lengths. In contrast, regions of structural variability are found on the protein surface, possibly owing to flexibility and disorder in the individual structures, differences in the chemical and crystalline environments or the different approaches used by different investigators to model weak or complicated electron-density maps. These findings suggest that care must be taken in interpreting structural details on protein surfaces on the basis of individual X-ray structures, even if atomic resolution data are available.

A knowledge-guided strategy for improving the accuracy of scoring functions in binding affinity prediction

BackgroundCurrent scoring functions are not very successful in protein-ligand binding affinity prediction albeit their popularity in structure-based drug designs. Here, we propose a general knowledge-guided scoring (KGS) strategy to tackle this problem. Our KGS strategy computes the binding constant of a given protein-ligand complex based on the known binding constant of an appropriate reference complex. A good training set that includes a sufficient number of protein-ligand complexes with known binding data needs to be supplied for finding the reference complex. The reference complex is required to share a similar pattern of key protein-ligand interactions to that of the complex of interest. Thus, some uncertain factors in protein-ligand binding may cancel out, resulting in a more accurate prediction of absolute binding constants.ResultsIn our study, an automatic algorithm was developed for summarizing key protein-ligand interactions as a pharmacophore model and identifying the reference complex with a maximal similarity to the query complex. Our KGS strategy was evaluated in combination with two scoring functions (X-Score and PLP) on three test sets, containing 112 HIV protease complexes, 44 carbonic anhydrase complexes, and 73 trypsin complexes, respectively. Our results obtained on crystal structures as well as computer-generated docking poses indicated that application of the KGS strategy produced more accurate predictions especially when X-Score or PLP alone did not perform well.ConclusionsCompared to other targeted scoring functions, our KGS strategy does not require any re-parameterization or modification on current scoring methods, and its application is not tied to certain systems. The effectiveness of our KGS strategy is in theory proportional to the ever-increasing knowledge of experimental protein-ligand binding data. Our KGS strategy may serve as a more practical remedy for current scoring functions to improve their accuracy in binding affinity prediction.

Nonspecific Protein−Carbohydrate Complexes Produced by Nanoelectrospray Ionization. Factors Influencing Their Formation and Stability

Factors influencing the formation of nonspecific protein-carbohydrate complexes during the nanoelectrospray (nanoES) process have been investigated. Protonated and deprotonated nonspecific complexes of ubiquitin (Ubq) and protonated complexes of carbonic anhydrase (CA) with carbohydrates, ranging in size from mono- to tetrasaccharide, were produced by nanoES and detected with a Fourier transform ion cyclotron resonance mass spectrometer. Both the fraction of protein engaged in nonspecific binding with the carbohydrates and the number of carbohydrates bound to the protein increase with increasing carbohydrate concentration. At a given concentration of protein and carbohydrate, nonspecific binding is favored for small (mono- and disaccharide) or hydrophilic carbohydrates over larger or more hydrophobic molecules, which tend to form gaseous monomer or cluster ions by nanoES. However, the extent of nonspecific binding is insensitive to the structure of the protein, with similar distributions of nonspecific complexes observed for both CA and Ubq. Nonspecific association is also insensitive to the charge state of the complex. A comparable degree of binding is observed for complexes in their protonated and deprotonated forms. Furthermore, the number of bound ligands can exceed significantly the charge state of the complex. Thermal dissociation experiments performed on the gaseous nonspecific complexes reveal that their kinetic stability is sensitive to both the structure of the carbohydrate (i.e., mono- < di- < tri- < tetrasaccharide) and the protein (Ubq < CA) and to the charge state, although no simple relationship between stability and charge state was identified. Taken together, the results of this study suggest that neutral protein-carbohydrate interactions (e.g., hydrogen bonds) contribute significantly and, perhaps, predominantly to the formation and stabilization of the nonspecific complexes. A strategy to minimize the formation of the nonspecific complexes, which is based on the enhancement of gaseous carbohydrate ion formation through the addition of metal salts (e.g., CaCl2) to the nanoES solution, is demonstrated.

Mutant carbonic anhydrase 4 impairs pH regulation and causes retinal photoreceptor degeneration

Retina and retinal pigment epithelium (RPE) belong to the metabolically most active tissues in the human body. Efficient removal of acid load from retina and RPE is a critical function mediated by the choriocapillaris. However, the mechanism by which pH homeostasis is maintained is largely unknown. Here, we show that a functional complex of carbonic anhydrase 4 (CA4) and Na+/bicarbonate co-transporter 1 (NBC1) is specifically expressed in the choriocapillaris and that missense mutations in CA4 linked to autosomal dominant rod-cone dystrophy disrupt NBC1-mediated HCO3- transport. Our results identify a novel pathogenic pathway in which a defect in a functional complex involved in maintaining pH balances, but not expressed in retina or RPE, leads to photoreceptor degeneration. The importance of a functional CA4 for survival of photoreceptors implies that CA inhibitors, which are widely used as medications, particularly in the treatment of glaucoma, may have long-term adverse effects on vision.

Nucleophilic reaction by carbonic anhydrase model zinc compound: characterization of intermediates for CO 2 hydration and phosphoester hydrolysis

The partially hydrophilic and hydrophobic tripodal ligands, tris(hydroxy-2-benzimidazolylmethyl)amine L1h and tris(2-benzimidazolyl)amine L1 were used for the preparation of biomimetic complex of carbonic anhydrase. The CO 2 hydration using [L1hZn(OH)]ClO 4 · 1.5H 2O provided the zinc-bound and free HCO 3 −s, which were formed by nucleophilic attack of Zn–OH toward CO 2 in dimethyl sulfoxide (DMSO). The phenolic OH in L1h can recognize water molecules through hydrogen bonds to facilitate the collection of the water molecules around a biomimetic zinc compound; the molecular structure of [L1hZn(OH)] + was revealed. The packing diagram has demonstrated the all the water molecules are hydrogen bonded to each phenolic OH. The nucleophilic attack of zinc-bound OH − to substrate is used to catalyze the CO 2 hydration and phosphoester hydrolysis. The carbonic anhydrase model compound [L1Zn(OH 2)] 2+ was applied for the hydrolysis of phosphoesters, parathion and bis( p-nitrophenyl)phosphate (BNPP −). The low reactivity of [L1Zn(OH)] + for parathion hydrolysis is attributed to the stability of the intermediate [L1Zn(OP(S)(OEt) 2)] +. Since the structures of the intermediates [L1Zn(OH 2)](BNPP) 2 ( 1) and [L1Zn(OP(S)(OEt) 2)]ClO 4 ( 2) formed on the way of hydrolysis are too stable to realize the catalytic cycle and are not active for hydrolysis, carbonic anhydrase model compound [L1Zn(OH 2)] 2+ was not suitable for phosphoester hydrolysis; the zinc model compound surrounded by three benzimidazolyl groups is used to have the steric hindrance for bulky substrate, such as parathion and BNPP −.

Kinetics and mechanism of the catalytic dehydration of HCO3− by zinc(II) complexes of tripod ligands

AbstractThe kinetics of the catalyzed dehydration of HCO3− by zinc(II) containing tripod complexes has been studied at 25°C using the stopped‐flow technique. The direction of reaction curve was changed in aqueous solution when the pH of the solution was greater than 7.5. The pH‐profile of rates of the dehydration reactions indicates that only the aqua complex catalyzes the dehydration of HCO3− via a ligand substitution process. The second‐order rate constants for the dehydration of HCO3− catalyzed by complexes Zn3L1, Zn3L2, Zn3L3, and Zn3L4 are 0.96, 2.53, 12.05, and 6.99 mol−1 dm3 s−1 respectively. At the same time, the pKa values 7.60, 7.16, 7.51, and 7.42 for the deprotonation of the Zn(II)‐bound water in the four catalysts were obtained, which are consistent with those that resulted from pH titrations, i.e. 7.47, 7.25, 7.52, and 7.38 respectively. The mechanism is proposed and the results are compared with other model complexes of carbonic anhydrase. © 2004 Wiley Periodicals, Inc. Int J Chem Kinet 36: 197–203, 2004

Probing the Energetics of Dissociation of Carbonic Anhydrase-Ligand Complexes in the Gas Phase

This paper describes the use of electrospray ionization-Fourier transform ion cyclotron mass spectrometry (ESI-FTICR-MS) to study the relative stabilities of noncovalent complexes of carbonic anhydrase II (CAII, EC 4.2.1.1) and benzenesulfonamide inhibitors in the gas phase. Sustained off-resonance irradiation collision-induced dissociation (SORI-CID) was used to determine the energetics of dissociation of these CAII-sulfonamide complexes in the gas phase. When two molecules of a benzenesulfonamide ( 1) were bound simultaneously to one molecule of CAII, one of them was found to exhibit significantly weaker binding (Δ E 50 = 0.4 V, where E 50 is defined as the amplitude of sustained off-resonance irradiation when 50% of the protein-ligand complexes are dissociated). In solution, the benzenesulfonamide group coordinates as an anion to a Zn(II) ion bound at the active site of the enzyme. The gas phase stability of the complex with the weakly bound inhibitor was the same as that of the inhibitor complexed with apoCAII (i.e., CAII with the Zn(II) ion removed from the binding site). These results indicate that specific interactions between the sulfonamide group on the inhibitor and the Zn(II) ion on CAII were preserved in the gas phase. Experiments also showed a higher gas phase stability for the complex of para-NO 2-benzenesulfonamide-CAII than that for ortho-NO 2-benzenesulfonamide-CAII complex. This result further suggests that steric interactions of the inhibitors with the binding pocket of CAII parallel those in solution. Overall, these results are consistent with the hypothesis that CAII retains, at least partially, the structure of its binding pocket in the gas phase on the time scale (seconds to minutes) of the ESI-FTICR measurements.

A ternary complex of carbonic anhydrase: X-ray crystallographic structure of the adduct of human carbonic anhydrase II with the activator phenylalanine and the inhibitor azide

The first X-ray structure of a ternary adduct of human carbonic anhydrase II with the activator phenylalanine and the inhibitor azide is reported. Confirming what is suggested by spectroscopic data on the Co(II) derivative of HCA II, the phenylalanine molecule does not interact with zinc and it is positioned in the hydrophobic region of the active site cavity, similarly to what is observed for histamine, the first activator for which the X-ray crystallographic structure has been reported. The aromatic ring of phenylalanine is pointed towards the cavity opening while its amino group is involved in a strong hydrogen bond with a water molecule which, on the other hand, interacts with a zinc-bound azide anion. This result provides further support for the existence of an ‘activator binding site’ in the HCA II cavity which allows the binding of compounds able to provide alternative proton transfer pathways from the zinc-bound water molecule to the bulk solvent.

Determination of sulfonamides with fluorescent inclusion complex of carbonic anhydrase and dansylamide

A new fluorimetric method for determination of sulfonamides was described based on the formation of a fluorescent inclusion complex of carbonic anhydrase(CAI with dansylamide(DNSA). The binding of DNSA to CA resulted in an enhancement in the fluorescence emission at 460 nrn with excitation at 280 nrn. Dissociation constants were determined for the carbonic anhydrase-sulfonamide complexes. Linear calibration graphs of sulfonamides were obtained within a concentration range of 0–0.058 μg/mL for DNSA; 0–0.344 pg/mL for sulfanilamide (SAN) and 0–0.286 μg/mL forP-toluene-sulfonamide (PTSN). The relative standard deviations were within 1.8%-4.2%. Limits of detection for DNSA, SAN and PTSN were 0.84, 19. 5 and 6.1 ng/mL, respectively. The method was applied to the determination of sulfonamides in cow milk at ng/mL level.

A new rapid fluorimetric method for the determination of carbonic anhydrase

A new assay procedure for carbonic anhydrase activity has been developed, based on the formation of a fluorescent inclusion complex of carbonic anhydrase with dansylamide. The excitation and emission wavelengths of the fluorescent complex are at 280 nm and 460 nm, respectively. The method can be used at 20 ± 1 ° C, with buffered media over a pH range of 7.0–8.5. The detection limit for carbonic anhydrase is 29 ng ml −1. The recommended method is very sensitive, simple and rapid, making it ideal for routine determinations of carbonic anhydrase activity.

Crystallographic analysis of Thr‐200 → His human carbonic anhydrase II and its complex with the substrate, HCO

A complex of carbonic anhydrase (CA) with one of its substrates, bicarbonate, has been studied crystallographically. Human isoenzyme II was mutated at position 200 from threonine to histidine, which results in higher affinity for bicarbonate. The HCO3- ion binds in the active site to the zinc ion as a pseudo-bidentate ligand which gives the metal a coordination geometry between tetrahedral and trigonal bipyramide. The water/hydroxide normally bound with tetrahedral coordination to the zinc is probably replaced by the OH group of the bicarbonate ion. The importance of residues Thr-199 and Glu-106 in controlling the binding orientation of HCO3- is discussed as well as the catalytic mechanism. Both the complex as well as the uncomplexed mutant were studied at 1.9 A resolution.

ChemInform Abstract: 15N NMR Studies of the Complex of Carbonic Anhydrase with the Novel Carbonyl Hydration Substrate Pyruvamide. Evidence for the Coordination of the Deprotonated Amide Group to the Active Site Zinc.

ChemInformVolume 19, Issue 10 Natural Products ChemInform Abstract: 15N NMR Studies of the Complex of Carbonic Anhydrase with the Novel Carbonyl Hydration Substrate Pyruvamide. Evidence for the Coordination of the Deprotonated Amide Group to the Active Site Zinc. J. MUKHERJEE, J. MUKHERJEE Res. Serv., Vet. Adm. Med. Cent., Kansas City, MO 64128, USASearch for more papers by this authorJ. I. ROGERS, J. I. ROGERS Res. Serv., Vet. Adm. Med. Cent., Kansas City, MO 64128, USASearch for more papers by this authorR. G. KHALIFAH, R. G. KHALIFAH Res. Serv., Vet. Adm. Med. Cent., Kansas City, MO 64128, USASearch for more papers by this authorG. W. JUN. EVERETT, G. W. JUN. EVERETT Res. Serv., Vet. Adm. Med. Cent., Kansas City, MO 64128, USASearch for more papers by this author J. MUKHERJEE, J. MUKHERJEE Res. Serv., Vet. Adm. Med. Cent., Kansas City, MO 64128, USASearch for more papers by this authorJ. I. ROGERS, J. I. ROGERS Res. Serv., Vet. Adm. Med. Cent., Kansas City, MO 64128, USASearch for more papers by this authorR. G. KHALIFAH, R. G. KHALIFAH Res. Serv., Vet. Adm. Med. Cent., Kansas City, MO 64128, USASearch for more papers by this authorG. W. JUN. EVERETT, G. W. JUN. EVERETT Res. Serv., Vet. Adm. Med. Cent., Kansas City, MO 64128, USASearch for more papers by this author First published: March 8, 1988 https://doi.org/10.1002/chin.198810388AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume19, Issue10March 8, 1988 RelatedInformation

Nitrogen-15 NMR studies of the complex of carbonic anhydrase with the novel carbonyl hydration substrate pyruvamide. Evidence for the coordination of the deprotonated amide group to the active site zinc

Nitrogen-15 NMR studies of the complex of carbonic anhydrase with the novel carbonyl hydration substrate pyruvamide. Evidence for the coordination of the deprotonated amide group to the active site zinc

Drug protein interaction at the molecular level: A study of sulphonamide carbonic anhydrase complexes

The molecular mechanism of drug action has been studied by X-ray diffraction analysis of human carbonic anhydrase I complexed with two different sulphonamides. The acetazolamide and amino benzene sulphonamide are found to bind to the catalytically essential zinc ion thereby inhibiting the function of the enzyme. The inhibitor molecules are stabilized in the active site of the protein by van der Waals interaction with a number of protein side chain groups.

Cyanide binding to carbonic anhydrase. A 13 C-nuclear-magnetic-resonance study.

From the similar 13C chemical shifts measured for the cyanide complexes of carbonic anhydrase B enzymes and inorganic zinc cyanide complexes, it is concluded that cyanide binds in its unprotonated form directly to the zinc ion in the enzyme. The cyanide 13C chemical shift of the enzyme · cyanide complex is pH independent indicating that neither the cyanide nor the atom to which it is bonded can undergo protolytic reactions.The 13C spectrum of HCN is reported and the C‐H directly‐bonded coupling constant measured as 275 ± 10 Hz.

Extrinsic Cotton effects in a metal chelator-bovine carbonic anhydrase complex.

Extrinsic Cotton effects in a metal chelator-bovine carbonic anhydrase complex.

Electron Spin Resonance of Metallocarbonic Anhydrases

Abstract Electron spin resonance (ESR) spectra of Cu(II) and Co(II) derivatives of human, monkey, and bovine carbonic anhydrases and their anion complexes are reported. The ESR signals of most of the Cu(II) derivatives indicate a coordination geometry with axial symmetry. Copper carbonic anhydrase complexes appear to have one coordination site filled by a monodentate ligand from solution. This ligand causes increasing electron delocalization from the copper ion in the order CH3COO- l OCN- l ethoxzolamide l HCO3- l azosulfamide l H2O l N3- l SH- l CN- as indicated by decreasing values of g||. The presence of highly resolved nitrogen superhyperfine structure in the spectra of the cyanide complexes of Cu(II) human carbonic anhydrases B and C identifies at least 2 equivalent nitrogen atoms as ligands to the metal ion. Carboxymethylation of the human B enzyme modifies the coordination site, but appears to leave the metal ligands unchanged. A new 2:1 cyanide complex of Co(II) carbonic anhydrase is described with an intense ESR signal typical of low spin Co(II) in an axially symmetrical environment. This is in contrast to the broad, weak absorption of the cobalt enzyme and its 1:1 anion complexes which we have examined and which appear to be high spin Co(II). While the ESR spectra of the Cu(II) and Co(II) carbonic anhydrase complexes which show intense signals are best explained by a square planar or five-coordinate square pyramidal geometry, the geometry of these inactive derivatives probably deviates from that of the catalytically active complex. These studies suggest that there is some flexibility of the ligands at the active site and that certain metal ions or anions may force deviation from the active highly distorted four-coordinate geometry.