The enzyme carbonic anhydrase (CA), catalysing the interconversion between CO2 and HCO3', has long been known to be present in plants as well as in animals. Several of the animal isozymes, but none of the plant CAs, have been extensively studied. When the first plant CA cDNA sequences were published in 1990, it was obvious that the animal and plant CAs represent evolutionarily distinct families with no significant sequence homology between the families. Pea CA is synthesised as a precursor and subsequently processed at the import into the chloroplast. When we purified CA from pea leaves two oligomeric forms with molecular masses around 230 kDa were obtained. One form was homogenous while the other form contained subunits of two different sizes. The larger subunit has an acidic and highly charged N-terminal extension, consisting of 37 residues. We propose that the sequence that precedes the cleavage site resulting in the large subunit represents the functional transit peptide, directing CA to the chloroplast. Neither the transit peptide nor the acidic 37-residue peptide were found to affect the folding, activity or oligomerisation of pea CA. Kinetic investigations showed that pea CA requires a reduced environment and high concentrations of buffer for maximal catalytic activity. High buffer concentrations result in a faster turnover of the enzyme (kcat) while the efficiency (kcatlKm) is not affected. This is consistent with a ping-pong mechanism with the buffer as the second substrate. Both kcat and kcatlKm increase with pH but the dependences cannot be described by simple titration curves. SCN' is an uncompetitive inhibitor at high pH and a noncompetitive inhibitor at neutral and low pH. This is in accordance with the mechanistic model, previously proposed for human CAM, involving a zincbound water molecule as a catalytic group. In this model, the carbon dioxide - bicarbonate interconversion, reflected by kcatlKm, is temporally separated from a rate limiting proton-transfer step. At high pH, solvent hydrogen isotope effects obtained for pea CA agree with this scheme, while they do not fit at neutral and low pH. Site-specific mutations of cysteine residues at positions 165, 269 and 272 were difficult to study, either because strong deviations from Michaelis-Menten kinetics were observed, or because the mutants were found in inclusion bodies. However, the mutant H208A was found to be a very efficient enzyme with the highest kcatlKm value obtained for any CA so far, 2.9-108 M'1s '1. With the H208A mutant an increased dependence on high buffer concentrations at low pH was obtained. At high pH, the mutant is more efficient than the unmutated enzyme. The H208A mutant is also more prone to oxidation than the wild-type enzyme. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 4 uppsatser</p> / digitalisering@umu

<p><p>The adsorption of proteins to solid surfaces has been extensively investigated during the past 20-30 years. The knowledge can be applied in biotechnological applications in for example immunoassays and biosensors. Human carbonic anhydrase II is a widely studied protein and the CO₂-activity makes it an interesting candidate for biotechnological purposes. To make this possible, the factors affecting the adsorption of proteins have to be mapped. The stability of the protein is under great influence of the adsorption and the protein tends to undergo conformational changes leading to a molten globule like state upon adsorption. The stability of a protein also affects the extent of conformational changes and the nature of the adsorption. A more stable protein, adsorbs with less structural changes as a consequence of adsorption, and desorbs from the surface more rapidly than a less stable one. Also the hydrophobicity, charge and area of the surface are affecting the interaction with the protein. Still, the same adsorption pattern is noticed for the same protein at different surfaces, leading to the conclusion that the properties of the protein affect the interaction, rather than the properties of the surface. Biosensors containing carbonic anhydrase have been developed. These make measurement and detection of zinc ions possible. To be able to use carbonic anhydrase as a potential agent in biotechnology, attached to solid surfaces, the protein has to be biotechnologically engineered to get a more stable structure, or else the denaturation will destroy this possibility.</p></p>

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories: Loss-of-function diseases Gain-of-toxic-function diseases Infectious misfolding diseases   Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.   The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease. Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

The adsorption of proteins to solid surfaces has been extensively investigated during the past 20-30 years. The knowledge can be applied in biotechnological applications in for example immunoassays and biosensors. Human carbonic anhydrase II is a widely studied protein and the CO2-activity makes it an interesting candidate for biotechnological purposes. To make this possible, the factors affecting the adsorption of proteins have to be mapped. The stability of the protein is under great influence of the adsorption and the protein tends to undergo conformational changes leading to a molten globule like state upon adsorption. The stability of a protein also affects the extent of conformational changes and the nature of the adsorption. A more stable protein, adsorbs with less structural changes as a consequence of adsorption, and desorbs from the surface more rapidly than a less stable one. Also the hydrophobicity, charge and area of the surface are affecting the interaction with the protein. Still, the same adsorption pattern is noticed for the same protein at different surfaces, leading to the conclusion that the properties of the protein affect the interaction, rather than the properties of the surface. Biosensors containing carbonic anhydrase have been developed. These make measurement and detection of zinc ions possible. To be able to use carbonic anhydrase as a potential agent in biotechnology, attached to solid surfaces, the protein has to be biotechnologically engineered to get a more stable structure, or else the denaturation will destroy this possibility.

No description available.

Šio darbo tikslas – pirimidino junginių – potencialių karboanhidrazių (CA) slopiklių –kūrimas. Teoriniai 4-[N-(pirimidin-4-il)]aminobenzensulfonamidų, turinčių pakaitus 2-, 5- ir 6-oje pirimidino žiedo padėtyje, sąveikos su aktyviuoju hCA centru tyrimai parodė, kad šie junginiai gali įsiterpti į aktyvųjį baltymo centrą ir su hCA turėtų sąveikauti kaip tipiški klasikiniai CA slopikliai. Tiksliniai 4-[N-(2,5,6-pakeisti pirimidin-4-il)amino]benzensulfonamidai sintetinti 4,6-dichlorpirimidinuose, 5-oje padėtyje turinčiuose cian-, formil- arba nitrogrupes, chloro atomą keičiant 4-aminobenzensulfonamidu. Bendradarbiaujant su Biotechnologijos instituto mokslininkais, kurie atliko hCA slopinimo susintetintais junginiais tyrimus, tobulintos šių junginių hCA slopinimo savybės. Slopiklių struktūros modifikuotos keičiant jungtuko tarp benzensulfonamido ir pirimidino fragmentų ilgį ir įvedant naujus pakaitus pirimidino žiede, kai kuriais atvejais taip sudarant naujas heterociklines sistemas. Šiuo tikslu ištirta pirimidin-5-karbaldehidų kondensacija su indolin-2-tionais. Nustatyta, kad 4-[N-(pirimidin-4-il)amino](metil-,etil-)benzensulfonamidai, 5-oje pirimidino žiedo padėtyje turintys cian-, formil- arba nitrogrupes, o 6-oje pirimidino žiedo padėtyje turintys benzilamino-, chlor-, metoksi- arba oksogrupes, yra nano- – mikromolinės eilės hCA slopikliai, galintys atrankiai slopinti hCAI, II ar XIII. Jų hCA slopinimo aktyvumą lemia sulfonamido grupės sąveika su katalitiniu cinko jonu ir... [toliau žr. visą tekstą] / The aim of the work was design of pyrimidine derivatives – potent carbonic anhydrase (CA) inhibitors. Theoretical investigation of interaction of 4-[N-(pyrimidin-4-yl)]aminobenzenesulfonamides containing substituents at 2-, 5- and 6- positions of pyrimidine ring with an active site of hCAs suggested that these compounds can fit into an active site of the enzymes and should interact with them as typical hCA inhibitors. Synthesis of target compounds was carried out by substitution of chloro group at 4,6-dichloropyrimidines containing cyano-, formyl- or nitro groups at position 5 of the pyrimidine ring with 4-aminobenzenesulfonamide. Regarding inhibition of hCAs with the synthesized compounds data acquired by the scientists of Institute of Biotechnology, structures of inhibitors were modified in order to improve their binding properties to hCA. It was performed by variation of a linker length between benzenesulfonamide and pyrimidine fragments and by introduction of new substituents at position 6 of the pyrimidine ring. In some cases these modifications led to the formation of new heterocyclic systems. For this purpose condensation of indoline-2-thiones with pyrimidine-5-carbaldehydes was investigated. It was determined that 4-[N-(pyrimidin-4-yl)amino](methyl-,ethyl-)benzenesulfonamides containing cyano-, formyl- or nitro groups at position 5 of the pyrimidine ring and benzylamino-, chloro-, methoxy- or oxo groups at position 6 of the pyrimidine ring inhibit hCA in... [to full text]

The aim of the work was design of pyrimidine derivatives – potent carbonic anhydrase (CA) inhibitors. Theoretical investigation of interaction of 4-[N-(pyrimidin-4-yl)]aminobenzenesulfonamides containing substituents at 2-, 5- and 6- positions of pyrimidine ring with an active site of hCAs suggested that these compounds can fit into an active site of the enzymes and should interact with them as typical hCA inhibitors. Synthesis of target compounds was carried out by substitution of chloro group at 4,6-dichloropyrimidines containing cyano-, formyl- or nitro groups at position 5 of the pyrimidine ring with 4-aminobenzenesulfonamide. Regarding inhibition of hCAs with the synthesized compounds data acquired by the scientists of Institute of Biotechnology, structures of inhibitors were modified in order to improve their binding properties to hCA. It was performed by variation of a linker length between benzenesulfonamide and pyrimidine fragments and by introduction of new substituents at position 6 of the pyrimidine ring. In some cases these modifications led to the formation of new heterocyclic systems. For this purpose condensation of indoline-2-thiones with pyrimidine-5-carbaldehydes was investigated. It was determined that 4-[N-(pyrimidin-4-yl)amino](methyl-,ethyl-)benzenesulfonamides containing cyano-, formyl- or nitro groups at position 5 of the pyrimidine ring and benzylamino-, chloro-, methoxy- or oxo groups at position 6 of the pyrimidine ring inhibit hCA in... [to full text] / Šio darbo tikslas – pirimidino junginių – potencialių karboanhidrazių (CA) slopiklių –kūrimas. Teoriniai 4-[N-(pirimidin-4-il)]aminobenzensulfonamidų, turinčių pakaitus 2-, 5- ir 6-oje pirimidino žiedo padėtyje, sąveikos su aktyviuoju hCA centru tyrimai parodė, kad šie junginiai gali įsiterpti į aktyvųjį baltymo centrą ir su hCA turėtų sąveikauti kaip tipiški klasikiniai CA slopikliai. Tiksliniai 4-[N-(2,5,6-pakeisti pirimidin-4-il)amino]benzensulfonamidai sintetinti 4,6-dichlorpirimidinuose, 5-oje padėtyje turinčiuose cian-, formil- arba nitrogrupes, chloro atomą keičiant 4-aminobenzensulfonamidu. Bendradarbiaujant su Biotechnologijos instituto mokslininkais, kurie atliko hCA slopinimo susintetintais junginiais tyrimus, tobulintos šių junginių hCA slopinimo savybės. Slopiklių struktūros modifikuotos keičiant jungtuko tarp benzensulfonamido ir pirimidino fragmentų ilgį ir įvedant naujus pakaitus pirimidino žiede, kai kuriais atvejais taip sudarant naujas heterociklines sistemas. Šiuo tikslu ištirta pirimidin-5-karbaldehidų kondensacija su indolin-2-tionais. Nustatyta, kad 4-[N-(pirimidin-4-il)amino](metil-,etil-)benzensulfonamidai, 5-oje pirimidino žiedo padėtyje turintys cian-, formil- arba nitrogrupes, o 6-oje pirimidino žiedo padėtyje turintys benzilamino-, chlor-, metoksi- arba oksogrupes, yra nano- – mikromolinės eilės hCA slopikliai, galintys atrankiai slopinti hCAI, II ar XIII. Jų hCA slopinimo aktyvumą lemia sulfonamido grupės sąveika su katalitiniu cinko jonu ir... [toliau žr. visą tekstą]

Karboanhidrazės (CA) yra metalofermentai, katalizuojantys virsmus tarp anglies dioksido ir bikarbonato. Jų slopinimas gali būti taikomas gydyti tokias skirtingas ligas kaip glaukoma, vėžys, nutukimas, epilepsija, osteoporozė ir kt. Šiuo metu yra beveik 30 mažamolekulinių junginių, kurie naudojami kaip vaistai, su padidėjusiu karboanhidrazių aktyvumu susijusioms ligoms gydyti. Darbe tirta rekombinantinių žmogaus karboanhidrazių I, II, VII, IX ir XIII sąveika su sulfonamidiniais ligandais. Įvertintas tirtų baltymų stabilumas skirtingomis eksperimentinėmis sąlygomis, nustatyta priešvėžinio taikinio CA IX oligomerinė būsena. Modeliniais baltymais naudojant karboanhidrazes, praplėstos terminio poslinkio metodo taikymo ribos. Išmatuoti 40 naujų susintetintų junginių sąveikos su karboanhidrazėmis termodinaminiai parametrai, išanalizuota CA XIII sąveikos su sulfonamidiniais slopikliais termodinamika, atskiriant tikruosius, nuo eksperimento sąlygų ir susijusių reakcijų nepriklausančius jungimosi parametrus. / Carbonic anhydrases (CAs) are metalloenzymes that catalyze the conversion between carbon dioxide and bicarbonate. Their inhibition can be applied for treatment of different diseases, such as glaucoma, cancer, obesity, epilepsy, osteoporosis, etc. There are nearly 30 small molecule ligands that are used as drugs for carbonic anhydrase related diseases. In this work interaction between recombinant human carbonic anhydrases I, II, VII, IX, XIII and sulfonamide ligands was analysed. Stability of selected carbonic anhydrases was evaluated in different experimental conditions. Oligomeric structure of anticancer target CA IX was determined. Using carbonic anhydrases as model proteins, the application range of thermal shift assay was extended. Binding parameters of 40 new compounds to human carbonic anhydrases were measured. The binding thermodynamics of sulfonamide ligands to CA XIII was analyzed and intrinsic binding parameters, independent of the experimental conditions and linked protonation reactions, were determined.

Carbonic anhydrases (CAs) are metalloenzymes that catalyze the conversion between carbon dioxide and bicarbonate. Their inhibition can be applied for treatment of different diseases, such as glaucoma, cancer, obesity, epilepsy, osteoporosis, etc. There are nearly 30 small molecule ligands that are used as drugs for carbonic anhydrase related diseases. In this work interaction between recombinant human carbonic anhydrases I, II, VII, IX, XIII and sulfonamide ligands was analysed. Stability of selected carbonic anhydrases was evaluated in different experimental conditions. Oligomeric structure of anticancer target CA IX was determined. Using carbonic anhydrases as model proteins, the application range of thermal shift assay was extended. Binding parameters of 40 new compounds to human carbonic anhydrases were measured. The binding thermodynamics of sulfonamide ligands to CA XIII was analyzed and intrinsic binding parameters, independent of the experimental conditions and linked protonation reactions, were determined. / Karboanhidrazės (CA) yra metalofermentai, katalizuojantys virsmus tarp anglies dioksido ir bikarbonato. Jų slopinimas gali būti taikomas gydyti tokias skirtingas ligas kaip glaukoma, vėžys, nutukimas, epilepsija, osteoporozė ir kt. Šiuo metu yra beveik 30 mažamolekulinių junginių, kurie naudojami kaip vaistai, su padidėjusiu karboanhidrazių aktyvumu susijusioms ligoms gydyti. Darbe tirta rekombinantinių žmogaus karboanhidrazių I, II, VII, IX ir XIII sąveika su sulfonamidiniais ligandais. Įvertintas tirtų baltymų stabilumas skirtingomis eksperimentinėmis sąlygomis, nustatyta priešvėžinio taikinio CA IX oligomerinė būsena. Modeliniais baltymais naudojant karboanhidrazes, praplėstos terminio poslinkio metodo taikymo ribos. Išmatuoti 40 naujų susintetintų junginių sąveikos su karboanhidrazėmis termodinaminiai parametrai, išanalizuota CA XIII sąveikos su sulfonamidiniais slopikliais termodinamika, atskiriant tikruosius, nuo eksperimento sąlygų ir susijusių reakcijų nepriklausančius jungimosi parametrus.

No description available.