Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues

Incorporation of the photoisomerizable amino acid phenylazophenylalanine(PAP) into enzyme structures has been proposed as a strategyfor photoswitching enzyme activity. To evaluate the strengthsand limitations of this approach to enzyme photo-control, weperformed a kinetic analysis of RNase S analogues containingPAP in positions 4, 7, 8, 10, 11 or 13. For an enzyme containinga single PAP group, the maximum extent of photoconversion (betweenapproximately 96% trans/4% cis and 10% trans/90% cis under standardconditions) sets a limit on the maximum fold change in the initialrate of ~25-fold, if the cis form is the more active isomer,and ~10-fold if the trans form is more active. This extent ofphotoswitching was not realized in the present case becausethe effects of photoisomerization on kinetic constants weresmall and distributed among effects on S-peptide binding, substratebinding and the rate of the chemical step. These results suggestthat photoisomerization could substantially alter enzyme kineticconstants but that a directed combinatorial approach might berequired for realizing maximal photo-control in such systems.The limit set by the extent of photoconversion might be overcomeby coupling multiple PAP groups to one enzyme or by alteringthe behaviour of a system that required oligomerization foractivity.     

Polypeptide conjugate binders that discriminate between two isoforms of human carbonic anhydrase in human blood

Two binder candidates 4‐C37L34‐B and 3‐C15L8‐B from a 16‐membered set of 42‐residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence‐based screening assay. Two carbonic anhydrase isoforms with 60 % homology exist in human blood with HCAI being present in five‐ to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16‐membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4‐C37L34‐B bound HCAII with a K_D of 17 nM and HCAI with a K_D of 470 nM , that is, with a 30‐fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3‐C15L8‐B and the two carbonic anhydrases were 60 and 390 nM , respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.     

Model studies for molecular recognition of carbonic anhydrase and carboxypeptidase

Model studies using Zn(2+) complexes of various derivatives of macrocyclic triamines ([12]aneN(3)) and tetraamines (cyclen) have been found to be useful in elucidating and understanding the intrinsic properties of substrate or inhibitor recognition by zinc ions at the active centers of carbonic anhydrase and carboxypeptidase.     

Partially folded conformations in the folding pathway of bovine carbonic anhydrase II: a fluorescence spectroscopic analysis

GdmCl-, urea-, and pH-induced unfolding pathways of bovine carbonic anhydrase II have been analyzed by using changes induced by different denaturing agents in intensity, anisotropy, life time, and parameter A value of intrinsic fluorescence as well as intensity and life time of ANS (ammonium salt of 8-anilinonaphthalene-1-sulfonic acid) fluorescence. The formation of several stable unfolding intermediates, some of which were not observed previously, has been established. This was further confirmed by representation of fluorescence data in terms of a "phase diagram", that is, I(lambda1) versus I(lambda2) dependence, where I(lambda1) and I(lambda2) are the fluorescence intensity values measured at wavelengths lambda(1) and lambda(2), respectively.     

Effects of surface charge on denaturation of bovine carbonic anhydrase

This work compares the denaturation of two proteins-bovine carbonic anhydrase II (BCA) and its derivative with all lysine groups acetylated (BCA-Ac(18))-by urea, guanidinium chloride (GuHCl), heat, and sodium dodecyl sulfate (SDS). It demonstrates that increasing the net negative charge of the protein by acetylation of lysines reduces its stability to urea, GuHCl, and heat, but increases its kinetic stability (its thermodynamic stability cannot be measured) towards denaturation by SDS. Increasing the ionic strength of the buffer improves the stability of BCA-Ac(18) to urea and heat, but still leaves it less stable than unacetylated BCA to those denaturants. In urea, the large change in electrostatic interactions not only modifies the free energy of denaturation, but also introduces a stable intermediate into the unfolding pathway. This work shows that modifications of charges on the surfaces of proteins can have a large effect--positive or negative, depending on the denaturant--on the stability of the proteins despite the exposure of these charges to high dielectric solvent and buffer ions.     

碳酸酐酶用于二氧化碳捕集的研究进展

碳酸酐酶(CA)可以加速捕集化石燃料燃烧产生的二氧化碳,从而降低CO2的排放量。主要介绍了CA的来源、活性、稳定性及作用。分析了使用新型生物方法对二氧化碳进行捕集和储存的优缺点,并对下一步的工作进行了展望。     

Mutagenesis, biochemical characterization and X-ray structural analysis of point mutants of bovine chymosin

Chymosin B point mutants, A115T and G243D (chymosin A), were expressed in Escherichia coli and Trichoderma reesei respectively, characterized biochemically, crystallized and studied by X-ray analysis at 2.3 and 2.8 angstroms resolutions respectively. The three-dimensional structures showed that the mutations gave rise to local conformational changes only when compared with that of chymosin B. Kinetic analysis of the A115T mutant with a six residue synthetic peptide revealed a reduction in Km with respect to the wild type, possibly caused by the small local changes in the vicinity of S1 and S3. Although, kinetic analyses of the G243D mutant using the short substrate showed reduced catalytic activity, use of a 15 residue substrate based on residues 98- 112 of kappa-casein, the natural substrate, revealed an increase in the kcat compared with chymosin B, probably a consequence of the charge introduced that may interact with the substrate between P4 and P8.      

Thienothiopyransulfonamides as complexing agents for the preparation of dual carbonic anhydrase inhibitors

Co(II); Zn(II) and Cu(II) complexes of two new sulfonamide carbonic anhydrase (CA) inhibitors, derivatives of thienothiopyran-2-sulfonamide, were prepared and characterized by analytic, spectroscopic, magnetic and conductimetric measurements. The new complexes are more potent CA inhibitors than the parent sulfonamides, with IC(50) values around 0.1 nM, against isozyme CA II.     

Carbonic anhydrase inhibitors: binding of indanesulfonamides to the human isoform II

Indanesulfonamides are interesting lead compounds for designing selective inhibitors of the different isoforms of the zinc enzyme Carbonic Anhydrase (CA). Herein, we report for the first time the X-ray crystal structure of two such derivatives, namely indane-5-sulfonamide and indane-2-valproylamido-5-sulfonamide, in complex with the physiologically dominant human isoform II. The structural analysis reveals that, although these two inhibitors have quite similar chemical structures, the arrangement of their indane ring within the enzyme active site is significantly diverse. Thus, our findings suggest that the introduction of bulky substituents on the indane-sulfonamide ring may alter the binding mode of this potent class of CA inhibitors, although retaining good inhibitory properties. Accordingly, the introduction of bulky tail moieties on the indane-sulfonamide scaffold may represent a powerful strategy to induce a desired physicochemical property to an aromatic sulfonamide or to obtain inhibitors with diverse inhibition profiles and selectivity for various mammalian CAs.     

Immobilization of carbonic anhydrase by embedding and covalent coupling into nanocomposite hydrogel containing hydrotalcite

Poly(acrylic acid-co-acrylamide)/hydrotalcite (PAA-AAm/HT) nanocomposite hydrogels activated by N-hydroxysuccinimide (NHS) in the presence of N, N'-dicyclohexylcarbodiimide (DCC) were used to immobilize carbonic anhydrase (CA) by embedding and covalent coupling. Cryo Scanning Electron Microscope (CryoSEM) proved the presence of free water in the porous network structures of the swollen hydrogels. Fluorescence microscopy indicated the existence of the immobilized enzyme in the hydrogels. Compared with un-activated hydrogels, activated hydrogels could improve the amount of the immobilization of enzyme, and maximum enzyme loading is about 4.6 mg/g of support for the activated hydrogels. The porous embedding and multi-point covalent linkage between enzyme and hydrogels strengthened the secondary structure stability of enzyme and thus enhanced enzyme stability in the presence of organic solvent and at high temperature. The immobilized enzyme in the activated hydrogel with enhanced structural stability offers great potential as a method to stabilize enzyme for various applications.     

Exploration of the binding mode of indanesulfonamides as selective inhibitors of human carbonic anhydrase type VII by targeting Lys 91

Convulsions are common neurological disorders in clinical medicine and are triggered by several mechanisms. The enhancement of neuronal excitability can be related, among other factors, to GABAergic depolarization. Carbonic anhydrase (CA) VII contributes to this electrophysiological behavior by providing bicarbonate anion, which can mediate current through channels coupled to GABA(A) receptors. Among the cytosolic CAs, the mechanism of action and inhibition of CA VII is less understood. We present herein the pharmacological evaluation of both enantiomers of an indanesulfonamide compound substituted by a pentafluorophenyl moiety against CA VII and five other human CA isoforms to evaluate their selectivity. The investigated compounds are powerful inhibitors of hCA VII, with K(i) values in the range of 1.7-3.3 nM, but their selectivity needs to be improved. A molecular modeling study was conducted to rationalize the structure-activity relationships and provide useful insight into the future design of selective hCA VII inhibitors.     

Chemical stability and use of thermostabilized polyacrylonitrile fibres

The chemical stability of thermostabilized polyacrylonitrile fibre was investigated. It was found that the material is resistant to acids with concentration of up to 30%, petroleum products, and salt water, and is less resistant to bases. The material is promising for fabrication of chemically stable gasket packing of different cross sections and densities.Translated from Khimicheskie Volokna, No. 1, pp. 45–46, January–February, 1995.     

碳酸酐酶固定及在二氧化碳捕集应用研究进展

化石燃料的使用是全球气候变暖的主要原因,二氧化碳捕集、利用及封存(CCUS)技术能够有效减少碳排放,缓解气候变化压力。化学吸收法是CO₂捕集的重要方法之一,具有分离效率高、成本低等优点,但存在解吸过程中消耗能量较多、长期使用造成设备腐蚀性等问题。利用碳酸酐酶(CA)强化化学方法吸收CO₂,可以提高CO₂吸收效率,有效解决传统工艺中的热能损失,逐渐成为CO₂捕集与封存研究中的热点。但CA自身热稳定性低、可重复性差,需对其进行固定化以提高稳定性和活性。重点介绍了CA的固定化方法及常用载体材料,总结了CA在强化CO₂捕集中的作用机理,讨论了其在CO₂捕集中的应用,并对该技术的未来发展方向作出了展望。固定化载体与方法会影响固定化酶的性质,故CA在固定化时要选择合适的载体与方法。CA固定化方法包括吸附法、包埋法、共价结合法和交联法4种,各有优劣,应根据酶所应用的领域来选择合适的固定化方法。常用固定化载体有天然高分子、无机载体材料等,选择时应综合考虑载体的理化性质和工业应用能力。CO₂捕集过程中,CA主要通过促进化学溶剂吸收CO₂和诱导CO₂矿化生成碳酸钙两方面强化捕集效率。未来研究方向应集中于开发具有更高活性和稳定性的新型CA、制备廉价高性能载体材料和进一步探究CA的内在因素和外界条件对其工业应用产生的阻碍。     

Structure of His94->Asp carbonic anhydrase II in a new crystalline form reveals a partially occupied zinc binding site

The structure of histidine 94aspartate (H94D) carbonic anhydraseII (CAII) crystallized in an orthorhombic space group has beendetermined to 2.5 Å resolution. This crystal form is notisomorphous with monoclinic wild-type enzyme crystals or withthe monoclinic crystal form of H94D CAII reported earlier [Kiefer,L.L.,Ippolito,J.A., Fierke,C.A. and Christianson,D.W. (1993) J. Am.Chem. Soc., 115, 12581–12582]. In monoclinic H94D CAII,a fully occupied zinc ion is tetrahedrally coordinated by D94,H96, H119 and a water molecule. In orthorhombic H94D CAII, apartially occupied zinc ion is coordinated by H96 and H119 andonly weakly coordinated by a disordered D94 side chain.Thesedifferences are particularly surprising given that the two crystalforms co-precipitate in the same drop in the same experiment.Re-refinement of the orthorhombic crystal form of H94C CAIIand comparison with its corresponding monoclinic crystal formyield similar results. It appears that partial—but notfull—zinc dissociation accompanies the crystallizationof CAII variants in the orthorhombic crystal form, and significantdifferences on the protein surface presumably affect the relativestability of each crystal lattice. These results underscoreanunexpected ambiguity in this protein engineering experiment:which crystal structure of H94D CAII should be correlated withfunctional measurements made in solution?     

Integrated approach using protein and ligand information to analyze selectivity- and affinity-determining features of carbonic anhydrase isozymes

The application and comparison of selected protein- and ligand-based approaches to elucidate factors important for affinity and selectivity towards the carbonic anhydrase isozymes I, II, and IV are described. Carbonic anhydrases are abundant in pro- and eukaryotes. These enzymes catalyze the reversible hydration of carbon dioxide to bicarbonate and H(+) ions and are thus involved in many important physiological and pathophysiological processes. Due to the fact that the human carbonic anhydrase family consists of 16 closely related isozymes, the design of selective inhibitors is a special challenge for medicinal chemists. In order to extract selectivity-determining features, we applied purely ligand-based 3D QSAR techniques as well as qualitative comparative molecular field analyses of the targets' binding sites using consensus principal component analysis (CPCA). The dataset for the QSAR studies was deliberately compiled from 1,748 inhibitors and comprises about 140 ligands, mainly of the sulfonamide type. Additionally, we employed the novel AFMoC approach, which intrinsically combines protein and ligand information. The simultaneous use of these different techniques gives deeper insight into selectivity and affinity-determining features and provides quantitative models for prediction.     

Kinetics of absorption of CO2 into buffer solutions containing carbonic anhydrase

The rate of absorption of pure CO₂ into equimolar phosphate (pH = 6.6 and 1 1) and carbonate (pH ≈ 9.6) buffers in the presence of crude carbonic anhydrase has been measured in a stirred cell at a temperature range of 5-35°C and at atmospheric pressure. Some experiments with carbonate buffer were also repeated in a wetted wall column.Experimental results were analysed using classical gas absorption with chemical reaction theory in order to extract information about the kinetics of the enzymic hydration of CO₂ and the catalytic power of carbonic anhydrase. At high pH-values (9.6-11.1) this reaction is first order in CO₂ the reaction rate constant being proportional to enzyme concentration with a rate constant of about 0.90 1/mg sec at 25°C and with an activation energy of 9.0 kcal/g mole.At low pH-values (pH ≈ 6.5-6.7) the catalytic power of the enzyme is considerably reduced and the results are not compatible with a simple first order reaction mechanism.     

Electrochemical reduction of carbon dioxide and the effect of the enzyme carbonic anhydrase 11 on iron corrosion

The corrosion of pure iron in carbon dioxide saturated solutions at low temperatures has been investigated in this study. Both dc polarisation and ac impedance techniques were used in conjunction with a rotating disc electrode to investigate the reaction sequence leading to hydrogen evolution at 30°C. The results indicate that protons from the low pH solution, the bicarbonate ion and the undissociated carbonic acid molecule contribute to the reaction sequence leading to eventual hydrogen evolution. The corrosion mechanism involves both activation and diffusion processes. A rotating disc electrode in combination with ac techniques and the enzyme carbonic anhydrate II, EC 4.2.1.1, were used to characterise the diffusion effects. A chemical–electrochemical cathodic reaction mechanism is proposed.     

The development of kinetics model for CO2 absorption into tertiary amines containing carbonic anhydrase

CO₂ absorption into aqueous solutions of two tertiary alkanolamines, namely, MDEA and DMEA with and without carbonic anhydrase (CA) was investigated with the use of the stopped‐flow technique at temperatures in the range of 293–313 K, CA concentration varying from 0 to 100 g/m³ in aqueous MDEA solution with the amine concentration ranging from 0.1 to 0.5 kmol/m³, and CA concentration varying from 0 to 40 g/m³ in aqueous DMEA solution with the amine concentration ranging from 0.05 to 0.25 kmol/m³. The results show that the pseudofirst‐order reaction rate (k_{0, amine}; s^?1) is significantly enhanced in the presence of CA as compared with that without CA. The enhanced values of the kinetic constant in the presence of CA has been calculated and a new kinetics model for reaction of CO₂ absorption into aqueous tertiary alkanolamine solutions catalyzed by CA has been established and used to make comparisons of experimental and calculated pseudo first‐order reaction rate constant (k_{0, with CA}) in CO₂‐MDEA‐H₂O and CO₂‐DMEA‐H₂O solutions. The AADs were 15.21 and 15.17%, respectively. The effect of pKa on the CA activities has also been studied by comparison of CA activities in different tertiary amine solutions, namely, TEA, MDEA, DMEA, and DEEA. The pKa trend for amines were: DEEA > DMEA > MDEA > TEA. In contrast, the catalyst enhancement in amines was in the order: TEA> MDEA> DMEA> DEEA. Therefore, it can be seen that the catalyst enhancement in the amines decreased with their increasing pKa values. © 2017 American Institute of Chemical Engineers AIChE J, 2017