Catalytic activity of a ζ-class zinc and cadmium containing carbonic anhydrase. Compared work mechanisms

The carbonic anhydrase is the enzyme that catalyzes the reversible hydration of carbon dioxide and represents one of the most ancient proteins to which a plethora of works was devoted. The three main classes rely on zinc ion for activity. Most recently a new class of CA was discovered in marine diatoms to use naturally a cadmium ion as catalytic metal. In the present investigation we focused our attention on a carbonic anhydrase cambialistic enzyme (CDCA1) belonging to this new class. The study was inspired by the discovery that the replacement of zinc ion with cadmium does not entail significant differences in the catalytic performance of the enzyme. Our aim was to give further insight of the enzymatic work mechanism. Different possible reaction paths were considered for both metallic forms of the enzyme and comparison with previous studies concerning other carbonic anhydrases was made. The effects of the solvent on the energetics of the catalytic process, was also taken into account by means of a polarizable continuum model. The results obtained from density functional calculations, using a well consolidated mixing of exchange-correlation potential and basis set, and performed with a model of the active site designed on the basis of the X-ray crystal structure, proposed for both metal ions similar reaction pathways consisting in the nucleophilic attack by the metal bound hydroxide to the carbon dioxide with bicarbonate formation, in a next internal rotation of this last fragment, and then in the formation of a species ready for the product removal. Similar activation barriers were found in the rate determining steps that confirm the experimental indication concerning the comparable efficiency of the enzyme in the presence of a zinc or cadmium metal ion.     

Structural studies of the [tris(imidazolyl)phosphine]metal nitrate complexes [[PimPrl,But]M(NO3)]+ (M = Co,Cu, Zn,Cd, Hg): comparison of nitrate-binding modes in synthetic analogues of carbonic anhydrase

X-ray diffraction studies on a series of cationic divalent metal nitrate complexes supported by the tris(1-isopropyl-4-tert-butylimidazolyl)phosphine ligand, [[PimPri,But]M(NO3)]+ (M = Co, Cu, Zn, Cd, Hg), demonstrate that the nitrate ligand coordination mode is strongly dependent upon the metal. With the exception of that for the HgII derivative, the nitrate ligand coordination modes correlate with the activities of metal-substituted carbonic anhydrases, such that the only MII-carbonic anhydrases which exhibit significant activity, i.e., the Zn and Co species, are those for which the [[PimPri,But]M(NO3)]+ complexes possess strongly asymmetric nitrate ligands. This trend supports the notion that access to a unidentate, rather than a bidentate, bicarbonate intermediate may be a critical requirement for significant carbonic anhydrase activity. Interestingly, the nitrate coordination modes in the series of group 12 complexes, [[PimPri,But]M(NO3)]+ (M = Zn, Cd, Hg), do not exhibit a monotonic periodic trend: the bidenticity is greater for the cadmium complex than for either the zinc or mercury complexes. Since HgII-carbonic anhydrase is inactive, the correlation between nitrate coordination mode and enzyme activity is anomalous for the mercury complex. Therefore, it is suggested that the inactivity of HgII-carbonic anhydrase may be consequence of the reduced tendency of the mercury center in HgII-carbonic anhydrase to bind water.     

静电纺丝制备中空纤维原位固定化碳酸酐酶用于二氧化碳的吸收

考察了游离碳酸酐酶吸收CO₂水合体系反应条件, 并通过同轴共纺静电纺丝技术制备出中空结构纤维, 实现了碳酸酐酶在中空纤维中的原位包埋, 提高了酶的稳定性并便于回收和重复利用. 实验结果表明, 固定化碳酸酐酶的热稳定性显著增强, 受Cu²⁺和Fe³⁺等金属离子的抑制作用大幅度降低. 连续使用11次后所生成的CaCO₃沉淀量仍能达到首次使用的81.9%. 固定化酶体系生成的CaCO₃沉淀包括方解石型和球文石型2种晶形, 而无酶和加入游离碳酸酐酶的反应体系则主要生成方解石型CaCO₃沉淀.     

Ultrahigh resolution crystal structures of human carbonic anhydrases I and II complexed with "two-prong" inhibitors reveal the molecular basis of high affinity

The atomic-resolution crystal structures of human carbonic anhydrases I and II complexed with "two-prong" inhibitors are reported. Each inhibitor contains a benzenesulfonamide prong and a cupric iminodiacetate (IDA-Cu(2+)) prong separated by linkers of different lengths and compositions. The ionized NH(-) group of each benzenesulfonamide coordinates to the active site Zn(2+) ion; the IDA-Cu(2+) prong of the tightest-binding inhibitor, BR30, binds to H64 of CAII and H200 of CAI. This work provides the first evidence verifying the structural basis of nanomolar affinity measured for two-prong inhibitors targeting the carbonic anhydrases.     

Dithiocarbamates: a new class of carbonic anhydrase inhibitors. Crystallographic and kinetic investigations

The zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) is inhibited by several classes of zinc-binders (sulfonamides, sulfamates, and sulfamides) as well as by compounds which do not interact with the metal ion (phenols, polyamines and coumarins). Here we report a new class of potent CA inhibitors which bind the zinc ion: the dithiocarbamates (DTCs). They coordinate to the zinc ion from the enzyme active site in monodentate manner and establish many favorable interactions with amino acid residues nearby. Several low nanomolar CA I, II and IX inhibitors were detected.     

Allene as the parent substrate in zinc-mediated biomimetic hydration reactions of cumulenes

The aim of our present investigation is to unravel the general mode of biomimetic activation of a wide variety of cumulenes by carbonic anhydrase (CA) models. Carbonic anhydrases allow the specific recognition, activation and transfer not only of CO2 but also of heteroallenes X=C=Y such as the polar or polarizable examples COS, CS2, H2CCO, and RNCS. Therefore, this enzyme class fulfils the requirements of excellent catalysts with a wide variety of important applications. Can this be extended to the isoelectronic but less reactive allene molecule, H2C=C=CH2 and extremely simplified models as mimetic concept for active center of the carbonic anhydrase? Allene is a waste product in the refinery, i.e. the C3-cut of the naphtha distillation; therefore, any addition product that can be obtained from allene in high yields will be of significant value. We investigated the complete catalytic cycle of a very simple model reaction, the hydration of allene, using density functional theory. Additionally, calculations were performed for the uncatalyzed reaction. There are two possible ways for the nucleophilic attack leading to different products. The zinc hydroxide complex and the water molecule can react at the central or the terminal carbon atoms (positional selectivity), the resulting products are 2-propen-1-ol and propen-2-ol, respectively, acetone. The calculations indicate a significant lower energy barrier for the rate determining step of the formation of propen-2-ol and therefore a well-expressed regioselectivity for the addition of such small molecules. The zinc complex has a pronounced catalytic effect and lowers the activation barrier from 262.5 to 123.9 kJ/mol compared with the uncatalyzed reaction. This work suggests the most probable paths for this reaction and discloses the necessity for the development of novel catalysts.     

Enhanced enzymatic activity exerted by a packed assembly of a single type of enzyme

In contrast to the dilute conditions employed for in vitro biochemical studies, enzymes are spatially organized at high density in cellular micro-compartments. In spite of being crucial for cellular functions, enzymatic reactions in such highly packed states have not been fully addressed. Here, we applied a protein adaptor to assemble a single type of monomeric enzyme on a DNA scaffold in the packed or dispersed states for carbonic anhydrase. The enzymatic reactions proceeded faster in the packed than in the dispersed state. Acceleration of the reaction in the packed assembly was more prominent for substrates with higher hydrophobicity. In addition, carbonic anhydrase is more tolerant of inhibitors in the packed assembly. Such an acceleration of the reaction in the packed state over the dispersed state was also observed for xylose reductase. We propose that the entropic force of water increases local substrate or cofactor concentration within the domain confined between enzyme surfaces, thus accelerating the reaction. Our system provides a reasonable model of enzymes in a packed state; this would help in engineering artificial metabolic systems.

The enzymatic reactions proceeded faster in the packed than in the dispersed state.     

In silico analysis of glutathione S-transferase supergene family revealed hitherto unreported insect specific δ- and ε-GSTs and mammalian specific μ-GSTs in Ixodes scapularis (Acari: Ixodidae)

The availability of whole genome sequence information of Ixodes scapularis (Acari: Ixodidae), an important disease vector of veterinary and public health importance, has opened up new opportunities to explore the vector species at genomic level. Use of acaricides is the mainstay in controlling the disease vector, as effective vaccines are not available for most of the diseases that are transmitted by ticks. The glutathione S-transferase (GST) enzymes are one of the important supergene families that are involved in protecting the organism from oxidative stress and xenobiotics including the acaricides. The analysis of GST supergene family from Ixodes identified all the three broad GST classes, viz. canonical, mitochondrial, and microsomal forms. In total, 35 GST genes belong to five different canonical GST classes, namely Delta (7 genes), Epsilon (5), Mu (14), Omega (3), and Zeta (3 genes) GST classes, and two mitochondrial Kappa class GST genes, and a single microsomal GST gene were found. Interestingly, Delta- and Epsilon-class members, which are thought to be specific to the class Insecta, were also identified in Ixodes. Further, vertebrate/mammalian specific Mu-GSTs (14 genes) were also identified in Ixodes. Analyses of the intron-exon organization revealed higher frequency of phase '0' and phase '2' introns. The comprehensive listing of the GST supergene family members from Ixodes may help in understanding molecular mechanisms of the acaricide resistance in mites and ticks. Cumulatively, these findings may provide an in-depth understanding of the complex evolution of GST supergene family, one of the oldest supergene families that exist in all the domains of life.     

A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis

A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02-3 microM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry.     

A comparative study of the catalytic mechanisms of the zinc and cadmium containing carbonic anhydrase

The catalytic mechanism for the conversion of carbon dioxide to hydrogen carbonate by a cadmium containing carbonic anhydrase was explored at density functional level employing two different models to simulate the active center of the enzyme. In the first model, the histidine residues around the metal ion were replaced with imidazole groups. Instead, in the second one, the simplest model was extended introducing two amino acidic residues generally present in the neighbor of enzyme and a deep water molecule. The results showed that cadmium carbonic anhydrase follows a reaction mechanism that is favored thermodynamically but not kinetically with respect to that of the most usual zinc-containing enzyme, both in a vacuum and in a protein environment.     

Synergistic Role of Bacterial Urease and Carbonic Anhydrase in Carbonate Mineralization

The investigation on the synergistic role of urease (UA) and carbonic anhydrase (CA) in biomineralization of calcium carbonate in Bacillus megaterium suggested that the precipitation of CaCO₃ is significantly faster in bacterial culture than in crude enzyme solutions. Calcite precipitation is significantly reduced when both the enzymes are inhibited in comparison with those of the individual enzyme inhibitions indicating that both UA and CA are crucial for efficient mineralization. Carbonic anhydrase plays a role in hydrating carbon dioxide to bicarbonate, while UA aids in maintaining the alkaline pH that promotes calcification process.     

Selective hydrophobic pocket binding observed within the carbonic anhydrase II active site accommodate different 4-substituted-ureido-benzenesulfonamides and correlate to inhibitor potency

4-Substituted-ureido benzenesulfonamides showing inhibitory activity against carbonic anhydrase (CA, EC 4.2.1.1) II between 3.3-226 nM were crystallized in complex with the enzyme. Hydrophobic interactions between the scaffold of the inhibitors in different hydrophobic pockets of the enzyme were observed, explaining the diverse inhibitory range of these derivatives.     

Phosphorus versus Sulfur: Discovery of Benzenephosphonamidates as Versatile Sulfonamide-Mimic Chemotypes Acting as Carbonic Anhydrase Inhibitors

The first zinc-binding group (ZBG) to have been identified as inhibitor of the metallo-enzymes carbonic anhydrases (CA, EC 4.2.1.1) was the sulfonamide. From then on several classes of zinc-binders have been described. This work reports the benzenephosponamidates as a new chiral aromatic sulfonamide-mimic ZBG able to meet the requirements for effectively binding the enzyme active site. Several low micromolar CA I, II, VII, IX inhibitors were thus detected. Kinetic studies, QM-polarized ligand docking, and MM-GBSA in silico methods were used to characterize this newly identified CA inhibitor chemotype.     

The Caenorhabditis elegans Y87G2A.14 Nudix hydrolase is a peroxisomal coenzyme A diphosphatase

Background  

The number of Nudix hydrolase family members varies widely among different organisms. In order to understand the reasons for the particular spectrum possessed by a given organism, the substrate specificity and function of different family members must be established.     

Bicarbonate as a proton donor in catalysis by Zn(II)- and Co(II)-containing carbonic anhydrases.

Catalysis of (18)O exchange between CO(2) and water catalyzed by a Co(II)-substituted mutant of human carbonic anhydrase II is analyzed to show the rate of release of H(2)(18)O from the active site. This rate, measured by mass spectrometry, is dependent on proton transfer to the metal-bound (18)O-labeled hydroxide, and was observed in a site-specific mutant of carbonic anhydrase II in which a prominent proton shuttle residue His64 was replaced by alanine, which does not support proton transport. Upon increasing the concentration of bicarbonate, the rate of release of H(2)(18)O increased in a saturable manner to a maximum of 4 x 10(5) s(-)(1), consistent with proton transfer from bicarbonate to the Co(II)-bound hydroxide. The same mutant of carbonic anhydrase containing Zn(II) had the rate of release of H(2)(18)O smaller by 10-fold, but rate of interconversion of CO(2) and HCO(3)(-) about the same as the Co(II)-containing enzyme. These data as well as solvent hydrogen isotope effects suggest that the bicarbonate transferring the proton is bound to the cobalt in the enzyme. The enhancement of (18)O exchange caused by increasing bicarbonate concentration during catalysis by the Zn(II)-containing carbonic anhydrase from the archaeon Methanosarcina thermophila suggests that a very similar mechanism for proton donation by bicarbonate occurs with this wild-type enzyme.     

Production of active human carbonic anhydrase II in E. coli

cDNA encoding human carbonic anhydrase II has been isolated and its nucleotide sequence determined. Expression of the isolated carbonic anhydrase gene in Escherichia coli from a plasmid containing the tac promoter yielded an active enzyme at a level of about 1% of total protein.     

脲变性牛碳酸酐酶B的稀释复性过程及其集聚作用研究

采用非变性聚丙烯酰胺凝胶电泳、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、高效凝胶排阻色谱以及激光光散射光谱研究了脲变性牛碳酸酐酶B的稀释复性过程及其集聚作用。在脲变性牛碳酸酐酶B的稀释复性过程中,当最终复性液中脲浓度大于2.0mol/L时,牛碳酸酐酶B在复性液中以单分子和二分子集聚体形式存在;当最终复性液中脲浓度小于2.0mol/L大于1.0mol/L时,牛碳酸酐酶B在复性液中以单分子、二分子集聚体和少量多分子集聚体形式存在;而当最终复性液中脲浓度小于等于1.0mol/L时,脲变性牛碳酸酐酶B复性时会形成均匀透明的上清和不透明的沉淀,牛碳酸酐酶B在上清和沉淀中达到动态解离平衡,且在两相中都以单分子、二分子集聚体和少量多分子集聚体形式存在。溶液中二分子和多分子牛碳酸酐酶B集聚体是通过牛碳酸酐酶B分子之间的疏水和静电相互作用力而形成的,当溶液中这些成分达到一定浓度并且溶液中脲的浓度小于某一个值时,它们之间会通过非共价形式形成沉淀。     

Kinetic model of development of acute viral infection in the human body. Critical conditions,control mechanisms, “thermoheliox”

A kinetic model of the development of acute viral infection is proposed and the dynamic behavior of key variables, including the concentrations of viral particles, infected cells, and pathogenic microorganisms, is described. The change in the hydrogen ion concentration in the lungs and pH dependence of the activity of carbonic anhydrase, a key respiration enzyme, are critical factors. An acute bifurcation transition determining either the life or collapse of the system is demonstrated. The transition is associated with exponential increase in the concentrations of participants in the process and with functioning of the key enzyme, carbonic anhydrase. A physicochemical interpretation is given for the therapeutic effect of temperature rise and potential therapeutic effect of “thermoheliox”, that is, breathing by heated helium-oxygen mixture.

     

Calculations of the electrostatic free energy contributions to the binding free energy of sulfonamides to carbonic anhydrase

The interactions between biologically important enzymes and drugs are of great interest. In order to address some aspects of these interactions we have initiated a program to investigate enzymedrug interactions. Specifically, the interactions between one of the isozymes of carbonic anhydrase and a family of drugs known as sulfonamides have been studied using computational methods. In particular the electrostatic free energy of binding of carbonic anhydrase II with acetazolamide, methazolamide,p-chlorobenzenesulfonamide,p-aminobenzenesulfonamide and three new compounds (MK1, MK2, and MK3) has been computed using finite-difference Poisson-Boltzmann (FDPB) [1] method and the semimacroscopic version [2, 3] of the protein dipole Langevin dipole (PDLD) method [4]. Both methods, FDPB and PDLD, give similar results for the electrostatic free energy of binding even though different charges and different treatments were used for the protein. The calculated electrostatic binding free energies are in reasonable agreement with the experimental data. The potential and the limitation of electrostatic models for studies of binding energies are discussed.     

Synthesis of 1-oxa-9-azaspiro[5.5]undecane-9-sulfonamides bearing a diverse molecular periphery and a rare zinc-binding group for carbonic anhydrase interrogation

A fundamentally novel type of molecular probes for the recognition by carbonic anhydrase zinc enzymes which constitute promising target family in diverse therapeutic areas has been designed and synthesized. To the best of our knowledge, these molecular tools of 1-oxa-9-azaspiro[5.5]-undecane-9-sulfonamide chemotype for the first time combine in their structure diversely substituted spirocyclic piperidines and an aminosulfamoyl moiety.