RESUMO
Carbonic anhydrase II (CA II) is a zinc metalloenzyme that catalyzes the reversible interconversion of water and CO2 to bicarbonate and a proton. CA II is abundant in most cells, and plays a role in numerous processes including gas exchange, epithelial ion transport, respiration, extra- and intracellular pH control, and vascular regulation. Beyond these CO2 and pH-linked roles, it has been postulated that CA II might also reduce nitrite (NO2-) to nitric oxide (NO), as bicarbonate and NO2- both exhibit sp2 molecular geometry and NO also plays an important role in vasodilation and regulation of blood pressure. Indeed, previous studies by Aamand et al. have shown that bovine CA II (BCA II) possesses nitrite dehydration activity and paradoxically demonstrated that CA inhibitors (CAIs) such as dorzolamide and acetazolamide significantly increased NO production (Aamand et al., 2009; Nielsen and Fago, 2015) [1,2]. Hence, the goal of this work was to revisit these studies using the same experimental conditions as Aamand et al. measuring NO generation by two methods, and to examine the structure of CA II in complex with NO2- in the presence and absence of dorzolamide. Our results contradict the previous findings and indicate that CA II does not exhibit nitrite reductase or dehydration activity, and that this is not enhanced in the presence of CA inhibitors. In addition, a structural examination of BCA II in complex with NO2- and superimposed with dorzolamide demonstrates that CA inhibitor binding at the active site to the zinc moiety blocks potential NO2- binding.
Assuntos
Anidrase Carbônica II/química , Nitrito Redutases/química , Oxirredutases/química , Animais , Bovinos , Cristalografia por Raios XRESUMO
Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser(161)-Glu(162)-Asn(163)-Ser(164) in the N-terminal domain of OxDC with the cognate residues Asp(161)-Ala(162)-Ser-(163)-Asn(164) of an evolutionarily related, Mn-dependent oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity has now been investigated using membrane inlet mass spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C-C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild-type enzyme, even though the Glu(162) side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn(II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from an increased level of access of the solvent to the active site during catalysis, implying that the functional role of Glu(162) is to control loop conformation. A 2.6 Å resolution X-ray crystal structure of a complex between oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu(162) has been deleted from the active site loop, reveals the likely mode by which the substrate coordinates the catalytically active Mn ion prior to C-C bond cleavage. The "end-on" conformation of oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to mediate the formation of Mn(III) for catalysis upon substrate binding.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Modelos Moleculares , Ácido Oxálico/metabolismo , Engenharia de Proteínas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Biocatálise , Carboxiliases/química , Carboxiliases/genética , Domínio Catalítico , Coriolaceae/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácido Glutâmico/química , Conformação Molecular , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ácido Oxálico/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de ProteínaRESUMO
Thiomicrospira crunogena XCL-2 expresses an α-carbonic anhydrase (TcruCA). Sequence alignments reveal that TcruCA displays a high sequence identity (>30%) relative to other α-CAs. This includes three conserved histidines that coordinate the active site zinc, a histidine proton shuttling residue, and opposing hydrophilic and hydrophobic sides that line the active site. The catalytic efficiency of TcruCA is considered moderate relative to other α-CAs (k(cat)/K(M)=1.1×10(7) M(-1) s(-1)), being a factor of ten less efficient than the most active α-CAs. TcruCA is also inhibited by anions with Cl(-), Br(-), and I(-), all showing Ki values in the millimolar range (53-361 mM). Hydrogen sulfide (HS(-)) revealed the highest affinity for TcruCA with a Ki of 1.1 µM. It is predicted that inhibition of TcruCA by HS(-) (an anion commonly found in the environment where Thiomicrospira crunogena is located) is a way for Thiomicrospira crunogena to regulate its carbon-concentrating mechanism (CCM) and thus the organism's metabolic functions. Results from this study provide preliminary insights into the role of TcruCA in the general metabolism of Thiomicrospira crunogena.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Gammaproteobacteria/enzimologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
By using a combination of liquid and solid-state NMR spectroscopy, (15) N-labeled 4-methylimidazole (4-MI) as a local probe of the environment has been studied: 1)â in the polar, wet Freon CDF3 /CDF2 Cl down to 130â K, 2)â in water at pHâ 12, and 3)â in solid samples of the mutant H64A of human carbonic anhydraseâ II (HCAâ II). In the latter, the active-site His64 residue is replaced by alanine; the catalytic activity is, however, rescued by the presence of 4-MI. For the Freon solution, it is demonstrated that addition of water molecules not only catalyzes proton tautomerism but also lifts its quasidegeneracy. The possible hydrogen-bond clusters formed and the mechanism of the tautomerism are discussed. Information about the imidazole hydrogen-bond geometries is obtained by establishing a correlation between published (1) H and (15) N chemical shifts of the imidazole rings of histidines in proteins. This correlation is useful to distinguish histidines embedded in the interior of proteins and those at the surface, embedded in water. Moreover, evidence is obtained that the hydrogen-bond geometries of His64 in the active site of HCAâ II and of 4-MI in H64A HCAâ II are similar. Finally, the degeneracy of the rapid tautomerism of the neutral imidazole ring His64 reported by Shimahara etâ al. (J. Biol. Chem.- 2007, 282, 9646) can be explained with a wet, polar, nonaqueous active-site conformation in the inward conformation, similar to the properties of 4-MI in the Freon solution. The biological implications for the enzyme mechanism are discussed.
Assuntos
Anidrase Carbônica II/química , Histidina/química , Imidazóis/química , Espectroscopia de Ressonância Magnética/métodos , Humanos , Hidrogênio , Ligação de HidrogênioRESUMO
The binding of anions to carbonic anhydrase II (CA II) has been attributed to high affinity for the active-site zinc. An anion of interest is cyanate, for which contrasting binding modes have been reported in the literature. Previous spectroscopic data have shown cyanate behaving as an inhibitor, directly binding to the zinc, in contrast to previous crystallographic data that implied that cyanate acts as a substrate mimic that is not directly bound to the zinc but overlaps with the binding site of the substrate CO2. Wild-type and the V207I variant of CA II have been expressed and X-ray crystal structures of their cyanate complexes have been determined to 1.7 and 1.5â Å resolution, respectively. The rationale for the V207I CA II variant was its close proximity to the CO2-binding site. Both structures clearly show that the cyanate binds directly to the zinc. In addition, inhibition constants (â¼40â µM) were measured using (18)O-exchange mass spectrometry for wild-type and V207I CA II and were similar to those determined previously (Supuran et al., 1997). Hence, it is concluded that under the conditions of these experiments the binding of cyanate to CA II is directly to the zinc, displacing the zinc-bound solvent molecule, and not in a site that overlaps with the CO2 substrate-binding site.
Assuntos
Anidrase Carbônica II/química , Inibidores da Anidrase Carbônica/química , Cianatos/química , Dióxido de Carbono/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação ProteicaRESUMO
Human carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the hydration and dehydration of CO2 and HCO3 (-), respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH(-)/H2O) in/out of the active site via His64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (â¼20-fold lower with respect to the wild type) of a variant of CA II in which His64 is replaced with Ala (H64A CA II) can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives (imidazole, 1--methylimidazole, 2--methylimidazole and 4-methylimidazole) have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the 'in' and 'out' rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations) the activity of H64A CA II.
RESUMO
The presence of aromatic clusters has been found to be an integral feature of many proteins isolated from thermophilic microorganisms. Residues found in aromatic cluster interact via π-π or C-Hâ¯π bonds between the phenyl rings, which are among the weakest interactions involved in protein stability. The lone aromatic cluster in human carbonic anhydrase II (HCA II) is centered on F226 with the surrounding aromatics F66, F95 and W97 located 12 Å posterior the active site; a location which could facilitate proper protein folding and active site construction. The role of F226 in the structure, catalytic activity and thermostability of HCA II was investigated via site-directed mutagenesis of three variants (F226I/L/W) into this position. The measured catalytic rates of the F226 variants via (18)O-mass spectrometry were identical to the native enzyme, but differential scanning calorimetry studies revealed a 3-4 K decrease in their denaturing temperature. X-ray crystallographic analysis suggests that the structural basis of this destabilization is via disruption and/or removal of weak C-Hâ¯π interactions between F226 to F66, F95 and W97. This study emphasizes the importance of the delicate arrangement of these weak interactions among aromatic clusters in overall protein stability.
Assuntos
Biocatálise , Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Mutação , Anidrase Carbônica II/genética , Domínio Catalítico , Estabilidade Enzimática , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , TemperaturaRESUMO
The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost-inefficient high-maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild-type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) was solved to 1.77â Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. Kinetic studies utilizing the measurement of (18)O-labeled CO2 by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14â K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.
Assuntos
Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Varredura Diferencial de Calorimetria , Anidrase Carbônica II/genética , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Dissulfetos , Estabilidade Enzimática , Humanos , Cinética , Oxirredução , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277-298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20-25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.
Assuntos
Acetazolamida/química , Anidrase Carbônica II/química , Inibidores da Anidrase Carbônica/química , Crioprotetores/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Varredura Diferencial de Calorimetria , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/metabolismo , Domínio Catalítico , Crioprotetores/química , Cristalografia por Raios X , Glicerol/química , Glicerol/metabolismo , Glicerol/farmacologia , Humanos , Cinética , Modelos Moleculares , Conformação Proteica , Sacarose/química , TemperaturaRESUMO
Although widely distributed in Nature, only two γ class carbonic anhydrases are reported besides the founding member (Cam). Although roles for active-site residues important for catalysis have been identified in Cam, second shell residues have not been investigated. Two residues (Trp19 and Tyr200), positioned distant from the catalytic metal, were investigated by structural and kinetic analyses of replacement variants. Steady-state k(cat)/K(m) and k(cat) values decreased 3- to 10-fold for the Trp19 variants whereas the Y200 variants showed up to a 5-fold increase in k(cat). Rate constants for proton transfer decreased up to 10-fold for the Trp19 variants, and an increase of ~2-fold for Y200F. The pK(a) values for the proton donor decreased 1-2 pH units for Trp19 and Y200 variants. The variant structures revealed a loop composed of residues 62-64 that occupies a different conformation than previously reported. The results show that, although Trp19 and Y200 are non-essential, they contribute to an extended active-site structure distant from the catalytic metal that fine tunes catalysis. Trp19 is important for both CO(2)/bicarbonate interconversion, and the proton transfer step of catalysis.
Assuntos
Proteínas Arqueais/química , Anidrases Carbônicas/química , Methanosarcina/enzimologia , Prótons , Triptofano/química , Tirosina/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Cinética , Methanosarcina/química , Modelos Moleculares , Mutação , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triptofano/metabolismo , Tirosina/metabolismoRESUMO
Variants of human carbonic anhydrase II (HCA II) with amino acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and (18)O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for transfer of a proton from His64 to the zinc-bound hydroxide during catalysis were 4 and 9 µs(-1) for Y7F and Y7F/N67Q, respectively, compared with a value of 0.8 µs(-1) for wild-type HCA II. These higher values observed for Y7F and Y7F/N67Q HCA II could not be explained by differences in the values of the pK(a) of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by the orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with a reduced level of branching in the networks of hydrogen-bonded water molecules between proton shuttle residue His64 and the zinc-bound solvent molecule as observed in crystal structures at 1.5-1.6 Å resolution. Moreover, Y7F/N67Q HCA II is unique among the variants studied in having a direct, hydrogen-bonded chain of water molecules between the zinc-bound solvent and N(ε) of His64. This study provides the clearest example to date of the relevance of ordered water structure to rate constants for proton transfer in catalysis by carbonic anhydrase.
Assuntos
Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Prótons , Água/química , Substituição de Aminoácidos , Anidrase Carbônica II/genética , Domínio Catalítico , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese Sítio-DirigidaRESUMO
This work examines the effect of perturbing the position of bound CO(2) in the active site of human carbonic anhydrase II (HCA II) on catalysis. Variants of HCA II in which Val143 was replaced with hydrophobic residues Ile, Leu, and Ala were examined. The efficiency of catalysis in the hydration of CO(2) for these variants was characterized by (18)O exchange mass spectrometry, and their structures were determined by X-ray crystallography at 1.7-1.5 Šresolution. The most hydrophobic substitutions, V143I and V143L, showed decreases in the level of catalysis, as much as 20-fold, while the replacement by the smaller V143A mutation showed an only moderate 2-fold decrease in activity. Structural data for all three variants show no significant change in the overall position of amino acid side chains in the active site compared with the wild type. However, V143A HCA II showed additional ordered water molecules in the active site compared to the number for the wild type. To further investigate the decrease in the catalytic efficiency of V143I HCA II, an X-ray crystallographic CO(2) entrapment experiment was performed to 0.93 Šresolution. This structure revealed an unexpected shift in the CO(2) substrate toward the zinc-bound solvent, placing it ~0.3 Ǻ closer than previously observed in the wild type in conjunction with the observed dual occupancy of the product bicarbonate, presumably formed during the acquisition of data. These data suggest that the Ile substitution at position 143 reduced the catalytic efficiency, which is likely due to steric crowding resulting in destabilization of the transition state for conversion of CO(2) into bicarbonate and a decreased product dissociation rate.
Assuntos
Dióxido de Carbono/metabolismo , Anidrase Carbônica II/química , Domínio Catalítico , Valina/química , Alanina/química , Anidrase Carbônica II/genética , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X , Humanos , Isoleucina/química , Cinética , Leucina/química , Mutagênese Sítio-DirigidaRESUMO
Carbonic anhydrases (CAs) catalyze the hydration of CO(2) forming HCO(3)(-) and a proton, an important reaction for many physiological processes including respiration, fluid secretion, and pH regulation. As such, CA isoforms are prominent clinical targets for treating various diseases. The clinically used acetazolamide (AZM) is a sulfonamide that binds with high affinity to human CA isoform II (HCA II). There are several X-ray structures available of AZM bound to various CA isoforms, but these complexes do not show the charged state of AZM or the hydrogen atom positions of the protein and solvent. Neutron diffraction is a useful technique for directly observing H atoms and the mapping of H-bonding networks that can greatly contribute to rational drug design. To this end, the neutron structure of H/D exchanged HCA II crystals in complex with AZM was determined. The structure reveals the molecular details of AZM binding and the charged state of the bound drug. This represents the first determined neutron structure of a clinically used drug bound to its target.
Assuntos
Acetazolamida/química , Anidrase Carbônica II/química , Hidrogênio/química , Preparações Farmacêuticas/química , Sítios de Ligação , Anidrase Carbônica II/metabolismo , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Difração de NêutronsRESUMO
The carbonic anhydrases (CAs) in the α class are zinc-dependent metalloenzymes. Previous studies have reported that recombinant forms of carbonic anhydrase IX (CAIX), a membrane-bound form of CA expressed in solid tumors, appear to be activated by low levels of zinc independent of its well-studied role at the catalytic site. In this study, we sought to determine if CAIX is stimulated by zinc in its native environment. MDA-MB-231 breast cancer cells express CAIX in response to hypoxia. We compared CAIX activity associated with membrane ghosts isolated from hypoxic cells with that in intact hypoxic cells. We measured CA activity directly using (18)O exchange from (13)CO(2) into water determined by membrane inlet mass spectrometry. In membrane ghosts, there was little effect of zinc at low concentrations on CAIX activity, although at high concentration zinc was inhibitory. In intact cells, zinc had no significant effect on CAIX activity. This suggests that there is an appreciable decrease in sensitivity to zinc when CAIX is in its natural membrane milieu compared to the purified forms.
Assuntos
Antígenos de Neoplasias/metabolismo , Anidrases Carbônicas/metabolismo , Zinco/metabolismo , Neoplasias da Mama/enzimologia , Anidrase Carbônica IX , Catálise , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Feminino , Humanos , Cinética , Zinco/farmacologiaRESUMO
The neutron structure of wild-type human carbonic anhydrase II at pH 7.8 has been determined to 2.0 Å resolution. Detailed analysis and comparison to the previously determined structure at pH 10.0 show important differences in the protonation of key catalytic residues in the active site as well as a rearrangement of the H-bonded water network. For the first time, a completed H-bonded network stretching from the Zn-bound solvent to the proton shuttling residue, His64, has been directly observed.
Assuntos
Anidrase Carbônica II/química , Domínio Catalítico , Água/química , Anidrase Carbônica II/metabolismo , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/metabolismo , Análise de Ativação de Nêutrons/métodos , Prótons , Soluções , Água/metabolismoRESUMO
The tryptophan residue Trp5, highly conserved in the α class of carbonic anhydrases including human carbonic anhydrase II (HCA II), is positioned at the entrance of the active site cavity and forms a π-stacking interaction with the imidazole ring of the proton shuttle His64 in its outward orientation. We have observed that replacement of Trp5 in HCA II caused significant structural changes, as determined by X-ray diffraction, in the conformation of 11 residues at the N-terminus and in the orientation of the proton shuttle residue His64. Most significantly, two variants W5H and W5E HCA II had His64 predominantly outward in orientation, while W5F and wild type showed the superposition of both outward and inward orientations in crystal structures. Although Trp5 influences the orientation of the proton shuttle His64, this orientation had no significant effect on the rate constant for proton transfer near 1µs(-1), determined by exchange of (18)O between CO(2) and water measured by mass spectrometry. The apparent values of the pK(a) of the zinc-bound water and the proton shuttle residue suggest that different active-site conformations influence the two stages of catalysis, the proton transfer stage and the interconversion of CO(2) and bicarbonate.
Assuntos
Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Anidrase Carbônica II/genética , Catálise , Domínio Catalítico , Cristalografia por Raios X , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triptofano/químicaRESUMO
Membrane inlet mass spectrometry (MIMS) uses diffusion across a permeable membrane to detect in solution uncharged molecules of small molecular weight. We point out here the application of MIMS to determine catalytic properties of decarboxylases using as an example catalysis by oxalate decarboxylase (OxDC) from Bacillus subtilis. The decarboxylase activity generates carbon dioxide and formate from the nonoxidative reaction but is accompanied by a concomitant oxidase activity that consumes oxalate and oxygen and generates CO(2) and hydrogen peroxide. The application of MIMS in measuring catalysis by OxDC involves the real-time and continuous detection of oxygen and product CO(2) from the ion currents of their respective mass peaks. Steady-state catalytic constants for the decarboxylase activity obtained by measuring product CO(2) using MIMS are comparable to those acquired by the traditional endpoint assay based on the coupled reaction with formate dehydrogenase, and measuring consumption of O(2) using MIMS also estimates the oxidase activity. The use of isotope-labeled substrate ((13)C(2)-enriched oxalate) in MIMS provides a method to characterize the catalytic reaction in cell suspensions by detecting the mass peak for product (13)CO(2) (m/z 45), avoiding inaccuracies due to endogenous (12)CO(2).
Assuntos
Carboxiliases/química , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Biocatálise , Dióxido de Carbono/análise , Dióxido de Carbono/metabolismo , Isótopos de Carbono , Cinética , Membranas Artificiais , Oxigênio/metabolismo , PermeabilidadeRESUMO
Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme whose expression is often considered a marker for hypoxia, an indicator of poor prognosis in the majority of cancer patients, and is associated with acidification of the tumor microenvironment. Here, we describe for the first time the catalytic properties of native CAIX in MDA-MB-231 breast cancer cells that exhibit hypoxia-inducible CAIX expression. Using (18)O exchange measured by membrane inlet mass spectrometry, we determined catalytic activity in membrane ghosts and intact cells. Exofacial carbonic anhydrase activity increases with exposure to hypoxia, an activity which is suppressed by impermeant sulfonamide CA inhibitors. Inhibition by sulfonamide inhibitors is not sensitive to reoxygenation. CAIX activity in intact cells increases in response to reduced pH. Data from membrane ghosts show that the increase in activity at reduced pH is largely due to an increase in the dehydration reaction. In addition, the kinetic constants of CAIX in membrane ghosts are very similar to our previous measurements for purified, recombinant, truncated forms. Hence, the activity of CAIX is not affected by the proteoglycan extension or membrane environment. These activities were measured at a total concentration for all CO(2) species at 25 mm and close to chemical equilibrium, conditions which approximate the physiological extracellular environment. Our data suggest that CAIX is particularly well suited to maintain the extracellular pH at a value that favors the survival fitness of tumor cells.
Assuntos
Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/enzimologia , Anidrases Carbônicas/metabolismo , Membrana Celular/enzimologia , Proteínas de Neoplasias/metabolismo , Antígenos de Neoplasias/química , Dióxido de Carbono/metabolismo , Anidrase Carbônica IX , Anidrases Carbônicas/química , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Oxigênio/metabolismoRESUMO
In human carbonic anhydrase II (HCA II), the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utilizing the multistate empirical valence bond (MS-EVB) methodology for H64A HCA II yields a PT free energy barrier significantly higher than that found in the wild-type enzyme. This high barrier, determined in the absence of exogenous buffer and assuming no additional ionizable residues in the PT pathway, indicates the likelihood of alternate enzyme pathways that utilize either ionizable enzyme residues (self-rescue) and/or exogenous buffers (chemical rescue). It has been shown experimentally that the catalytic activity of H64A HCA II can be chemically rescued to near wild-type levels by the addition of the exogenous buffer 4-methylimidazole (4MI). Crystallographic studies have identified two 4MI binding sites, yet site-specific mutations intended to disrupt 4MI binding have demonstrated these sites to be nonproductive. In the present work, MS-EVB simulations show that binding of 4MI near Thr199 in the H64A HCA II mutant, a binding site determined by NMR spectroscopy, results in a viable chemical rescue pathway. Additional viable rescue pathways are also identified where 4MI acts as a proton transport intermediary from the active site to ionizable residues on the rim of the active site, revealing a probable mode of action for the chemical rescue pathway.
Assuntos
Anidrase Carbônica II/metabolismo , Prótons , Biocatálise , Anidrase Carbônica II/genética , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-DirigidaRESUMO
Membrane inlet (or introduction) mass spectrometry (MIMS) was used to detect nitroxyl (HNO) in aqueous solution for the first time. The common HNO donors Angeli's salt (AS) and Piloty's acid (PA), along with a newly developed donor, 2-bromo-N-hydroxybenzenesulfonamide (2-bromo-Piloty's acid, 2BrPA), were examined by this technique. MIMS experiments revealed that under physiological conditions 2BrPA is an essentially pure HNO donor, but AS produces a small amount of nitric oxide (NO). In addition, MIMS experiments also confirmed that PA is susceptible to oxidation and NO production, but that 2BrPA is not as prone to oxidation.