RESUMEN
The maximal velocity in the hydration of CO(2) catalyzed by the carbonic anhydrases in well-buffered solutions is limited by an intramolecular proton transfer from zinc-bound water to acceptor groups of the enzyme and hence to buffer in solution. Stopped-flow spectrophotometry was used to accumulate evidence that this maximal velocity is affected by residues of basic pK(a), near 8 to above 9, in catalysis of the hydration of CO(2) by carbonic anhydrases III, IV, V, and VII. A mutant of carbonic anhydrase II containing the replacement His-64-->Ala, which removes the prominent histidine proton shuttle (with pK(a) near 7), allows better observation of these basic groups. We suggest this feature of catalysis is general for the human and animal carbonic anhydrases and is due to residues of basic pK(a), predominantly lysines and tyrosines more distant from the zinc than His-64, that act as proton acceptors. These groups supplement the well-studied proton transfer from zinc-bound water to His-64 in the most efficient of the carbonic anhydrases, isozymes II, IV, and VII.
Asunto(s)
Anhidrasas Carbónicas/química , Protones , Animales , Tampones (Química) , Dióxido de Carbono/química , Catálisis , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/química , Cinética , SolucionesRESUMEN
Free radical-mediated mechanisms of cellular damage have been implicated in the early stages of spinal cord injury (SCI). Manganese superoxide dismutase (MnSOD) is a potent scavenger of superoxide radicals and likely serves an important cytoprotective role in preventing cellular damage after SCI. We have evaluated the expression of MnSOD to address its role during the early events of SCI using a well-established rat contusion model. Northern analysis showed a rapid induction of MnSOD mRNA between 2 and 6 h post injury. Observed time-dependent increases in MnSOD message was maximal 6 h post injury over that of MnSOD mRNA levels induced by laminectomy alone. Immunoblot and immunohistochemical analysis demonstrated increased expression of MnSOD protein 24 h after SCI with localization primarily within neurons. Interestingly, laminectomy alone also caused an induction of MnSOD gene and protein expression. To evaluate one potential mechanism of MnSOD induction, we microinjected the naive spinal cord with IL-1beta, which caused a similar fold induction of MnSOD mRNA levels by 6 h as observed with SCI, thus implicating it as a potential inducer of MnSOD during SCI. In summary, these results demonstrate that this potent cytoprotective antioxidant enzyme is rapidly and significantly induced as a consequence of SCI.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Traumatismos de la Médula Espinal/enzimología , Médula Espinal/enzimología , Superóxido Dismutasa/biosíntesis , Animales , Northern Blotting , Inducción Enzimática , Femenino , Immunoblotting , Inmunohistoquímica , Interleucina-1/administración & dosificación , Interleucina-1/farmacología , Laminectomía , Microinyecciones , ARN Mensajero/biosíntesis , Ratas , Ratas Long-Evans , Médula Espinal/efectos de los fármacos , Médula Espinal/cirugía , Superóxido Dismutasa/efectos de los fármacos , Superóxido Dismutasa/genética , Factores de TiempoRESUMEN
Maximal turnover rates for the hydration of CO2 and the depletion of 18O from CO2 catalyzed by carbonic anhydrase III (CA III) and carbonic anhydrase V (CA V) are limited by proton transfer involving zinc-bound water or hydroxide in the active site. We have investigated the capacity of glutamic and aspartic acids at position 64 in human CA III and murine CA V to act as proton shuttles in this pathway. The distance from the Calpha of position 64 to the zinc is near 9.5 A in the crystal structures of both CA III and CA V. Rates of intramolecular proton transfer between these proton shuttle groups and the zinc-bound water molecule were estimated as the predominant rate-contributing step in the catalytic turnover kcat in the hydration of CO2 measured by stopped flow and in the 18O exchange between CO2 and water measured by mass spectrometry. We found that both glutamate and aspartate residues at position 64 are efficient proton shuttles in HCA III. The rate constant for intramolecular proton transfer from either residue to zinc-bound hydroxide is 4 x 10(4) s-1, about 20-fold greater than that of the wild type which has lysine at position 64. When the active site residue Phe 198 in human CA III was replaced with Leu, measurement of catalysis showed that Glu 64 retained but Asp 64 lost its capacity to act as a proton shuttle. These observations were supported in studies of catalysis by murine CA V which contains Leu 198; here again, Glu 64 acted as a proton shuttle, but Asp 64 did not. Phe 198 in HCA III is thus a significant factor in the capacity of the active site to sustain proton transfer, possibly through its stabilization of hydrogen-bonded water bridges that enhance proton translocation from both Glu and Asp at position 64 to the zinc-bound hydroxide.
Asunto(s)
Ácido Aspártico/metabolismo , Anhidrasas Carbónicas/metabolismo , Ácido Glutámico/metabolismo , Protones , Animales , Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/genética , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ratones , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometría , Zinc/metabolismoRESUMEN
The hydration of CO2 catalyzed by carbonic anhydrase requires proton transfer from the zinc-bound water at the active site to solution for each cycle of catalysis. In the most efficient of the mammalian carbonic anhydrases, isozyme II, this transfer is facilitated by a proton shuttle residue, His 64. Murine carbonic anhydrase V (mCA V) has a sterically constrained tyrosine at the analogous position; it is not an effective proton shuttle, yet catalysis by this isozyme still achieves a maximal turnover in CO2 hydration of 3 x 10(5) s-1 at pH > 9. We have investigated the source of proton transfer in a truncated form of mCA V and identified several basic residues, including Lys 91 and Tyr 131, located near the mouth of the active-site cavity that contribute to proton transfer. Intramolecular proton-transfer rates between these shuttle groups and the zinc-bound water were estimated as the rate-determining step in kcat for hydration of CO2 measured by stopped-flow spectrophotometry and in the exchange of 18O between CO2 and water measured by mass spectrometry. Comparison of kcat in catalysis by Lys 91 and Tyr 131 and the corresponding double mutant showed a strong antagonistic interaction between these sites, suggesting a cooperative behavior in facilitating the proton-transfer step of catalysis. Replacing four potential proton shuttle residues produced a multiple mutant that had 10% of the catalytic turnover kcat of the wild type, suggesting that the main proton shuttles have been accounted for in mCA V. These replacements caused relatively small changes in kcat/Km for hydration, which measures the interconversion of CO2 and HCO3- in a stage of catalysis that is separate and distinct from the proton transfers; these measurements serve as a control indicating that the replacements of proton shuttles have not caused structural changes that affect reactivity at the zinc.
Asunto(s)
Anhidrasas Carbónicas/química , Protones , Animales , Sitios de Unión , Dióxido de Carbono , Anhidrasas Carbónicas/genética , Catálisis , Cinética , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Isótopos de Oxígeno , EspectrofotometríaRESUMEN
We investigated the efficiency of glutamic acid 64 and aspartic acid 64 as proton donors to the zinc-bound hydroxide in a series of site-specific mutants of human carbonic anhydrase III (HCA III). Rate constants for this intramolecular proton transfer, a step in the catalyzed dehydration of bicarbonate, were determined from the proton-transfer-dependent rates of release of H2 18O from the enzyme measured by mass spectrometry. The free energy plots representing these rate constants could be fit by the Marcus rate theory, resulting in an intrinsic barrier for the proton transfer of deltaG0++ = 2.2 +/- 0.5 kcal/mol, and a work function or thermodynamic contribution to the free energy of reaction wr = 10.8 +/- 0.1 kcal/mol. These values are very similar in magnitude to the Marcus parameters describing intramolecular proton transfer from His64 and His67 to the zinc-bound hydroxide in mutants of HCA III. That result and the equivalent efficiency of Glu64 and Asp64 as proton donors in the catalysis by CA III demonstrate a lack of specificity in proton transfer from these sites, which is indirect evidence of a number of proton conduction pathways through different structures of intervening water chains. The dominance of the thermodynamic contribution or work function for all of these proton transfers is consistent with the view that formation and breaking of hydrogen bonds in such water chains is a limiting factor for proton translocation.
Asunto(s)
Ácido Aspártico , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Ácido Glutámico , Sustitución de Aminoácidos , Calorimetría , Humanos , Concentración de Iones de Hidrógeno , Cinética , Mutagénesis Sitio-Dirigida , Isótopos de Oxígeno , Mutación Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
Carbonic anhydrase VII (CA VII) appears to be the most highly conserved of the active mammalian carbonic anhydrases. We have characterized the catalytic activity and inhibition properties of a recombinant murine CA VII. CA VII has steady-state constants similar to two of the most active isozymes of carbonic anhydrase, CA II and IV; also, it is very strongly inhibited by the sulfonamides ethoxzolamide and acetazolamide, yielding the lowest Ki values measured by the exchange of 18O between CO2 and water for any of the mammalian isozymes of carbonic anhydrase. The catalytic measurements of the hydration of CO2 and the dehydration of HCO3- were made by stopped-flow spectrophotometry and the exchange of 18O using mass spectrometry. Unlike the other isozymes of this class of CA, for which Kcat/K(m) is described by the single ionization of zinc-bound water, CA VII exhibits a pH profile for Kcat/K(m) for CO2 hydration described by two ionizations at pKa 6.2 and 7.5, with a maximum approaching 8 x 10(7) M-1 s-1. The pH dependence of kcat/K(m) for the hydrolysis of 4-nitrophenyl acetate could also be described by these two ionizations, yielding a maximum of 71 M-1 s-1 at pH > 9. Using a novel method that compares rates of 18O exchange and dehydration of HCO3-, we assigned values for the apparent pKa at 6.2 to the zinc-bound water and the pKa of 7.5 to His 64. The magnitude of Kcat, its pH profile, 18O-exchange data for both wild-type and a H64A mutant, and inhibition by CuSO4 and acrolein suggest that the histidine at position 64 is functioning as a proton-transfer group and is responsible for one of the observed ionizations. A truncation mutant of CA VII, in which 23 residues from the amino-terminal end were deleted, has its rate constant for intramolecular proton transfer decreased by an order of magnitude with no change in Kcat/K(m). This suggests a role for the amino-terminal end in enhancing proton transfer in catalysis by carbonic anhydrase.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Isoenzimas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/genética , Catálisis , Transporte de Electrón/genética , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/química , Isoenzimas/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Protones , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The rate-limiting step in the catalysis of the hydration of CO2 by carbonic anhydrase involves transfer of protons between zinc-bound water and solution. This proton transfer can be enhanced by proton shuttle residues within the active-site cavity of the enzyme. We have used chemical modulation to provide novel internal proton transfer groups that enhance catalysis by murine carbonic anhydrase V (mCA V). This approach involves the site-directed mutation of a targeted residue to a cysteine which is then subsequently reacted with an imidazole analog containing an appropriately positioned leaving group. Compounds examined include 4-bromoethylimidazole (4-BEI), 2-chloromethylimidazole (2-CMI), 4-chloromethylimidazole (4-CMI), and a triazole analog. Two sites in mCA V, Lys 91 and Tyr 131, located on the rim of the active-site cavity have been targeted for the introduction of these imidazole analogs. Modification of the introduced Cys 131 with 4-BEI and 4-CMI resulted in enhancements of up to threefold in catalytic activity. The pH profiles indicate the presence of a new proton shuttle residue of pKa near 5.8, consistent with the introduction of a functional proton transfer group into the active site. This is the first example of incorporation by chemical modification of an unnatural amino acid analog of histidine that can act as a proton shuttle in an enzyme.