RESUMO
20-Carboxyeicosatetraenoic acid (20-COOH-AA) is a bioactive metabolite of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid that produces vasoconstriction in the cerebral circulation. We found that smooth muscle (MSMC) and endothelial (MEC) cultures obtained from mouse brain microvessels convert [3H]20-HETE to 20-COOH-AA, indicating that the cerebral vasculature can produce this metabolite. The [3H]20-COOH-AA accumulated primarily in the culture medium, together with additional radiolabeled metabolites identified as the chain-shortened dicarboxylic acids 18-COOH-18:4, 18-COOH-18:3, and 16-COOH-16:3. N-Heptylformamide, a potent inhibitor of alcohol dehydrogenase (ADH), decreased the conversion of [3H]20-HETE to 20-COOH-AA by the MSMC and MEC and also by isolated mouse brain microvessels. Purified mouse and human ADH4, human ADH3, and horse liver ADH1 efficiently oxidized 20-HETE, and ADH4 and ADH3 were detected in MSMC and MEC by Western blotting. N-Heptylformamide inhibited the oxidation of 20-HETE by mouse and human ADH4 but not by ADH3. These results demonstrated that cerebral microvessels convert 20-HETE to 20-COOH-AA and that ADH catalyzes the reaction. Although ADH4 and ADH3 are expressed in MSMC and MEC, the inhibition produced by N-heptylformamide suggests that ADH4 is primarily responsible for 20-COOH-AA formation in the cerebral microvasculature.
Assuntos
Álcool Desidrogenase/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Microcirculação/enzimologia , Músculo Liso Vascular/metabolismo , Telencéfalo/irrigação sanguínea , Álcool Desidrogenase/genética , Álcool Desidrogenase/isolamento & purificação , Animais , Western Blotting , Catálise , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/análise , Inibidores Enzimáticos/farmacologia , Formamidas/farmacologia , Cinética , Lipídeos/análise , Camundongos , Microcirculação/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Oxirredução , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Trítio/metabolismoRESUMO
ADH2 is a member of one of the six classes of mammalian alcohol dehydrogenases, which catalyze the reversible oxidation of alcohols using NAD(+) as a cofactor. Within the ADH2 class, the rodent enzymes form a subgroup that exhibits low catalytic activity with all substrates that were examined, as compared to other groups, such as human ADH2. The low activity can be ascribed to the rigid nature of the proline residue at position 47 as the activity can be increased by approximately 100-fold by substituting Pro47 with either His (as found in human ADH2), Ala, or Gln. Mouse ADH2 follows an ordered bi-bi mechanism, and hydride transfer is rate-limiting for oxidation of benzyl alcohols catalyzed by the mutated and wild-type enzymes. Structural studies suggest that the mouse enzyme with His47 has a more closed active site, as compared to the enzyme with Pro47, and hydride transfer can be more efficient. Oxidation of benzyl alcohol catalyzed by all forms of the enzyme is strongly pH dependent, with pK values in the range of 8.1-9.3 for turnover numbers and catalytic efficiency. These pK values probably correspond to the ionization of the zinc-bound water or alcohol. The pK values are not lowered by the Pro47 to His substitution, suggesting that His47 does not act as a catalytic base in the deprotonation of the zinc ligand.
Assuntos
Álcool Desidrogenase/química , Alanina/genética , Álcool Desidrogenase/antagonistas & inibidores , Álcool Desidrogenase/genética , Substituição de Aminoácidos/genética , Animais , Álcool Benzílico/química , Catálise , Medição da Troca de Deutério , Ativação Enzimática/genética , Glutamina/genética , Histidina/química , Histidina/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Mutagênese Sítio-Dirigida , Prolina/química , Prolina/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato/genéticaRESUMO
The steady-state kinetics of the recombinant human alcohol dehydrogenase (ADH) 1C*2 with steroids were studied in order to determine substrate and inhibitor specificity. The assays were carried out under conditions of pH and temperature that are similar to those found in vivo. The enzyme has measurable activity on 5beta-androstan-17beta-ol-3-one, 5beta-androstan-3beta-ol-17-one, 5beta-pregnan-3beta-ol-20-one and 5beta-pregnan-3,20-dione, but much less activity with 5beta-cholanic acid-3-one or 5alpha-pregnan-3beta-ol-20-one. The determinants of specificity appear to include a 5beta configuration (cis A/B ring fusion) and a 3beta-hydroxy or 3-keto group. None of the reactive steroids has a known function in vivo. The activities with the human ADH1C*2 are <10% of those found with the recombinant horse ADH1S, but higher than the activities with recombinant horse ADH1E, which has an active site very similar to human ADH1C. 5alpha-Dihydrotestosterone is a ketone and a competitive inhibitor against varied concentrations of the substrate cyclohexanone, whereas it is an uncompetitive inhibitor against ethanol or NAD(+). Such patterns are expected for the binding of the steroid as a dead-end inhibitor to the enzyme-NADH complex. Thus, it does not appear that 5alpha-dihydrotestosterone is an allosteric inhibitor of the enzyme. Another dead-end inhibitor that gives uncompetitive inhibition of alcohol oxidation, 3-butylthiolane 1-oxide, is a potent inhibitor of alcohol metabolism in rats and mice. Simulation of the kinetics of ethanol elimination in rats with varied concentrations of the inhibitor is shown to yield the in vivo inhibition constant and an estimate of the rate of elimination of the inhibitor.