Catalytic properties of phenol carboxylase. In vitro study of CO2: 4-hydroxybenzoate isotope exchange reaction.
Phenol is metabolized in a denitrifying bacterium in the absence of molecular oxygen via para-carboxylation to 4-hydroxybenzoate (biological Kolbe-Schmitt synthesis). The enzyme system catalyzing the presumptive carboxylation of phenol, tentatively named 'phenol carboxylase', catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate (specific activity 0.1 mumol 14CO2 incorporated into 4-hydroxybenzoate x min-1 x mg-1 cell protein) which is considered a partial reaction of the overall enzyme catalysis; 14C from [14C]phenol was not exchanged into 4-hydroxybenzoate ring positions to a significant extent. The 14CO2 isotope exchange reaction was studied in vitro. The reaction was dependent on the substrates CO2 and 4-hydroxybenzoate and required K+ and Mn2+. The actual substrate was CO2 rather than HCO3-. The apparent Km values were 1 mM dissolved CO2, 0.2 mM 4-hydroxybenzoate, 2 mM K+, and 0.1 mM Mn2+. The cationic cocatalysts could be substituted by ions of similar ionic radius: K+ could be replaced to some extent by Rb+, but not by Li+, Na+, Cs+, or NH4+; Mn2+ could be replaced to some extent by Fe2+ greater than Mg2+, Co2+, but not by Ni2+, Zn2+, Ca2+, or Cu2+. The exchange reaction was not strictly specific for 4-hydroxybenzoate, however it required a p-hydroxyl group; derivatives of 4-hydroxybenzoate with OH, CH3 or Cl substituents in m-position did react, whereas those with substitutions in the o-position were inactive or were inhibitory. The enzyme was induced when cells were grown on phenol, but not on 4-hydroxybenzoate. Comparison of SDS/PAGE protein patterns of cells grown on phenol or 4-hydroxybenzoate revealed several additional protein bands in phenol-grown cells. The possible role of similar enzymes in the anaerobic metabolism of phenolic compounds is discussed.
Lack
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Abteilung Angewandte Mikrobiologie, University of Ulm, Federal Republic of Germany.
Tommasi
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I
Aresta
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Fuchs
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D002245:Carbon Dioxide / Q000737:chemistry*; D002262:Carboxy-Lyases / Q000737:chemistry*; D002384:Catalysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007554:Isotopes; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D010226:Parabens / Q000737:chemistry*; D010636:Phenols / Q000737:chemistry*; D011549:Pseudomonas / Q000378:metabolism; D013379:Substrate Specificity
D007554:Isotopes; D010226:Parabens; D010636:Phenols; D002245:Carbon Dioxide; D008345:Manganese; D002262:Carboxy-Lyases; C065828:4-hydroxybenzoate decarboxylase