Lack A, Fuchs G
Angewandte Mikrobiologie, University of Ulm, Germany.
J Bacteriol. 1992 Jun;174(11):3629-36. doi: 10.1128/jb.174.11.3629-3636.1992.
Several lines of evidence indicate that the first step in the anaerobic metabolism of phenol is phenol carboxylation to 4-hydroxybenzoate; this reaction is considered a biological Kolbe-Schmitt carboxylation. A phenol carboxylase system was characterized by using a denitrifying Pseudomonas strain, K 172, which catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate. The enzymatic isotope exchange activity (100 nmol min-1 mg-1 of protein) requires Mn2+ and K+. We show that this system also catalyzes the carboxylation of phenylphosphate (the phosphoric acid monophenyl ester) to 4-hydroxybenzoate and phosphate. The specific activity of phenylphosphate carboxylation at the optimal pH of 6.5 is 12 nmol of CO2 fixed min-1 mg-1 of protein. Phenylphosphate cannot be replaced by Mg(2+)-ATP and phenol. The carboxylase activity requires Mn2+ but, in contrast to the isotope exchange activity, does not require K+. The apparent Km values are 1.5 mM dissolved CO2 and 0.2 mM phenylphosphate. Several convenient assays for phenylophosphate carboxylation are described. The isotope exchange reaction and the net carboxylation reaction are catalyzed by the same oxygen-sensitive enzyme, which has a half-life in an air-saturated solution of less than 1 min. Both activities cochromatographed with a protein with a Mr of 280,000, and both activities were induced only after anaerobic growth on phenol. The carboxylation of phenylphosphate suggests that phenylphosphate itself is the physiological CO2 acceptor molecular of this novel CO2 fixation reaction. Alternatively, phenylphosphate could simulate the unknown natural precursor. It is suggested that the formation of an enzyme-bound phenolate anion from the activated phenolic compound is the rate-determining step in the carboxylation reaction.
几条证据线索表明，苯酚厌氧代谢的第一步是苯酚羧化生成4-羟基苯甲酸；该反应被认为是生物科尔贝-施密特羧化反应。通过使用反硝化假单胞菌菌株K 172对苯酚羧化酶系统进行了表征，该菌株催化14CO2与4-羟基苯甲酸羧基之间的同位素交换。酶促同位素交换活性（100 nmol·min-1·mg-1蛋白质）需要Mn2+和K+。我们表明，该系统还催化磷酸苯酯（磷酸单苯酯）羧化生成4-羟基苯甲酸和磷酸盐。在最佳pH 6.5下，磷酸苯酯羧化的比活性为12 nmol CO2固定·min-1·mg-1蛋白质。磷酸苯酯不能被Mg(2+)-ATP和苯酚替代。羧化酶活性需要Mn2+，但与同位素交换活性不同，不需要K+。表观Km值分别为1.5 mM溶解CO2和0.2 mM磷酸苯酯。描述了几种方便的磷酸苯酯羧化测定方法。同位素交换反应和净羧化反应由同一种对氧敏感的酶催化，该酶在空气饱和溶液中的半衰期小于1分钟。两种活性都与分子量为280,000的蛋白质共色谱，并且两种活性仅在以苯酚为底物厌氧生长后才被诱导。磷酸苯酯的羧化表明磷酸苯酯本身是这种新型CO2固定反应的生理CO2受体分子。或者，磷酸苯酯可以模拟未知的天然前体。有人认为，从活化的酚类化合物形成酶结合的酚盐阴离子是羧化反应中的速率决定步骤。
