INHIBITION OF CATECHOL 2,3-DIOXYGENASE FROM PSEUDOMONAS-PUTIDA BY 3-CHLOROCATECHOL
INHIBITION OF CATECHOL 2,3-DIOXYGENASE FROM PSEUDOMONAS-PUTIDA BY 3-CHLOROCATECHOL
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DOI：
10.1128/aem.41.5.1159-1165.1981
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发表时间：
1981-01-01
影响因子：
4.4
通讯作者：
GIBSON, DT
中科院分区：
文献类型：
--
作者：
KLECKA, GM;GIBSON, DT
恶臭假单胞菌甲苯生长细胞制备的部分纯化的邻苯二酚2，3-双加氧酶催化3-甲基邻苯二酚化学计量比氧化生成2-羟基-6-氧杂-2，4-二烯酸酯。其他被酶制剂氧化的底物是邻苯二酚、4-甲基邻苯二酚和4-氟邻苯二酚。3-甲基儿茶酚和邻苯二酚的表观Km值分别为10.6和22.0µm。4位取代降低了酶对底物的亲和力和活性。邻苯二酚2，3-双加氧酶制剂不氧化3-氯代儿茶酚。该酶与3-氯代儿茶酚孵育会导致该酶失活。动力学分析表明，3-氯代儿茶酚和4-氯代儿茶酚都是该酶的非竞争性或混合型抑制剂。3-氯代酚(Ki=0.14µm)是比4-氯代酚(Ki=50µm)更有效的缓蚀剂。比较了铁络合剂TIron和邻菲咯啉与3-氯代儿茶酚对酶失活的影响。每种抑制剂似乎都能去除酶中的铁，因为用亚铁和还原剂处理失活的酶制剂可以完全重新激活。
Partially purified preparations of catechol 2,3-dioxygenase from toluene-grown cells of P. putida catalyzed the stoichiometric oxidation of 3-methylcatechol to 2-hydroxy-6-oxohepta-2,4-dienoate. Other substrates oxidized by the enzyme preparation were catechol, 4-methylcatechol and 4-fluorocatechol. The apparent Km for 3-methylcatechol and catechol were 10.6 and 22.0 .mu.M, respectively. Substitution at the 4-position decreases the affinity and activity of the enzyme for the substrate. Catechol 2,3-dioxygenase preparations did not oxidize 3-chlorocatechol. Incubation of the enzyme with 3-chlorocatechol led to inactivation of the enzyme. Kinetic analyses revealed that both 3-chlorocatechol and 4-chlorocatechol were noncompetitive or mixed-type inhibitors of the enzyme. 3-Chlorocatechol (Ki [inhibition constant] = 0.14 .mu.M) was a more potent inhibitor than 4-chlorocatechol (Ki = 50 .mu.M). The effect of the Fe-chelating agents Tiron and o-phenanthrolene were compared with that of 3-chlorocatechol on the inactivation of the enzyme. Each inhibitor appeared to remove Fe from the enzyme, since inactive enzyme preparations could be fully reactivated by treatment with ferrous iron and a reducing agent.