Löffler F, Lingens F, Müller R
Technische Universität Hamburg-Harburg, Arbeitsbereich Biotechnologie II, Hamburg, Germany.
Biodegradation. 1995 Sep;6(3):203-12. doi: 10.1007/BF00700458.
Pseudomonas sp. CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy. The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins. A 4-halobenzoate-coenzyme A ligase activates 4-chlorobenzoate in a coenzyme A, ATP and Mg2+ dependent reaction to 4-chlorobenzoyl-coenzyme A. This thioester intermediate is dehalogenated by the 4-chlorobenzoyl-coenzyme A dehalogenase. Finally coenzyme A is split off by a 4-hydroxybenzoyl-CoA thioesterase to form 4-hydroxybenzoate. The involved 4-chlorobenzoyl-coenzyme A dehalogenase was purified to apparent homogeneity by a five-step purification procedure. The native enzyme had an apparent molecular mass of 120,000 and was composed of four identical polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.7. The maximal initial rate of catalysis was achieved at pH 10 at 60 degrees C. The apparent Km value for 4-chlorobenzoyl-coenzyme A was 2.4-2.7 microM. Vmax was 1.1 x 10(-7) M sec-1 (2.2 mumol min-1 mg-1 of protein). The NH2-terminal amino acid sequence was determined. All 4-halobenzoyl-coenzyme A thioesters, except 4-fluorobenzoyl-coenzyme A, were dehalogenated by the 4-chlorobenzoyl-CoA dehalogenase.
假单胞菌属CBS3能够以4-氯苯甲酸作为唯一碳源和能源生长。在第一步降解反应中，由三种蛋白质组成的酶系统负责去除4-氯苯甲酸中的氯。一种4-卤代苯甲酸辅酶A连接酶在辅酶A、ATP和Mg2+依赖的反应中，将4-氯苯甲酸激活为4-氯苯甲酰辅酶A。这种硫酯中间体由4-氯苯甲酰辅酶A脱卤酶进行脱卤反应。最后，辅酶A被4-羟基苯甲酰辅酶A硫酯酶裂解，形成4-羟基苯甲酸。通过五步纯化程序，将所涉及的4-氯苯甲酰辅酶A脱卤酶纯化至表观均一。天然酶的表观分子量为120,000，由四个31 kDa的相同多肽亚基组成。该酶的等电点为6.7。在60℃、pH 10条件下实现最大初始催化速率。4-氯苯甲酰辅酶A的表观Km值为2.4 - 2.7 μM。Vmax为1.1×10(-7) M sec-1（2.2 μmol min-1 mg-1蛋白质）。测定了其NH2末端氨基酸序列。除4-氟苯甲酰辅酶A外，所有4-卤代苯甲酰辅酶A硫酯均能被4-氯苯甲酰辅酶A脱卤酶脱卤。
