Walsh C T, Erion M D, Walts A E, Delany J J, Berchtold G A
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1987 Jul 28;26(15):4734-45. doi: 10.1021/bi00389a021.
Chorismate is converted by regiospecific amination/aromatization sequences to o-aminobenzoate and p-aminobenzoate (PABA) by anthranilate synthase (AS) and PABA synthase (PABS), respectively. We report here the first partial purification of the large subunit of Escherichia coli PABA synthase, previously reported to be quantitatively inactivated in purification attempts. The subunit encoded by the pabB gene was overexpressed from a T7 promoter and purified 9-fold to 25-30% homogeneity. The pabB subunit appears unusually sensitive to inactivation by glycerol so this cosolvent is contraindicated. The Km for chorismate is 42 microM in the ammonia-dependent conversion to PABA, and we estimate a turnover number of 2.6 min-1. A variety of chorismate analogues have been prepared and examined. Of these compounds, cycloheptadienyl analogue 11 has been found to be the most potent inhibitor of Serratia marcescens anthranilate synthase (Ki = 30 microM for an RS mixture) and of the E. coli pabB subunit of PABA synthase (Ki = 226 microM). Modifications in the substituents at C-3 [enolpyruyl ether, (R)- or (S)-lactyl ether, glycolyl ether] or C-4 (O-methyl) of chorismate lead to alternate substrates. The Vmax values for (R)- and (S)-lactyl ethers are down 10-20-fold for each enzyme, and V/K analyses show the (S)-lactyl chorismate analogue to be preferred by 12/1 over (R)-lactyl for anthranilate synthase while a 3/1 preference was observed for (R)-/(S)-lactyl analogues by PABA synthase. The glycolyl ether analogue of chorismate shows 15% Vmax vs. chorismate for anthranilate synthase but is actually a faster substrate (140%) than chorismate with PABA synthase, suggesting the elimination/aromatization step from an aminocyclohexadienyl species may be rate limiting with AS but not with PABS. Indeed, studies with (R)-lactyl analogue 14 and anthranilate synthase led to accumulation of an intermediate, isolable by high-performance liquid chromatography and characterized by NMR and UV-visible spectroscopy as 6-amino-5-[(1-carboxyethyl)oxy]-1,3-cyclohexadiene-1-carboxylic acid (17). This is the anticipated intermediate predicted by our previous work with conversion of synthetic trans-6-amino-5-[(1-carboxyethenyl)oxy]-1,3-cyclohexadiene-1-carbo xylic acid (2) to anthranilate by the enzyme. Compound 17 is quantitatively converted to anthranilate on reincubation with enzyme, but at a 1.3-10-fold lower Vmax than starting lactyl substrate 14 under the conditions investigated; the basis for this kinetic variation is not yet determined.
分支酸分别通过邻氨基苯甲酸合酶（AS）和对氨基苯甲酸合酶（PABS）的区域特异性胺化/芳构化序列转化为邻氨基苯甲酸和对氨基苯甲酸（PABA）。我们在此报告了大肠杆菌对氨基苯甲酸合酶大亚基的首次部分纯化，此前报道该大亚基在纯化过程中会被定量灭活。由pabB基因编码的亚基从T7启动子过量表达，并纯化了9倍，达到25 - 30%的纯度。pabB亚基似乎对甘油灭活异常敏感，因此这种助溶剂不宜使用。在氨依赖转化为PABA的过程中，分支酸的Km为42微摩尔，我们估计其周转数为2.6分钟⁻¹。已经制备并检测了多种分支酸类似物。在这些化合物中，环庚二烯基类似物11已被发现是粘质沙雷氏菌邻氨基苯甲酸合酶（RS混合物的Ki = 30微摩尔）和大肠杆菌对氨基苯甲酸合酶的pabB亚基（Ki = 226微摩尔）的最有效抑制剂。分支酸C-3位[烯醇丙酮酸醚、（R）-或（S）-乳酰醚、乙醇酰醚]或C-4位（O-甲基）取代基的修饰会产生替代底物。对于每种酶，（R）-和（S）-乳酰醚的Vmax值降低了10 - 20倍，V/K分析表明，邻氨基苯甲酸合酶对（S）-乳酰分支酸类似物的偏好是对（R）-乳酰的12/1，而对氨基苯甲酸合酶对（R）- /（S）-乳酰类似物的偏好为3/1。分支酸的乙醇酰醚类似物与分支酸相比，邻氨基苯甲酸合酶的Vmax为15%，但实际上它是比对氨基苯甲酸合酶更快的底物（140%），这表明从氨基环己二烯基物种进行的消除/芳构化步骤可能是AS的限速步骤，但不是PABS的限速步骤。事实上，对（R）-乳酰类似物14和邻氨基苯甲酸合酶的研究导致了一种中间体的积累，该中间体可通过高效液相色谱分离，并通过核磁共振和紫外 - 可见光谱表征为6-氨基-5-[(1-羧乙基)氧基]-1,3-环己二烯-1-羧酸（17）。这是我们之前关于该酶将合成的反式-6-氨基-5-[(1-羧乙烯基)氧基]-1,3-环己二烯-1-羧酸（2）转化为邻氨基苯甲酸的工作所预测的预期中间体。在与酶再孵育时，化合物17定量转化为邻氨基苯甲酸，但在所研究的条件下，其Vmax比起始乳酰底物14低1.3 - 10倍；这种动力学变化的基础尚未确定。
