13C NMR studies of the enzyme-product complex of Bacillus subtilis chorismate mutase.
13C NMR studies of the enzyme-product complex of Bacillus subtilis chorismate mutase.
复制标题
枯草芽孢杆菌分支酸变位酶酶-产物复合物的 13C NMR 研究。
DOI：
10.1021/bi00066a017
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Jaffe,EK
中科院分区：
文献类型：
--
作者：
Rajagopalan,JS;Taylor,KM;Jaffe,EK
1993年2月2日收到的修订稿摘要：分支酸变位酶反应是一种罕见的酶催化的3，3-S型重排分支酸预苯酸酯。利用Gray等人(Gray，J.V.，Grolinelli-Pimpaneau，B.，&Knowles，J.R.(1990年)BioChemical 29,376-383)的改进程序，从大肠杆菌XL 1-Blue(PBSCM2)中高产并纯化了分支酸变位酶；修改后的预苯酸脱水酶污染最小(<；0.001%)。用凝胶过滤的方法测得枯草杆菌分支酸变位酶的天然相对分子质量为~44 kDa，用电喷雾-质谱法测定了14.5 kDa亚基的同源三聚体。用~(13)C核磁共振研究了枯草杆菌分支酸变位酶活性部位结合的[U-~(13)C]预苯酸酯的结构。[U-13C]预苯酸酯和[2，6，9-*3C]预苯酸酯分别合成了[L，3，5，8-13C]预苯酸酯和[2，6，9-*3C]预苯酸酯。酶结合的预苯酸酯相对于游离的预苯酸酯表现出明显不同的化学位移；化学位移的变化范围从C6共振的-6.6ppm到C5共振的5.6ppm，表明C5-C6键有强烈的扰动。*模型化合物在不同pH值和不同溶剂中的3C核磁共振研究表明，观察到的酶结合预苯酸酯的*3C化学位移变化不能仅仅基于活性中心羧基pKa的变化或疏水溶剂化来解释。关于分支酸变位酶催化反应的化学机制，这些核磁共振研究没有提供任何涉及离散中间体的解离机制的证据。氯酸是合成氨基酸酪氨酸、苯丙氨酸和色氨酸所必需的分支点中间体(Gibson&Gibson，1964；Edwards&Jackman，1965)。分支酸转化为预苯酯，酪氨酸和苯丙氨酸生物合成的第一步，如图1所示；这一反应由分支酸变位酶催化(CM，EC 5.4。99.5)，是一种罕见的酶催化的3，3-S重排反应。CM存在于植物和微生物中(Ganem，1978；Poulsen&Verpoorte，1991)，不存在于动物体内。设计高效的无毒的CM抑制剂，作为抗生素或除草剂，需要了解反应机理和结合在活性部位的配体的结构。
Revised Manuscript Received February 2, 1993 abstract: The chorismate mutase reaction is a rare enzyme-catalyzed 3, 3-sigmatropic rearrangement of chorismate to prephenate. Bacillus subtilis chorismate mutase was overproduced and purified from Escherichia coli XL 1-Blue (pBSCM2) using a modification of the procedure of Gray et al.(Gray, J. V., Grolinelli-Pimpaneau, B., & Knowles, J. R.(1990) Biochemistry 29, 376-383); the modification leads to minimal contaminating prephenate dehydratase activity (< 0.001%). The native molecular mass of B. subtilis chorismate mutase was determined by gel filtration to be~ 44 kDa, indicative of a homotrimer of the 14.5-kDa subunits as determined by electrospray mass spectrometry. 13C NMR was used to study the structure of [U-13C] prephenate bound at the active site of B. subtilis chorismate mutase. All the enzyme-bound* 3C NMR resonances of [U-13C] prephenate were assigned, and where possible,'/c. cs were quantified;[l, 3, 5, 8-13C] prephenate and [2, 6, 9-13C] prephenate, prepared respectively from [1, 3, 5, 8-'3C]-chorismate and [2, 6, 9-* 3C] chorismate, aided the* 3C NMR resonance assignments. Enzyme-bound prephenate exhibits remarkablydifferent chemical shifts relative to free prephenate; the chemicalshift changes range from-6.6 ppm for the C6 resonance to 5.6 ppm for the C5 resonance, suggesting a strong perturbation of the C5-C6 bond.* 3C NMR studies of model compounds at various pH values and in various solvents suggest that the observed* 3C chemical shift changes of enzyme-bound prephenate cannot be rationalized solely on the basis of changes in the pKas of the carboxylic acidgroups or hydrophobic solvation at the active site. With regard to the chemical mechanism of the chorismate mutase-catalyzed reaction, these NMR studies do not provide any evidence for a dissociative mechanism which involves discrete intermediates.Chorismate is the branch-point intermediate necessary for the biosynthesis of the amino acids tyrosine, phenylalanine, and tryptophan (Gibson & Gibson, 1964; Edwards & Jackman, 1965). The conversion of chorismate to prephenate, the first committed step in the biosynthesis of tyrosine and phenylalanine, is illustrated in Figure 1; this reaction, catalyzed by chorismate mutase (CM, EC 5.4. 99.5), is a rare enzyme-catalyzed 3, 3-sigmatropic rearrangement. CM occurs in plants and microorganisms (Ganem, 1978; Poulsen & Verpoorte, 1991) and is not found in animals. Design of efficient nontoxic inhibitorsof CM, which might serve as antibiotics or herbicides, requires an understanding of the reaction mechanism and the structures of the ligands bound at the active site.