Brevibacterium fuscum protocatechuate 3,4-dioxygenase. Purification, crystallization, and characterization.
Brevibacterium fuscum protocatechuate 3,4-dioxygenase. Purification, crystallization, and characterization.
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DOI：
10.1016/s0021-9258(17)43071-7
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发表时间：
1984-04
期刊：
影响因子：
--
通讯作者：
J. W. Whittaker;J. Lipscomb;T. Kent;E. Münck
中科院分区：
文献类型：
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作者：
J. W. Whittaker;J. Lipscomb;T. Kent;E. Münck
原儿茶酸3，4-双加氧酶是从革兰氏阳性菌中提纯并结晶出来的一种具有特殊光谱特征和新的亚基组成的酶。EPR谱表明，催化必需的Fe~(3+)位于几乎具有最大菱形的位置(E/D=0.333+/-0.003)。在g=9.67时的谱线宽度(1.4毫微秒)是已报道的生物高自旋Fe3+络合物中最小的，这表明该酶在Fe中心附近是相当均匀的。用57Fe富集液中培养的细胞制备的酶测得的穆斯堡尔谱，以及共振拉曼光谱和光学光谱也得出了同样的结论。相比之下，原儿茶酸盐(PCA)厌氧复合体的EPR和穆斯堡尔谱比较复杂，表明存在多种电子对称性明显不同的物种，并且存在正负零场分裂。MR=315,000酶的组成为(α-β-Fe)5(MR(α)=22,500；MR(β)=40,000)。氨基酸分析表明，两个亚基都不含半胱氨酸，从而排除了该氨基酸可能的铁配体。这种酶的结构、光谱和催化反应的一般特征似乎与从革兰氏阴性生物中分离的原儿茶酸3，4-双加氧酶非常相似。然而，在无限大的PCA和O2浓度下，动力学参数(Km(PCA)=125微米，Km(O2)=800微米，周转次数=25,000分钟-1)高出5-50倍。锐利的光谱和动力学性质促进了本报告和后续报告中描述的机理研究。
A protocatechuate 3,4-dioxygenase with exceptionally sharp spectral features and a new subunit composition has been purified and crystallized from the Gram-positive organism Brevibacterium fuscum. EPR spectra show that the catalytically essential Fe3+ resides in a site of almost the maximal rhombicity (E/D = 0.333 +/- 0.003). The spectral line widths (1.4 milliTesla at g = 9.67) are the smallest reported for any biological high spin Fe3+ complex and suggest that the enzyme is quite homogeneous in the vicinity of the Fe site. The same conclusion is drawn from Mössbauer spectra measured with enzyme prepared from cells cultured in 57Fe-enriched media as well as from resonance Raman and optical spectra. In contrast, EPR and Mössbauer spectra of the anaerobic complex with protocatechuate (PCA) are complex and demonstrate that multiple species with markedly different electronic symmetries and both positive and negative zero field splittings are present. The Mr = 315,000 enzyme has a composition of (alpha beta Fe)5 (Mr(alpha) = 22,500; Mr (beta) = 40,000). Amino acid analysis shows that neither subunit contains cysteine, thus eliminating this amino acid as a possible Fe ligand. The general features of the structure, spectra, and catalyzed reaction of this enzyme appear to be very similar to those of protocatechuate 3,4-dioxygenase isolated from Gram-negative organisms. However, the kinetic parameters (Km(PCA) = 125 microM, Km(O2) = 800 microM, turnover number = 25,000 min-1 at infinite PCA and O2 concentrations) are 5- to 50-fold higher. The sharp spectra and the kinetic properties facilitate mechanistic studies described in this and the following reports.