Doyle W A, Blodig W, Veitch N C, Piontek K, Smith A T
School of Biological Sciences, University of Sussex, Falmer, Brighton, UK.
Biochemistry. 1998 Oct 27;37(43):15097-105. doi: 10.1021/bi981633h.
It has been shown recently that Trp171 of lignin peroxidase (LiP) is hydroxylated at the Cbeta position [Blodig, W., Doyle, W. A., Smith, A. T., Winterhalter, K., Choinowski, T., and Piontek, K. (1998) Biochemistry 37, 8832-8838]. Comparative experiments, carried out on both wild-type fungal and recombinant LiP isoenzyme H8 (LiPH8), indicate that the process of hydroxylation is autocatalytic and that Trp171 may be implicated in catalysis. The role of this residue has therefore been examined using site-directed mutagenesis to obtain recombinant enzymes with Trp171 substituted by Phe or Ser (W171F and W171S LiPH8, respectively). The wild-type recombinant enzyme (LiPH8) was analyzed in solution using 1H NMR spectroscopy and its integrity confirmed prior to the kinetic and spectroscopic characterization of LiPH8 mutants. A charge neutralization mutation in the "classical heme edge" substrate access channel of LiP, in which Glu146 was substituted by Gly (E146G LiPH8), showed substantial activity with respect to veratryl alcohol (VA) oxidation and a marked (2.4 pH units) increase in pKa for the oxidation of a negatively charged difluoroazo dye. More surprisingly, the Trp171 LiPH8 mutants W171F and W171S LiPH8 were found to have lost all activity with VA as substrate, and compounds I and II were unable to react with VA. Both mutants, however, retained substantial activity with two dye substrates. These data provide the first direct evidence for the existence of two distinct substrate interaction sites in LiP, a heme-edge site typical of those encountered in other peroxidases and a second, novel site centered around Trp171 which is required for the oxidation of VA. Stopped-flow kinetic studies showed that all the mutants examined reacted normally with hydrogen peroxide to give a porphyrin cation radical (compound I). However, the rapid phase of spontaneous compound I reduction (2.3 s-1), typical of wild-type LiP, was absent in the Trp171 mutants, strongly suggesting that an electron-transfer pathway must exist within the protein leading from the heme to a surface site in close proximity to Trp171. The kinetic competence of such a pathway is dependent on interaction of the enzyme with VA, at or near Trp171.
最近研究表明，木质素过氧化物酶（LiP）的色氨酸171（Trp171）在β位被羟基化[布洛迪格，W.，多伊尔，W. A.，史密斯，A. T.，温特哈尔特，K.，乔伊诺夫斯基，T.，和皮翁泰克，K.（1998年）《生物化学》37卷，8832 - 8838页]。对野生型真菌LiP和重组LiP同工酶H8（LiPH8）进行的对比实验表明，羟基化过程是自催化的，并且Trp171可能参与催化作用。因此，通过定点诱变获得了色氨酸171被苯丙氨酸或丝氨酸取代的重组酶（分别为W171F LiPH8和W171S LiPH8），以此来研究该残基的作用。利用1H核磁共振光谱对溶液中的野生型重组酶（LiPH8）进行了分析，并在对LiPH8突变体进行动力学和光谱表征之前确认了其完整性。在LiP的“经典血红素边缘”底物进入通道中进行的电荷中和突变，即将谷氨酸146（Glu146）替换为甘氨酸（E146G LiPH8），该突变体对藜芦醇（VA）氧化显示出显著活性，并且对于带负电荷的二氟偶氮染料氧化的pKa显著升高（2.4个pH单位）。更令人惊讶的是，发现Trp171 LiPH8突变体W171F LiPH8和W171S LiPH8以VA作为底物时完全失去了活性，并且化合物I和II无法与VA反应。然而，这两个突变体对两种染料底物仍保留显著活性。这些数据首次直接证明了LiP中存在两个不同的底物相互作用位点，一个是其他过氧化物酶中常见的血红素边缘位点，另一个是以Trp171为中心的新位点，VA氧化需要该位点。停流动力学研究表明，所有检测的突变体与过氧化氢正常反应生成卟啉阳离子自由基（化合物I）。然而，Trp171突变体中不存在野生型LiP典型的化合物I快速自发还原相（2.3 s-1），这强烈表明蛋白质内部必定存在一条从血红素到紧邻Trp171的表面位点的电子传递途径。这种途径的动力学能力取决于酶与Trp171处或其附近的VA的相互作用。
