...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>The FEBS journal >Aromatic stacking interactions govern catalysis in aryl-alcohol oxidase
【24h】

Aromatic stacking interactions govern catalysis in aryl-alcohol oxidase

机译：芳香堆积相互作用控制芳基醇氧化酶中的催化作用

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Aryl-alcohol oxidase (AAO, ) generates H2O2 for lignin degradation at the expense of benzylic and other system-containing primary alcohols, which are oxidized to the corresponding aldehydes. Ligand diffusion studies on Pleurotuseryngii AAO showed a T-shaped stacking interaction between the Tyr92 side chain and the alcohol substrate at the catalytically competent position for concerted hydride and proton transfers. Bi-substrate kinetics analysis revealed that reactions with 3-chloro- or 3-fluorobenzyl alcohols (halogen substituents) proceed via a ping-pong mechanism. However, mono- and dimethoxylated substituents (in 4-methoxybenzyl and 3,4-dimethoxybenzyl alcohols) altered the mechanism and a ternary complex was formed. Electron-withdrawing substituents resulted in lower quantum mechanics stacking energies between aldehyde and the tyrosine side chain, contributing to product release, in agreement with the ping-pong mechanism observed in 3-chloro- and 3-fluorobenzyl alcohol kinetics analysis. In contrast, the higher stacking energies when electron donor substituents are present result in reaction of O-2 with the flavin through a ternary complex, in agreement with the kinetics of methoxylated alcohols. The contributionof Tyr92 to the AAO reaction mechanism was investigated by calculation of stacking interaction energies and site-directed mutagenesis. Replacement of Tyr92 by phenylalanine does not alter the AAO kinetic constants (on 4-methoxybenzyl alcohol), most probably because the stacking interaction is still possible. However, introduction of a tryptophan residue at this position strongly reduced the affinity for the substrate (i.e. the pre-steady state K-d and steady-state K-m increase by 150-fold and 75-fold, respectively), and therefore the steady-state catalytic efficiency, suggesting that proper stacking is impossible with this bulky residue. The above results confirm the role of Tyr92 in substrate binding, thus governing the kinetic mechanism in AAO.
机译：芳醇氧化酶（AAO，）生成用于木质素降解的H2O2，代价是苄基和其他含系统的伯醇被氧化为相应的醛。在杏鲍菇AAO上的配体扩散研究表明，在Tyr92侧链与醇底物之间在催化协同作用的氢化物和质子转移催化位置上呈T形堆积相互作用。双底物动力学分析表明，与3-氯-或3-氟苄醇（卤素取代基）的反应是通过乒乓机制进行的。但是，单和二甲氧基取代基（在4-甲氧基苄基和3,4-二甲氧基苄醇中）改变了机理，并形成了三元络合物。吸电子取代基导致醛和酪氨酸侧链之间的量子力学能量降低，从而导致产物释放，这与在3-氯-和3-氟苄醇动力学分析中观察到的乒乓机理相一致。相反，当存在电子给体取代基时，较高的堆积能导致O-2与黄素通过三元络合物反应，这与甲氧基化醇的动力学相符。通过计算堆积相互作用能和定点诱变研究了Tyr92对AAO反应机理的贡献。用苯丙氨酸代替Tyr92不会改变AAO动力学常数（在4-甲氧基苄醇上），这很可能是因为堆积相互作用仍然可能。但是，在该位置引入色氨酸残基会大大降低对底物的亲和力（即稳态前Kd和稳态Km分别增加150倍和75倍），因此稳态催化效率高，这说明使用这种庞大的残留物无法正确堆叠。以上结果证实了Tyr92在底物结合中的作用，从而控制了AAO中的动力学机理。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号