黄孢原毛平革菌木质素过氧化物酶氧化中间体的光谱和动力学性质。
Spectroscopic and kinetic properties of the oxidized intermediates of lignin peroxidase from Phanerochaete chrysosporium.
作者信息
Marquez L, Wariishi H, Dunford H B, Gold M H
机构信息
Department of Chemistry, University of Alberta, Edmonton, Canada.
出版信息
J Biol Chem. 1988 Aug 5;263(22):10549-52.
Stopped-flow rapid scan techniques were used to obtain a spectrum of nearly homogeneous lignin peroxidase compound I (LiPI) under pseudo-first order conditions at the unusually low pH optimum (3.0) for the enzyme. The LiPI spectrum had a Soret band at 407 nm with approximately 60% reduced intensity and a visible maximum at 650 nm. Under steady-state conditions a Soret spectrum for lignin peroxidase compound II (LiPII) was also obtained. The Soret maximum of LiPII at 420 nm was only approximately 15% reduced in intensity compared to native LiP. Transient state kinetic results confirmed the pH independence of LiPI formation over the pH range 3.06-7.39. The rate constant was (6.5 +/- 0.2) x 10(5) M-1 S-1. Addition of excess veratryl alcohol to LiPI resulted in its reduction to LiPII with subsequent reduction of LiPII to the native enzyme. Reactions of LiPI and LiPII with veratryl alcohol exhibited marked pH dependencies. For the LiPI reaction the rate constants ranged from 2.5 x 10(6) M-1 S-1 at pH 3.06 to 4.1 x 10(3) M-1 S-1 at pH 7.39; for the LiPII reaction, 1.6 x 10(5) M-1 S-1 (pH 3.06) to 2.3 x 10(3) M-1 S-1 (pH 5.16). These single turnover experiments demonstrate directly that the pH dependence of these reactions dictates the overall pH dependence of this novel enzyme. These results are consistent with the one-electron oxidation of veratryl alcohol to an aryl cation radical by LiPI and by LiPII.
采用停流快速扫描技术，在该酶异常低的最适pH值（3.0）下，于准一级条件下获得了几乎均一的木质素过氧化物酶化合物I（LiPI）的光谱。LiPI光谱在407 nm处有一个Soret带，强度降低约60%，在650 nm处有一个可见最大值。在稳态条件下，还获得了木质素过氧化物酶化合物II（LiPII）的Soret光谱。LiPII在420 nm处的Soret最大值与天然LiP相比，强度仅降低约15%。瞬态动力学结果证实，在3.06 - 7.39的pH范围内，LiPI的形成与pH无关。速率常数为(6.5 ± 0.2) × 10⁵ M⁻¹ s⁻¹。向LiPI中加入过量的藜芦醇会导致其还原为LiPII，随后LiPII再还原为天然酶。LiPI和LiPII与藜芦醇的反应表现出明显的pH依赖性。对于LiPI反应，速率常数在pH 3.06时为2.5 × 10⁶ M⁻¹ s⁻¹，在pH 7.39时为4.1 × 10³ M⁻¹ s⁻¹；对于LiPII反应，为1.6 × 10⁵ M⁻¹ s⁻¹（pH 3.06）至2.3 × 10³ M⁻¹ s⁻¹（pH 5.16）。这些单周转实验直接表明，这些反应的pH依赖性决定了这种新型酶的整体pH依赖性。这些结果与LiPI和LiPII将藜芦醇单电子氧化为芳基阳离子自由基一致。
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