Glenn J K, Gold M H
Arch Biochem Biophys. 1985 Nov 1;242(2):329-41. doi: 10.1016/0003-9861(85)90217-6.
A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified by DEAE-Sepharose ion-exchange chromatography, Blue Agarose chromatography, and gel filtration on Sephadex G-100. Sodium dodecyl sulfate-gel electrophoresis indicated that the homogeneous protein has an Mr of 46,000. The absorption spectrum of the enzyme indicates the presence of a heme prosthetic group. The pyridine hemochrome absorption spectrum indicates that the enzyme contained one molecule of heme as iron protoporphyrin IX. The absorption maximum of the native enzyme (406 nm) shifted to 433 nm in the reduced enzyme and to 423 nm in the reduced-CO complex. Both CN- and N-3 readily bind to the native enzyme, indicating an available coordination site and that the heme iron is high spin. The absorption spectrum of the H2O2 enzyme complex, maximum at 420 nm, is similar to that of horseradish peroxidase compound II. P. chrysosporium peroxidase activity is dependent on Mn(II), with maximal activity attained above 100 microM. The enzyme is also stimulated to varying degrees by alpha-hydroxy acids (e.g., malic, lactic) and protein (e.g., gelatin, albumin). The peroxidase is capable of oxidizing NADH and a wide variety of dyes, including Poly B-411 and Poly R-481. Several of the substrates (indigo trisulfonate, NADH, Poly B-411, variamine blue RT salt, and Poly R-481) are oxidized by this Mn(II)-dependent peroxidase at considerably faster rates than those catalyzed by horseradish peroxidase. The enzyme rapidly oxidizes Mn(II) to Mn(III); the latter was detected by the characteristic absorption spectrum of its pyrophosphate complex. Inhibition of the oxidation of the substrate diammonium 2,2-azino-bis(3-ethyl-6-benzothiazolinesulfonate) (ABTS) by Na-pyrophosphate suggests that Mn(III) plays a role in the enzyme mechanism.
在白腐真菌黄孢原毛平革菌木质素分解培养物的细胞外培养基中发现的一种依赖锰（II）的过氧化物酶，通过DEAE-琼脂糖离子交换色谱、蓝色琼脂糖色谱以及Sephadex G-100凝胶过滤进行纯化。十二烷基硫酸钠-凝胶电泳表明该纯蛋白的相对分子质量为46,000。该酶的吸收光谱表明存在血红素辅基。吡啶血色原吸收光谱表明该酶含有一分子作为铁原卟啉IX的血红素。天然酶的最大吸收峰（406 nm）在还原酶中移至433 nm，在还原一氧化碳复合物中移至423 nm。氰根离子和叠氮根离子都能迅速与天然酶结合，表明存在一个可用的配位位点且血红素铁为高自旋态。过氧化氢酶复合物的吸收光谱在420 nm处有最大值，与辣根过氧化物酶化合物II的吸收光谱相似。黄孢原毛平革菌过氧化物酶活性依赖于锰（II），在100 microM以上达到最大活性。该酶也受到α-羟基酸（如苹果酸、乳酸）和蛋白质（如明胶、白蛋白）不同程度的刺激。该过氧化物酶能够氧化NADH和多种染料，包括聚B-411和聚R-481。几种底物（靛蓝三磺酸盐、NADH、聚B-411、变胺蓝RT盐和聚R-481）被这种依赖锰（II）的过氧化物酶氧化的速度比辣根过氧化物酶催化的速度快得多。该酶能迅速将锰（II）氧化为锰（III）；后者通过其焦磷酸复合物的特征吸收光谱检测到。焦磷酸钠对底物2,2-联氮-双（3-乙基苯并噻唑啉-6-磺酸）二铵盐（ABTS）氧化的抑制作用表明锰（III）在酶促机制中起作用。
