Protein control of prosthetic heme reactivity. Reaction of substrates with the heme edge of horseradish peroxidase.
Protein control of prosthetic heme reactivity. Reaction of substrates with the heme edge of horseradish peroxidase.
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DOI：
10.1016/s0021-9258(19)75669-5
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发表时间：
1987-02
期刊：
影响因子：
--
通讯作者：
M. Ator;P. Ortiz de Montellano
中科院分区：
文献类型：
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作者：
M. Ator;P. Ortiz de Montellano
辣根过氧化物酶与苯肼和H2 O2一起孵育可显着抑制Soret带的催化活性和强度，但不会抑制Soret带的位置。每摩尔酶需要大约11-13摩尔苯肼和25摩尔H2 O2以使发色团强度最小化。酶在这种处理后保留一些活性，但如果将酶分离并与苯肼重新孵育，则该活性被消除。该酶的辅血红素不像其他血红素蛋白那样与苯肼反应生成σ-键合的苯基-铁复合物，而是转化为δ-mesophenyl和8-羟甲基衍生物。然而，活性的丧失更多地是由于蛋白质而不是血红素修饰。失活的酶与H2 O2反应，得到光谱可检测的化合物I。结果表明，基板与血红素边缘，而不是与化合物I和II的活性氧相互作用，并具体确定周围的δ-中位碳和8-甲基作为血红素的暴露部门的区域。辣根过氧化物酶，与细胞色素P-450相反，通常不催化氧转移反应。目前的结果表明，氧转移反应不会发生，因为活性氧和底物的物理分离的蛋白质施加的障碍，辣根过氧化物酶。
Incubation of horseradish peroxidase with phenylhydrazine and H2O2 markedly depresses the catalytic activity and the intensity, but not position, of the Soret band. Approximately 11-13 mol of phenylhydrazine and 25 mol of H2O2 are required per mol of enzyme to minimize the chromophore intensity. The enzyme retains some activity after such treatment, but this activity is eliminated if the enzyme is isolated and reincubated with phenylhydrazine. The prosthetic heme of the enzyme does not react with phenylhydrazine to give a sigma-bonded phenyl-iron complex, as it does in other hemoproteins, but is converted instead to the delta-mesophenyl and 8-hydroxymethyl derivatives. The loss of activity is due more to protein than heme modification, however. The inactivated enzyme reacts with H2O2 to give a spectroscopically detectable Compound I. The results imply that substrates interact with the heme edge rather than with the activated oxygen of Compounds I and II and specifically identify the region around the delta-meso-carbon and 8-methyl group as the exposed sector of the heme. Horseradish peroxidase, in contrast to cytochrome P-450, generally does not catalyze oxygen-transfer reactions. The present results indicate that oxygen-transfer reactions do not occur because the activated oxygen and the substrate are physically separated by a protein-imposed barrier in horseradish peroxidase.