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首页> 外文期刊>Biochemistry >ALTERING SUBSTRATE SPECIFICITY AT THE HEME EDGE OF CYTOCHROME C PEROXIDASE
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ALTERING SUBSTRATE SPECIFICITY AT THE HEME EDGE OF CYTOCHROME C PEROXIDASE

机译：在细胞色素C过氧化物酶血红素边缘改变底物特异性

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摘要

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site, This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while V-max/e for oxidation of other small molecules is less affected by either mutation. However, the ''specificity'' of aniline oxidation by A147M, i.e., (V-max/e)/K-m, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H2O2, but not to an extent that it becomes rate limiting for the oxidation of-the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than with wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. These results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidases with engineered function.
机译：据报道，细胞色素C过氧化物酶（CCP）的两个突变体表现出对小底物氧化的独特特异性。 CCP中的Ala-147位于血红素的δ-内消旋边缘附近，沿着溶剂进入通道，H2O2被认为通过该通道接近活性位点，该残基被Met和Tyr取代，以研究小分子底物是在血红素暴露的δ-中观边缘被氧化。 X射线晶体学分析证实，A147M和A147Y的侧链位于δ-内消旋血红素位置上方，因此可能会修饰小分子接近氧化血红素辅因子的途径。稳态动力学测量表明，相对于野生型，A147Y的细胞色素c氧化增强了3倍，而小分子氧化则根据底物和突变体而不同程度地改变。例如，相对于野生型酶，A147Y对苯酚的氧化减少至小于20％，而其他小分子氧化的V-max / e受任一突变的影响较小。但是，A147M对苯胺氧化的“特异性”，即（V-max / e）/ Km，比野生型酶高43倍，这表明苯胺已经引入了对苯胺的特异性相互作用。突变。停止流动的动力学数据表明，在A147Y或A147M中限制的血红素进入会减慢酶与H2O2之间的反应，但不会在一定程度上限制所检测底物的氧化速度。用A147Y形成化合物ES的速率常数比使用野生型CCP慢2.5倍。这些观察结果强烈支持小分子氧化发生在酶上不同于细胞色素c所利用的酶的位点的建议，并且小分子氧化的特异性可以通过操纵对血红素边缘的访问而得到显着调节。这些结果有助于确定替代电子转移途径在细胞色素c过氧化物酶中的作用，并且在提高具有工程功能的过氧化物酶的特异性方面可能具有有用的应用。

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