细胞色素c过氧化物酶改造为锰过氧化物酶：色氨酸在过氧化物酶活性中的作用
Redesign of cytochrome c peroxidase into a manganese peroxidase: role of tryptophans in peroxidase activity.
作者信息
Gengenbach A, Syn S, Wang X, Lu Y
机构信息
Department of Chemistry, University of Illinois at Urbana-Champaign 61801, USA.
出版信息
Biochemistry. 1999 Aug 31;38(35):11425-32. doi: 10.1021/bi990666+.
Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome c peroxidase (CcP) containing a Mn(II)-binding site reported previously (MnCcP; see Yeung, B. K.-S., et al. (1997) Chem. Biol. 5, 215-221). The goal of the present study is to elucidate the role of tryptophans in peroxidase activity since CcP contains both Trp51 and Trp191 while manganese peroxidase (MnP) contains phenylalanine residues at the corresponding positions. The presence of Trp191 in CcP allows formation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical called compound I'. The absence of a tryptophan residue at this position in MnP is the main reason for the formation of an intermediate called compound I which contains a ferryl oxo and porphyrin pi-cation radical. In this study, we showed that introduction of the Trp191Phe mutation to MnCcP did not improve MnP activity (specific activity: MnCcP, 0.750 micromol min-1 mg-1; MnCcP(W191F), 0.560 micromol min-1 mg-1. k(cat)/K(m): MnCcP, 0.0517 s-1 mM-1; MnCcP(W191F), 0.0568 s-1 mM-1) despite the fact that introduction of the same mutation to WTCcP caused the formation of a transient compound I (decay rate, 60 s-1). However, introducing both the Trp191Phe and Trp51Phe mutations not only resulted in a longer lived compound I in WTCcP (decay rate, 18 s-1), but also significantly improved MnP activity in MnCcP (MnCcP(W51F, W191F): specific activity, 8.0 micromol min-1 mg-1; k(cat)/K(m), 0. 599 s-1 mM-1). The increase in activity can be attributed to the Trp51Phe mutation since MnCcP(W51F) showed significantly increased MnP activity relative to MnCcP (specific activity, 3.2 micromol min-1 mg-1; k(cat)/K(m), 0.325 s-1 mM-1). As with MnP, the activity of MnCcP(W51F, W191F) was found to increase with decreasing pH. Our results demonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizing compound I, only the Trp51Phe mutation contributes significantly to increasing the MnP activity because this mutation increases the reactivity of compound II, whose oxidation of Mn(II) is the rate-determining step in the reaction mechanism.
色氨酸191苯丙氨酸（Trp191Phe）和色氨酸51苯丙氨酸（Trp51Phe）突变已被引入到先前报道的含有锰（II）结合位点的工程化细胞色素c过氧化物酶（CcP）中（锰细胞色素c过氧化物酶，MnCcP；见Yeung，B.K.-S.等人（1997年）《化学生物学》5，215 - 221）。本研究的目的是阐明色氨酸在过氧化物酶活性中的作用，因为CcP同时含有色氨酸51和色氨酸191，而锰过氧化物酶（MnP）在相应位置含有苯丙氨酸残基。CcP中色氨酸191的存在使得能够形成一种独特的高价中间体，该中间体含有一个铁氧代和色氨酸自由基，称为化合物I'。MnP在该位置不存在色氨酸残基是形成含有铁氧代和卟啉π - 阳离子自由基的中间体化合物I的主要原因。在本研究中，我们表明将Trp191Phe突变引入MnCcP并未提高MnP活性（比活性：MnCcP，0.750微摩尔·分钟⁻¹·毫克⁻¹；MnCcP（W191F），0.560微摩尔·分钟⁻¹·毫克⁻¹。k（cat）/K（m）：MnCcP，0.0517秒⁻¹·毫摩尔⁻¹；MnCcP（W191F），0.0568秒⁻¹·毫摩尔⁻¹），尽管将相同突变引入野生型CcP（WTCcP）会导致形成一种瞬态化合物I（衰减速率，60秒⁻¹）。然而，同时引入Trp191Phe和Trp51Phe突变不仅导致WTCcP中化合物I的寿命延长（衰减速率，18秒⁻¹），而且还显著提高了MnCcP中的MnP活性（MnCcP（W51F，W191F）：比活性，8.0微摩尔·分钟⁻¹·毫克⁻¹；k（cat）/K（m），0.599秒⁻¹·毫摩尔⁻¹）。活性的增加可归因于Trp51Phe突变，因为相对于MnCcP，MnCcP（W51F）显示出显著增加的MnP活性（比活性，3.2微摩尔·分钟⁻¹·毫克⁻¹；k（cat）/K（m），0.325秒⁻¹·毫摩尔⁻¹）。与MnP一样，发现MnCcP（W51F，W191F）的活性随着pH值的降低而增加。我们的结果表明，虽然Trp191Phe和Trp51Phe突变在稳定化合物I方面都起着重要作用，但只有Trp51Phe突变对增加MnP活性有显著贡献，因为该突变增加了化合物II的反应性，其对Mn（II）的氧化是反应机制中的速率决定步骤。
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