Redesign of cytochrome c peroxidase into a manganese peroxidase:: Role of tryptophans in peroxidase activity-猫眼课题宝

DOI：

10.1021/bi990666

发表时间：

1999-08-31

期刊：

BIOCHEMISTRY

影响因子：

2.9

通讯作者：

中科院分区：

生物学3区

文献类型：

--

作者：

Gengenbach, A;Syn, S;Lu, Y

Trp 191 Phe和Trp 51 Phe突变已经被引入到含有先前报道的Mn（II）结合位点的工程化细胞色素c过氧化物酶（CcP）中（MnCcP;参见Yeung，B. K.-美国，等人，（1997）Chem. Biol.，5，215-221）。本研究的目的是阐明在过氧化物酶活性的作用，因为CcP含有Trp 51和Trp 191，而锰过氧化物酶（MNP）含有苯丙氨酸残基在相应的位置。Trp 191在CcP中的存在允许形成一种独特的高价中间体，其含有铁氧和色氨酸基团，称为化合物I '。在MnCcP中，在该位置处缺少色氨酸残基是形成称为化合物I的中间体的主要原因，所述化合物I含有铁基氧代和卟啉π-阳离子自由基。在本研究中，我们表明，将Trp 191 Phe突变引入MnCcP并不提高MnP活性（比活度：MnCcP，0.750 μ mol min（-1）mg（-1）; MnCcP（W191 F），0.560 μ mol min（-1）mg（-1）。k（cat）/K-m：MnCcP，0.0517 s（-1）mM（-1）; MnCcP（W191 F）0.0568 s（-1）mM（-1））），尽管事实上向WTCcP引入相同的突变会导致瞬时化合物I的形成（衰变率，60 s（-1））。然而，引入Trp 191 Phe和Trp 51 Phe突变不仅导致WTCcP中化合物I的寿命更长（衰减速率，18 s（-1）），而且显著提高了MnCcP中MnP的活性（MnCcP（W51 F，W191 F）：比活性，8.0 μ mol min（-1）mg（-1）; k（cat）/K-m，0.599 s（-1）mM（-1））。活性的增加可归因于Trp 51 Phe突变，因为MnCcP（W51 F）显示出相对于MnCcP显著增加的MNP活性（比活性，3.2 μ mol min（-1）mg（-1）; k（cat）/K-m，0.325 s（-1）mM（-1））。与MnP一样，MnCcP的活性我们的结果表明，虽然Trp 191 Phe和Trp 51 Phe突变都在稳定化合物I中起重要作用，但只有Trp 51 Phe突变显著有助于增加MNP活性，因为该突变增加了化合物II的反应性，其锰（II）的氧化是反应机制中的速率决定步骤。

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 mu mol min(-1) mg(-1); MnCcP(W191F), 0.560 mu mol 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 mu mol 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 mu mol 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.