Redesign of Cytochrome c Peroxidase into a Manganese Peroxidase: Role of Tryptophans in Peroxidase Activity

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• 作者：Alan Gengenbach ; Sung Syn ; Xiaotang Wang ; and Yi Lu
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：August 31, 1999
• 年：1999
• 卷：38
• 期：35
• 页码：11425 - 11432
• 全文大小：87K
• 年卷期：v.38,no.35(August 31, 1999)
• ISSN：1520-4995

文摘

Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome cperoxidase (CcP) containing a Mn(II)-binding site reported previously (MnCcP; see Yeung, B. K.-S., etal. (1997) Chem. Biol. 5, 215-221). The goal of the present study is to elucidate the role of tryptophansin 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 allowsformation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical calledcompound I'. The absence of a tryptophan residue at this position in MnP is the main reason for theformation of an intermediate called compound I which contains a ferryl oxo and porphyrin -cation radical.In this study, we showed that introduction of the Trp191Phe mutation to MnCcP did not improve MnPactivity (specific activity: MnCcP, 0.750 mol min^-1 mg^-1; MnCcP(W191F), 0.560 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 introductionof 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 livedcompound I in WTCcP (decay rate, 18 s^-1), but also significantly improved MnP activity in MnCcP(MnCcP(W51F, W191F): specific activity, 8.0 mol min^-1 mg^-1; k_cat/K_m, 0.599 s^-1 mM^-1). The increasein activity can be attributed to the Trp51Phe mutation since MnCcP(W51F) showed significantly increasedMnP activity relative to MnCcP (specific activity, 3.2 mol min^-1 mg^-1; k_cat/K_m, 0.325 s^-1 mM^-1). Aswith MnP, the activity of MnCcP(W51F, W191F) was found to increase with decreasing pH. Our resultsdemonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizingcompound I, only the Trp51Phe mutation contributes significantly to increasing the MnP activity becausethis mutation increases the reactivity of compound II, whose oxidation of Mn(II) is the rate-determiningstep in the reaction mechanism.