1JCI

Stabilization of the Engineered Cation-binding Loop in Cytochrome c Peroxidase (CcP)

1JCI の概要

• エントリーDOI: 10.2210/pdb1jci/pdb
• 分子名称: Cytochrome C Peroxidase, POTASSIUM ION, PROTOPORPHYRIN IX CONTAINING FE, ... (4 entities in total)
• 機能のキーワード: cation-binding loop, trp191 cationic radical, open/closed conformer, oxidoreductase
• 由来する生物種: Saccharomyces cerevisiae (baker's yeast)
• 細胞内の位置: Mitochondrion matrix: P00431
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 34298.89

構造登録者

Bhaskar, B.,Bonagura, C.A.,Li, H.,Poulos, T.L. (登録日: 2001-06-09, 公開日: 2002-03-06, 最終更新日: 2024-04-03)

主引用文献

Bhaskar, B.,Bonagura, C.A.,Li, H.,Poulos, T.L.
Cation-induced stabilization of the engineered cation-binding loop in cytochrome c peroxidase (CcP).
Biochemistry, 41:2684-2693, 2002

PubMed Abstract: We have previously shown that the K(+) site found in the proximal heme pocket of ascorbate peroxidase (APX) could be successfully engineered into the closely homologous cytochrome c peroxidase (CcP) [Bonagura et al., (1996) Biochemistry 35, 6107-6115; Bonagura et al. (1999) Biochemistry 38, 5538-5545]. In addition, specificity could be switched to binding Ca(2+) as found in other peroxidases [Bonagura et al. (1999) J. Biol. Chem. 274, 37827-37833]. The introduction of a proximal cation-binding site also promotes conversion of the Trp191 containing cation-binding loop from a "closed" to an "open" conformer. In the present study we have changed a crucial hinge residue of the cation-binding loop, Asn195, to Pro which stabilizes the loop, albeit, only in the presence of bound K(+). The crystal structure of this mutant, N195PK2, has been refined to 1.9 A. As predicted, introduction of this crucial hinge residue stabilizes the cation-binding loop in the presence of the bound K(+). As in earlier work, the characteristic EPR signal of Trp191 cation radical becomes progressively weaker with increasing [K(+)] and the lifetime of the Trp191 radical also has been considerably shortened in this mutant. This mutant CcP exhibits reduced enzyme activity, which could be titrated to lower levels with increasing [K(+)] when horse heart cytochrome c is the substrate. However, with yeast cytochrome c as the substrate, the mutant was as active as wild-type at low ionic strength, but 40-fold lower at high ionic strength. We attribute this difference to a change in the rate-limiting step as a function of ionic strength when yeast cytochrome c is the substrate.

PubMed: 11851415
DOI: 10.1021/bi011599y

実験手法

X-RAY DIFFRACTION (1.9 Å)