Distal Site Aspartate Is Essential in the Catalase Activity of Catalase-Peroxidases
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- 作者：Christa Jakopitsch ; Markus Auer ; Gü ; nther Regelsberger ; Walter Jantschko ; Paul Georg Furtmü ; ller ; Florian Rü ; ker ; and Christian Obinger
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：May 13, 2003
- 年：2003
- 卷：42
- 期：18
- 页码：5292 - 5300
- 全文大小：191K
- 年卷期：v.42,no.18(May 13, 2003)
- ISSN：1520-4995
文摘
Structural and biochemical characterization of aspartate 152 at the distal heme side of catalase-peroxidase (KatG) from Synechocystis PCC 6803 reveals an important functional role for this residue. Inthe wild-type protein, the side chain carboxyl group of Asp152 is 7.8 Å apart from the heme iron and ishydrogen-bonded to two water molecules and a KatG-specific large loop. We have prepared the site-specific variants Asp152Asn, Asp152Ser, Asp152Trp, and Pro151Ala. Exchange of Asp152 exhibiteddramatic consequences on the bifunctional activity of this unique peroxidase. The turnover number ofcatalase activity of Asp152Asn is 2.7%, Asp152Ser 5.7%, and Asp152Trp is 0.6% of wild-type activity.By contrast, the peroxidase activity of the Asp152 variants was 2-7 times higher than that of wild-typeKatG or Pro151Ala. The KatG-specific pH profile of the catalase activity was completely different inthese variants and exchange of Asp152 made it possible to follow the transition of the ferric enzyme tothe redox intermediate compound I by hydrogen peroxide spectroscopically and to determine thecorresponding bimolecular rate constant to be 7.5 × 106 M-1 s-1 (pH 7 and 15 
C). The reactivity ofcompound I toward aromatic one-electron donors was enhanced in the Asp152 variants compared withthe wild-type protein, whereas the reactivity toward hydrogen peroxide was dramatically decreased. Amechanism for the hydrogen peroxide oxidation, which is different from monofunctional catalases andinvolves the distal residues Trp122 and Asp152, is proposed.
C). The reactivity ofcompound I toward aromatic one-electron donors was enhanced in the Asp152 variants compared withthe wild-type protein, whereas the reactivity toward hydrogen peroxide was dramatically decreased. Amechanism for the hydrogen peroxide oxidation, which is different from monofunctional catalases andinvolves the distal residues Trp122 and Asp152, is proposed.