Identification of Two Electron-Transfer Sites in Ascorbate Peroxidase Using Chemical Modification, Enzyme Kinetics, and Crystallography
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- 作者：David Mandelman ; Joumana Jamal ; and Thomas L. Poulos
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：December 15, 1998
- 年：1998
- 卷：37
- 期：50
- 页码：17610 - 17617
- 全文大小：182K
- 年卷期：v.37,no.50(December 15, 1998)
- ISSN：1520-4995
文摘
Chemical and mutagenic modification combined with X-ray crystallography has been used toprobe the ascorbate binding site in ascorbate peroxidase (APX). Chemical modification of the single Cysresidue in APX with Ellman's reagent (DTNB) blocks the ability of APX to oxidize ascorbate but notother small aromatic phenolic substrates. DTNB-modified APX (APX-TNB) exhibits only 1.3% wild-type activity when ascorbate is used as the substrate but full activity when aromatic substrates, guaiacolor pyrogallol, are used. Stopped-flow studies show that APX-TNB reacts normally with peroxide to givecompound I but that the rates of reduction of both compounds I and II by ascorbate are dramaticallyslowed. Conversion of Cys32 to Ser leads to 
70% drop in ascorbate peroxidase activity with no effecton guaiacol peroxidase activity. These results indicate that uncharged aromatic substrates and the anionicascorbate molecule interact with different sites on APX. The 2.0 Å X-ray crystal structure of APX-TNBshows clear electron density for the TNB group covalently attached to Cys32 in all four molecules of theasymmetric unit, indicating complete and specific modification. It appears that the ascorbate site is blockedby DTNB modification which is well removed from the exposed 
-heme edge where aromatic substratesare thought to bind. This is the first experimental evidence indicating that ascorbate oxidation does notoccur at the exposed heme edge but at an alternate binding site in the vicinity of Cys32 near Arg172 andthe heme propionates.
70% drop in ascorbate peroxidase activity with no effecton guaiacol peroxidase activity. These results indicate that uncharged aromatic substrates and the anionicascorbate molecule interact with different sites on APX. The 2.0 Å X-ray crystal structure of APX-TNBshows clear electron density for the TNB group covalently attached to Cys32 in all four molecules of theasymmetric unit, indicating complete and specific modification. It appears that the ascorbate site is blockedby DTNB modification which is well removed from the exposed -heme edge where aromatic substratesare thought to bind. This is the first experimental evidence indicating that ascorbate oxidation does notoccur at the exposed heme edge but at an alternate binding site in the vicinity of Cys32 near Arg172 andthe heme propionates.