Journal Article

Involvement of the Binuclear Copper Site in the Proteolytic Activity of Polyphenol Oxidase

Tomohiko Kuwabara and Yuji Katoh

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 40, issue 10, pages 1029-1035
Published in print January 1999 | ISSN: 0032-0781
Published online January 1999 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/oxfordjournals.pcp.a029484
Involvement of the Binuclear Copper Site in the Proteolytic Activity of Polyphenol Oxidase

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Plant polyphenol oxidase (PPO) is apt to degrade during and even after purification. We developed a method to stabilize PPO by 0.3 M NaCl, 0.1% (w/v) Tween 20, and 50% (w/v) ethylene glycol at pH 6.5. The protein slowly degraded by itself when the stabilizing reagents were removed. Ascorbate and/or H2O2 accelerated the degradation. The ascorbate-induced degradation was inhibited by catalase, suggesting that H2O2 is generated through reduction of PPO by ascorbate. It is likely that dissolved oxygen is converted to peroxide through two-electron reduction by the reaction center of PPO, binuclear Cu site, and a Fenton-type reaction occurred on it. This understanding was supported by the finding that the H2O2-induced degradation was inhibited by metal-chelators as well as by polyphenolic substrate of PPO. Considering the postulated mechanism of the self-degradation of PPO, we re-examined the degradation of the 23-kDa protein of PSII by PPO [Kuwabara et al. (1997) Plant Cell Physiol. 38: 179]. The obtained results suggested that the 23-kDa protein triggers the active oxygen production by the binuclear Cu site, probably as reductant, and receives the radical species preferentially to the polypeptide moiety of PPO.

Keywords: Active oxygen; Binuclear Cu site; Dithiothreitol-sensitive tetrameric protease; Fenton reaction; Polyphenol oxidase (E.C. 1.10.3.1); Spinach

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Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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