A Fluoro Analogue of the Menadione Derivative 6-[2'-(3'-Methyl)-1',4'-naphthoquinolyl]hexanoic Acid Is a Suicide Substrate of Glutathione Reductase. Crystal Structure of the Alkylated Human Enzyme
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- 作者：Holger Bauer ; Karin Fritz-Wolf ; Andreas Winzer ; Sebastian Kü ; hner ; Susan Little ; Vanessa Yardley ; Hervé ; Vezin ; Bruce Palfey ; R. Heiner Schirmer ; Elisabeth Davioud-Charvet
- 刊名：Journal of the American Chemical Society
- 出版年：2006
- 出版时间：August 23, 2006
- 年：2006
- 卷：128
- 期：33
- 页码：10784 - 10794
- 全文大小：206K
- 年卷期：v.128,no.33(August 23, 2006)
- ISSN：1520-5126
文摘
Glutathione reductase is an important housekeeping enzyme for redox homeostasis both in humancells and in the causative agent of tropical malaria, Plasmodium falciparum. Glutathione reductase inhibitorswere shown to have anticancer and antimalarial activity per se and to contribute to the reversal of drugresistance. The development of menadione chemistry has led to the selection of 6-[2'-(3'-methyl)-1',4'-naphthoquinolyl]hexanoic acid, called M5, as a potent reversible and uncompetitive inhibitor of both humanand P. falciparum glutathione reductases. Here we describe the synthesis and kinetic characterization ofa fluoromethyl-M5 analogue that acts as a mechanism-based inhibitor of both enzymes. In the course ofenzymatic catalysis, the suicide substrate is activated by one- or two-electron reduction, and then a highlyreactive quinone methide is generated upon elimination of the fluorine. Accordingly the human enzymewas found to be irreversibly inactivated with a kinact value of 0.4 ± 0.2 min-1. The crystal structure of thealkylated enzyme was solved at 1.7 Å resolution. It showed the inhibitor to bind covalently to the active siteCys58 and to interact noncovalently with His467', Arg347, Arg37, and Tyr114. On the basis of the crystalstructure of the inactivated human enzyme and stopped-flow kinetic studies with two- and four-electron-reduced forms of the unreacted P. falciparum enzyme, a mechanism is proposed which explainsnaphthoquinone reduction at the flavin of glutathione reductase.