Mechanism-Based Inactivation of Thioredoxin Reductase from Plasmodium falciparum by Mannich Bases. Implication for Cytotoxicity
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- 作者：Elisabeth Davioud-Charvet ; Michael J. McLeish ; Donna M. Veine ; David Giegel ; L. David Arscott ; Adriano D. Andricopulo ; Katja Becker ; Sylke Mü ; ller ; R. Heiner Schirmer ; Charles H. Williams ; Jr. ; an
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：November 18, 2003
- 年：2003
- 卷：42
- 期：45
- 页码：13319 - 13330
- 全文大小：212K
- 年卷期：v.42,no.45(November 18, 2003)
- ISSN：1520-4995
文摘
Thioredoxin reductase (TrxR) is the homodimeric flavoenzyme that catalyzes reduction ofthioredoxin disulfide (Trx). For Plasmodium falciparum, a causative agent of tropical malaria, TrxR is anessential protein which has been validated as a drug target. The high-throughput screening of 350000compounds has identified Mannich bases as a new class of TrxR mechanism-based inhibitors. Duringcatalysis, TrxR conducts reducing equivalents from the NADPH-reduced flavin to Trx via the two redox-active cysteine pairs, Cys88-Cys93 and Cys535'-Cys540', referred to as N-terminal and C-terminal cysteinepairs. The structures of unsaturated Mannich bases suggested that they could act as bisalkylating agentsleading to a macrocycle that involves both C-terminal cysteines of TrxR. To confirm this hypothesis,different Mannich bases possessing one or two electrophilic centers were synthesized and first studied indetail using glutathione as a model thiol. Michael addition of glutathione to the double bond of anunsaturated Mannich base (3a) occurs readily at physiological pH. Elimination of the amino group, promotedby base-catalyzed enolization of the ketone, is followed by addition of a second nucleophile. Theintermediate formed in this reaction is an 
,
-unsaturated ketone that can react rapidly with a secondthiol. When studying TrxR as a target of Mannich bases, we took advantage of the fact that the charge-transfer complex formed between the thiolate of Cys88 and the flavin in the reduced enzyme can beobserved spectroscopically. The data show that it is the C-terminal Cys 535'-Cys540' pair rather than theN-terminal Cys88-Cys93 pair that is modified by the inhibitor. Although alkylated TrxR is unable to turnover its natural substrate Trx, it can reduce low Mr electron acceptors such as methyl methanethiolsulfonateby using its unmodified N-terminal thiols. On the basis of results with chemically distinct Mannich bases,a detailed mechanism for the inactivation of TrxR is proposed.
,-unsaturated ketone that can react rapidly with a secondthiol. When studying TrxR as a target of Mannich bases, we took advantage of the fact that the charge-transfer complex formed between the thiolate of Cys88 and the flavin in the reduced enzyme can beobserved spectroscopically. The data show that it is the C-terminal Cys 535'-Cys540' pair rather than theN-terminal Cys88-Cys93 pair that is modified by the inhibitor. Although alkylated TrxR is unable to turnover its natural substrate Trx, it can reduce low Mr electron acceptors such as methyl methanethiolsulfonateby using its unmodified N-terminal thiols. On the basis of results with chemically distinct Mannich bases,a detailed mechanism for the inactivation of TrxR is proposed.