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J. Biol. Chem., Vol. 280, Issue 28, 26121-26128, July 15, 2005

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Cooperativity and Pseudo-cooperativity in the Glutathione S-Transferase from Plasmodium falciparum^*

Eva Liebau, Francesca De Maria¶, Cora Burmeister, Markus Perbandt||, Paola Turella¶, Giovanni Antonini**, Giorgio Federici, Francesco Giansanti**, Lorenzo Stella¶, Mario Lo Bello, Anna Maria Caccuri¶, and Giorgio Ricci¶¶¶

From the Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany, ^¶Department of Chemical Sciences and Technologies and Department of Biology, University of Rome "Tor Vergata," 00133 Rome, Italy, ^||Department of Biochemistry and Molecular Biology, University of Hamburg, 20146 Hamburg, Germany, ^**Department of Biology, University of Rome "Roma Tre," 00146 Rome, and Children's Hospital IRCCS "Bambino Gesù," 00165 Rome, Italy

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K_D for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK_a value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n_H = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the -amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Received for publication, April 11, 2005 , and in revised form, May 11, 2005.

^* This work was supported in part by a grant from the Ministero della Pubblica Istruzione, Progetto Ministero dell'Istruzione dell'Università e della Ricerca (MIUR). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both of these authors contributed equally to this work.

^¶¶ To whom correspondence should be addressed: Dept. of Chemical Sciences and Technologies, University of Rome "Tor Vergata," 00133 Rome, Italy. E-mail: riccig{at}uniroma2.it.

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