Journal of Biological Chemistry
Volume 277, Issue 2, 11 January 2002, Pages 1158-1165
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ENZYME CATALYSIS AND REGULATION
Drosophila melanogaster Glutamate-Cysteine Ligase Activity Is Regulated by a Modifier Subunit with a Mechanism of Action Similar to That of the Mammalian Form*

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Glutamate-cysteine ligase (GCL) plays an important role in regulating glutathione homeostasis. In mammals, it comprises a catalytic (GCLC) and modifier (GCLM) subunit. The existence of a modifier subunit in invertebrates has not been described to date. We now demonstrate that GCL from Drosophila melanogasterhas a functional modifier subunit (DmGCLM). A putative DmGCLM was obtained as an expressed sequence tag with 27% identity to human GCLM at the amino acid level. D. melanogaster GCLC (DmGCLC) and the candidate DmGCLM were expressed separately inEscherichia coli, purified, mixed, and then subjected to gel filtration, where they eluted as an ∼140-kDa complex. DmGCLC co-immunoprecipitated with DmGCLM from S2 cell extracts, suggesting that they also associate in vivo. Enzyme kinetic analyses showed that DmGCLC has a Km for glutamate of 2.88 mm, but when complexed with DmGCLM, theKm for glutamate is 0.45 mm. Inhibition of DmGCLC activity by glutathione was found to be competitive with respect to glutamate (Ki = 0.03 mm), whereas inhibition of the GCL complex was mixed (Ki= 0.67 mm), suggesting allosteric effects. In accordance with this, DmGCLC and DmGCLM have the ability to form reversible intermolecular disulfide bridges. A further mechanism for control ofD. melanogaster GCL was found to be induction of DmGCLC bytert-butylhydroquinone in S2 cells. DmGCLM levels were, however, unaffected by tert-butylhydroquinone.

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Published, JBC Papers in Press, November 6, 2001, DOI 10.1074/jbc.M106683200

*

This work was supported in part by Grants 94/G15091 and 108/G15090 from the Biotechnology and Biological Sciences Research Council and by Royal Society Research Grant 21511.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supported by Biotechnology and Biological Sciences Research Council Studentship 98/A/C/04147.