Structural Basis for Feedback and Pharmacological Inhibition of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Ekaterina I Biterova,Joseph J Barycki

doi:10.1074/jbc.m110.104802

Ekaterina I Biterova, Joseph J Barycki

Open Access

https://doi.org/10.1074/jbc.m110.104802

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Abstract

Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 A; R = 19.9%, R(free) = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine-binding site and disrupts the previously observed Mg(2+) coordination in the ATP-binding site. l-Buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 A; R = 18.1%, R(free) = 23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme.

Highlights

Increased glutamate cysteine ligase catalytic subunit mRNA levels and GCL activity have been frequently observed in cells derived from human tumors resistant to chemotherapeutic agents (14 –16)
Subsequent ATP-dependent phosphorylation of the sulfoximine nitrogen by GCL leads to the formation of a tightly bound transition state analogue [20, 23]
Kinetic Characterization of Saccharomyces cerevisiae GCL (ScGCL)—Previously reported structural and biochemical data indicate that ScGCL likely functions as a monomer both in vitro and in vivo [24]

Summary

Introduction

Glutathione is a competitive inhibitor with respect to the glutamate substrate (supplemental Fig. S1), with an apparent Ki(GSH) of 2.12 Ϯ 0.13 mM, similar to other Group 2 GCL holoenzymes [11,12,13, 33]. As glutathione is positioned by hydrogen bonds with the side discussed above, BSO is a potent mechanism-based inhibitor of chains of Tyr362 and Arg313 as well as with an ordered water ScGCL.

Results

Conclusion