Insights into the inhibition of E. coli gamma‐glutamylcysteine ligase by sulfoximines

Gamma‐glutamylcysteine ligase (γ‐GCL) is the rate‐limiting enzyme in the biosynthesis of glutathione, a biological molecule implicated in chemotherapeutic resistance. Transition‐state analogs including buthionine sulfoximine (BSO) and its derivatives (methionine sulfoximine, ethionine sulfoximine, pentathionine sulfoximine, and hexathionine sulfoximine) are hypothesized to be irreversible inhibitors of gamma‐GCL, yet little is known about the binding site for these compounds. Using pre‐incubation studies, we have confirmed that the sulfoximines are binding irreversibly to E. coli gamma‐GCL in the glutamate region of the active site, but not the cysteine site. The rate of the irreversible covalent modification of each sulfoximine is dependent on both the size and stereochemistry of the sulfoximine alkyl tail. Our results indicate the buthionine sulfoximine irreversibly inhibits the enzyme 2‐ to 400‐times faster than the other sulfoximines. Current and future work includes detecting conformational changes in the enzyme active site upon sulfoximine binding using fluorescence spectroscopy and electron paramagnetic resonance. This work has been supported by the Howard Hughes Medical Institute and Gustavus Adolphus College.