Explaining the inhibition of glyoxalase II by 9-fluorenylmethoxycarbonyl-protected glutathione derivatives

In an effort to probe the inhibition of glyoxalase II (GLX2-2) from Arabidopsis thaliana, a series of N- and S-blocked glutathione compounds containing 9-fluorenylmethoxycarbonyl (FMOC) and Cbz protecting groups were synthesized and tested. The di-FMOC and di-Cbz compounds were the best inhibitors of GLX2-2 with Ki values of 0.89 ± 0.05 and 2.3 ± 0.5 μM, respectively. The removal of protecting groups from either position resulted in comparable, diminished binding affinities. Analyses of site-directed mutants of GLX2-2 demonstrated that tight binding of these inhibitors is not due to interactions of the protecting groups with hydrophobic amino acids on the surface of the enzyme. Instead, MM2 calculations predict that the lowest energy structures of the unbound, doubly substituted inhibitors are similar to those of a bound inhibitor. These studies represent the first systematic attempt to understand the peculiar inhibition of GLX2 by N- and S-blocked glutathiones.