Inhibition of glyceraldehyde-3-phosphate dehydrogenase by phosphorylated epoxides and alpha-enones.

Pentalenolactone and koningic acid are antibiotics known for their potent inhibition of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. The reactive groups present in these antibiotics are, respectively, an epoxide and an alpha-enone, which form covalent bonds with an active-site cysteine residue of the enzyme. This information was used for the design of two series of glyceraldehyde 3-phosphate analogues with similar reactive groups that could function as potential irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase. Inactivation kinetics, NMR analysis, protection experiments, and titration of free cysteine residues together indicate that the inhibitors bind to the active site of the enzyme and form a covalent bond with the active-site cysteine residue of the enzyme. Binding probably takes place at the inorganic phosphate site of the enzyme and may lead to a conformational change. Comparison of the reactivities of the inhibitors for the glycosomal enzyme from the protozoan parasite Trypanosoma brucei and the rabbit muscle enzyme revealed that some of them had a preference for the trypanosome enzyme. When their effect was measured on the multiplication of trypanosome in vitro cultures, one inhibitor appeared to exhibit an inhibitory effect at a concentration significantly lower than the trypanocidal drugs, pentamidin and (difluoromethyl)ornithine, that are routinely used in the treatment of African sleeping sickness.