Trichosanthin is a ribosome-inactivating protein that cleaves specifically the N-glycosidic bond of A-4324 of 28S rRNA. Trichosanthin and its variant [E160A-E189A]-trichosanthin were found to bind an adenine base with a K(d) value of approximately 0.2mM. To determine how this doubly mutated variant of trichosanthin interacts with adenine, the co-crystal structure of [E160A-E189A]-trichosanthin and adenine was resolved to 0.193nm which revealed that the active site conformation of the doubly mutated variant is isomorphous to wild-type trichosanthin. Water molecules were found at locations corresponding to the eliminated side chain of Glu-160 and Glu-189. On the other hand, the adenine base interacted with [E160A-E189A]-trichosanthin in a manner similar to that in wild-type trichosanthin. Our structural analysis illustrates that Glu-160 and Glu-189 in trichosanthin do not play an important role in maintaining the active site conformation and binding adenine, an essential step for substrate-enzyme interaction. On the other hand, removal of two glutamate residues changed a large patch of negatively charged surface to a positive charge, which may account for the destabilization of the oxocarbenium-like transition-state and the significant decrease in ribosome-inactivating activity in [E160A-E189A]-trichosanthin.