STRUCTURE FUNCTION RELATIONSHIP STUDY OF GLN156, GLU160 AND GLU189 INTHE ACTIVE-SITE OF TRICHOSANTHIN/

Trichosanthin is a protein used medicinally in China for abortifacient purposes. It is also an RNA N-glycosidase which inactivates eukaryoti c ribosomes by removing adenine4324 from 28S rRNA. Site-directed mutag enesis was performed to probe the role of Gln156, Glu160 and Glu189 in the active site of trichosanthin. The purified altered proteins were assayed for their potency in inhibiting in vitro protein synthesis. Th e data indicate Glu160 is involved in the catalytic reaction. Kinetics studies suggest the carboxylate group of Glu160 serves to stabilize t he transition-state complex. Similar to ricin A, the variant [E160A]tr ichosanthin is more potent than [E160D]trichosanthin. This is because Glu189 serves as a back-up of the carboxylate group in case Glu160 is mutated to alanine. However, removal of Glu189 in the presence of Glu1 60 does not affect the ID50 value drastically. An activity of 1800-fol d less than that of the wild-type protein was found when both Glu160 a nd Glu189 were changed to alanine, indicating that some other residues in the active site are also taken part in the lowering of energy barr ier for the catalytic reaction. Although Gln156 is highly conserved in related proteins, its mutation to alanine only slightly decreases the activity, showing that this residue does not participate directly in catalysis.