Structure/function relationship study of Gln156, Glu160 and Glu189 in the active site of trichosanthin
| Autor: | Yicheng Dong, Yan‐Wen Guo, Kam-Bo Wong, H.W. Yeung, Yi‐Bao Ke, Xiao‐Bing Li, Pang-Chui Shaw |
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| Rok vydání: | 1994 |
| Předmět: |
Trichosanthin
Glycoside Hydrolases Stereochemistry Glutamine Molecular Sequence Data Glycine Biochemistry Catalysis Structure-Activity Relationship Escherichia coli Protein biosynthesis Structure–activity relationship Amino Acid Sequence Binding site Site-directed mutagenesis Alanine Binding Sites Sequence Homology Amino Acid biology Ribosome-inactivating protein Active site Kinetics Protein Biosynthesis Mutagenesis Site-Directed biology.protein Ribosomes Sequence Alignment Plasmids |
| Zdroj: | European Journal of Biochemistry. 221:787-791 |
| ISSN: | 1432-1033 0014-2956 |
| DOI: | 10.1111/j.1432-1033.1994.tb18792.x |
| Popis: | Trichosanthin is a protein used medicinally in China for abortifacient purposes. It is also an RNA N-glycosidase which inactivates eukaryotic ribosomes by removing adenine4324 from 28S rRNA. Site-directed mutagenesis 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. The data indicate Glu160 is involved in the catalytic reaction. Kinetics studies suggest the carboxylate group of Glu160 serves to stabilize the transition-state complex. Similar to ricin A, the variant [E160A]trichosanthin 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 Glu160 does not affect the ID50 value drastically. An activity of 1800-fold less than that of the wild-type protein was found when both Glu160 and Glu189 were changed to alanine, indicating that some other residues in the active site are also taken part in the lowering of energy barrier 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. |
| Databáze: | OpenAIRE |
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