Structure/function relationship study of Tyr14 and Arg22 in trichosanthin, a ribosome-inactivating protein.
Amino acids Tyr14 and Arg22 in trichosanthin are residues on helix A1 close to the active-site cleft. They are invariant in various type-I and type-II ribosome-inactivating proteins. In this study, Tyr14 was changed to Phe and Arg22 to Lys and Leu. Modified proteins were purified, and activities compared by assaying their median inhibitory concentration (ID50) on a rabbit-reticulocyte-lysate protein-synthesis system. While the ID50 of wild-type trichosanthin was 0.02 nM, those for [Phe14], [Lys22], [Leu22] and [Phe14, Leu22]trichosanthin were 0.10, 0.03, 0.25 and 0.15 nM, respectively. Therefore, compared with Tyr14, Arg22 appears to play a more important role in trichosanthin. Structural studies on [Leu22]trichosanthin showed that two water molecules occupy the space left by the side chain of Arg22, and hydrogen bonds exist between these water molecules and nearby residues to retain the conformation. The use of intermolecular rather than intramolecular hydrogen bonds may have an adverse effect on stability or folding of the protein and results in a mild decrease in activity.
Shaw
P C
PC
Department of Biochemistry, The Chinese University of Hong Kong, Shatin.
Mulot
S
S
Ma
S K
SK
Xu
Q F
QF
Yao
H B
HB
Wu
S
S
Lu
X H
XH
Dong
Y C
YC
D000020:Abortifacient Agents, Nonsteroidal / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D019380:Anti-HIV Agents / Q000737:chemistry / Q000494:pharmacology*; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D007930:Leucine / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011817:Rabbits; D012270:Ribosomes / Q000187:drug effects*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D015978:Trichosanthin / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism*
D000020:Abortifacient Agents, Nonsteroidal; D019380:Anti-HIV Agents; D011500:Protein Synthesis Inhibitors; D014443:Tyrosine; D010649:Phenylalanine; D015978:Trichosanthin; D001120:Arginine; D007930:Leucine