Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes.
The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.
Frankel
A
A
Florida Hospital Cancer, Altamonte Springs.
Welsh
P
P
Richardson
J
J
Robertus
J D
JD
D000818:Animals; D001120:Arginine; D004926:Escherichia coli / Q000235:genetics*; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D014176:Protein Biosynthesis / Q000187:drug effects; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects* / Q000378:metabolism; D012276:Ricin / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development
D005971:Glutamates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012276:Ricin; D001120:Arginine