EFFECTS OF CHEMICAL MODIFICATION OF ARGININE RESIDUES OUTSIDE THE ACTIVE-SITE CLEFT OF RICIN A-CHAIN ON ITS RNA N-GLYCOSIDASE ACTIVITY FOR RIBOSOMES

Ricin A-chain, a protein that inactivates ribosomes by a specific RNA N-glycosidase activity, has been shown to be inactivated by chemical m odification of a few arginine residues. When two or fewer arginine res idues in the A-chain were modified with [C-14]phenylglyoxal, arginines at positions of 193, 196, 213, and 234/235 were found to be modified, from amino acid compositions and radioactivities of the modified pept ides that were obtained by cyanogen bromide cleavage followed by trypt ic and chymotryptic digestion. All these arginines have side chains ou tside the active site cleft; the side chain of Arg213 is adjacent to t he edge of the cleft, while other modified arginines are located on th e opposite side of the cleft. Kinetic analysis showed that the modific ation of two arginine residues caused a 8-fold loss in k(cat) with a 3 -fold increase in K-m, suggesting that this modification mainly decrea se the rate of depurination with an additional effect on the affinity for ribosomes. Neither the environment of tryptophan 211 at the bottom of the cleft nor an interaction of adenine with the cleft was changed by this modification, as judged by fluorescence spectroscopy, suggest ing that a conformational change of the catalytic site does not occur upon the modification. These results, taken together with other works, suggest that some of the above arginine residues outside the active s ite cleft may additively contribute to the catalysis of depurination a nd/or the initial formation of the A-chain/ribosome complex.