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 modification of a few arginine residues. When two or fewer arginine residues in the A-chain were modified with [14C]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 peptides that were obtained by cyanogen bromide cleavage followed by tryptic and chymotryptic digestion. All these arginines have side chains outside the active site cleft; the side chain of Arg213 is adjacent to the edge of the cleft, while other modified arginines are located on the opposite side of the cleft. Kinetic analysis showed that the modification of two arginine residues caused a 8-fold loss in kcat with a 3-fold increase in Km, suggesting that this modification mainly decrease 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, suggesting 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 site cleft may additively contribute to the catalysis of depurination and/or the initial formation of the A-chain/ribosome complex.
Watanabe
K
K
Department of Applied Biological Sciences, Saga University, Japan.
Dansako
H
H
Asada
N
N
Sakai
M
M
Funatsu
G
G
D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D003488:Cyanogen Bromide; D007700:Kinetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D054788:Ribosome Inactivating Proteins; D012270:Ribosomes / Q000378:metabolism*; D012276:Ricin / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D012276:Ricin; D001120:Arginine; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins; D002918:Chymotrypsin; D014357:Trypsin; D000225:Adenine; D003488:Cyanogen Bromide