Amino acid specific ADP-ribosylation: substrate specificity of an ADP-ribosylarginine hydrolase from turkey erythrocytes

Abstract

An ADP-ribosylarginine hydrolase, which catalyzes the degradation of ADP-ribosyl[14C]arginine to ADP-ribose plus arginine, was separated by ion exchange, hydrophobic, and gel permation chromatography from NAD:arginine ADP-ribosyltransferases, which are responsible for the stereospecific formation of alpha-ADP-ribosylarginine. As determined by NMR, the specific substrate for the hydrolase was alpha-ADP-ribosylarginine, the product of the transferase reaction. The ADP-ribose moiety was critical for substrate recognition; (phosphoribosyl) [14C]arginine and ribosyl[14C]arginine were poor substrates and did not significantly inhibit ADP-ribosyl[14C]arginine degradation. In contrast, ADP-ribose was a potent inhibitor of the hydrolase and significantly more active than ADP greater than AMP greater than adenosine. In addition to ADP-ribosyl[14C]arginine, both ADP-ribosyl[14C]guanidine and (2'-phospho-ADP-ribosyl)[14C]arginine were also substrates; at pH greater than 7, ADP-ribosyl[14C]guanidine was degraded more readily than the [14C]arginine derivative. Neither arginine, guanidine, nor agmatine, an arginine analogue, was an effective hydrolase inhibitor. Thus, it appears that the ADP-ribosyl moiety but not the arginine group is critical for substrate recognition. Although the hydrolase requires thiol for activity, dithiothreitol accelerated loss of activity during incubation at 37 degrees C. Stability was enhanced by Mg2+, which is also necessary for optimal enzymatic activity. The findings in this paper are consistent with the conclusion that different enzymes catalyze ADP-ribosylarginine synthesis and degradation. Furthermore, since the hydrolase and transferases possess a compatible stereospecificity and substrate specificity, it would appear that the two enzymatic activities may serve as opposing arms in an ADP-ribosylation cycle.

Hydrolysis of O-Acetyl-ADP-ribose Isomers by ADP-ribosylhydrolase 3

“…In addition, mammalian cells possess hydrolases that cleave the ADPr-protein linkage, releasing ADPr and regenerating the unmodified protein (5,6). An ADP-ribosyl(arginine) hydrolase, termed ARH1, catalyzes in a stereospecific manner, hydrolysis of the ␣-linkage of arginine-ribose found in ADP-ribosyl(arginine)-protein to ADPr and (arginine)-protein (7,8), consistent with the regulation of ADP-ribosyl(arginine)-protein levels by opposing activities of transferases and hydrolases, participating in an ADP-ribosylation cycle (4,9).…”

“…As noted above, ARH1 catalyzes the hydrolysis of ADP-ribose-arginine and also hydrolyzes ADP-ribose linkages to guanidine. The reaction is stereospecific, and only the ␣-anomer at the C-1Љ position of ADP-ribose-arginine is hydrolyzed (8). ARH1 also hydrolyzed the stereospecific C-1Љ-C-2Ј linkage in poly(ADP-ribose).…”

“…The ARH1 reaction is inhibited by ADP-ribose, but not by phosphoribose, suggesting that the catalytic site recognizes the adenosine moiety (8). The fact that only the guanidine group of arginine is necessary for hydrolysis further supports a critical role for the ADP-ribose as opposed to the arginine in catalysis (8). ARH2, which has considerable structural similarity to ARH1, has no reported activity.…”

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

“…1). The hydrolases only cleave the ␣-anomer of the ADP- ribose linked amino acid, the stereospecific product of the transferase reaction (13).…”

Purification, Characterization, and Localization of an ADP-ribosylactin Hydrolase That Uses ADP-ribosylated Actin from Rat Brains as a Substrate

“…Little is known about the de-ADP-ribosylation of modified proteins. An ADP-ribosylprotein hydrolase that hydrolyzes ADPribosylated arginine has been purified from turkey erythrocytes (18). The immunoreactivity of certain proteins was identified by the use of a rat ADP-ribosylhydrolase antibody found in the most abundance in the brain, spleen, and testis (19).…”