ADP-ribosylarginine glycohydrolase catalyzing the release of ADP-ribose from the cholera toxin-modified alpha-subunits of GTP-binding proteins.
A rat glycohydrolase which catalyzes the hydrolysis of ADP-ribosylarginine was expressed in Escherichia coli and purified to homogeneity for characterization of its enzymatic properties. The purified glycohydrolase catalyzed the hydrolysis of N-glycoside linked ADP-ribosylarginine on the alpha-subunits of stimulatory GTP-binding proteins (Gs) and cholera toxin A1-subunit that had been modified by cholera toxin and NAD. Nonmuscle actin of which an arginine residue was ADP-ribosylated by botulinum C2 toxin also served as a substrate of the glycohydrolase. On the other hand, the glycohydrolase did not hydrolyze ADP-ribosylated cysteine on the alpha-subunits of pertussis toxin-substrate GTP-binding proteins, ADP-ribosylated diphthamide on elongation factor 2, or ADP-ribosylated asparagine on rho GTP-binding proteins. The rate of the reaction catalyzed by the glycohydrolase was affected by nucleotide-binding form of the ADP-ribosylated substrate proteins; the GDP-bound form of the modified Gs-alpha was more rapidly hydrolyzed than the guanosine 5'-(3-O-thio)triphosphate-bound form. Interestingly, the glycohydrolase activity was markedly inhibited by mM order concentration of ATP in addition to ADP-ribose, the product of the enzyme reaction, though ADP had no inhibitory effect on the activity. Moreover, alpha NAD, but not beta NAD, inhibited the enzyme activity, suggesting that the glycohydrolase reaction was stereospecific for the alpha-anomer.
Maehama
T
T
Department of Life Science, Tokyo Institute of Technology, Yokohama.
Nishina
H
H
Katada
T
T
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001483:Base Sequence; D002772:Cholera Toxin; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006026:Glycoside Hydrolases; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D015398:Signal Transduction; D013379:Substrate Specificity
D000227:Adenine Nucleotides; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D006867:Hydrolases; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C046422:ADP-ribosylarginine hydrolase; D019204:GTP-Binding Proteins