Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase.
CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.
Tohgo
A
A
Department of Biochemistry, Tohoku University School of Medicine, Sendai 980-77, Miyagi, Japan.
Munakata
H
H
Takasawa
S
S
Nata
K
K
Akiyama
T
T
Hayashi
N
N
Okamoto
H
H
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000255:Adenosine Triphosphate / Q000378:metabolism*; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008562:Membrane Glycoproteins; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1; D008239:Lysine