Tohgo A, Munakata H, Takasawa S, Nata K, Akiyama T, Hayashi N, Okamoto H
Department of Biochemistry, Tohoku University School of Medicine, Sendai 980-77, Miyagi, Japan.
J Biol Chem. 1997 Feb 14;272(7):3879-82. doi: 10.1074/jbc.272.7.3879.
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.
CD38不仅催化由NAD⁺形成环ADP核糖（cADPR），还催化cADPR水解为ADP核糖（ADPR），并且ATP抑制这种水解作用（高泽幸男、东古晃、野口直树、小久间隆、名田佳代、杉本智、米仓博、冈本博，（1993年）《生物化学杂志》268卷，26052 - 26054页）。在本研究中，我们使用纯化的重组CD38表明，CD38的cADPR水解酶活性受到ATP的竞争性抑制，其竞争对象是cADPR。为了确定ATP和/或cADPR的结合位点，我们用FSBA标记纯化的CD38。对标记的CD38经赖氨酰内肽酶消化后的片段进行序列分析表明，FSBA标记的残基是Lys - 129。我们引入定点突变，将CD38的Lys - 129分别突变为Ala和Arg。这两种突变体都未被FSBA标记，也不催化cADPR水解为ADPR。此外，突变体不结合cADPR，然而它们仍将NAD⁺用作底物来形成cADPR和ADPR。这些结果表明，CD38的Lys - 129参与cADPR的结合，并且ATP与cADPR竞争结合位点，从而导致CD38的cADPR水解酶活性受到抑制。
