Identification of the enzymatic active site of CD38 by site-directed mutagenesis

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca2+ messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Apl ysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia c yclase were mutagenized. Changing Glu226, which corresponded to the catalyt ic residue of the cyclase, to Asp, Asn, Gin, Leu, or Gly eliminated essenti ally all enzymatic activities of CD38, indicating it is most likely the cat alytic residue. Photoaffinity labeling showed that E226G, nevertheless, ret ained substantial NAD binding activity. The secondary structures of these i nactive mutants as measured by circular dichroism were essentially unpertur bed as compared with the wild type. Other nearby residues were also investi gated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADas e activity, respectively. The cADPR hydrolase activity of the two mutants w as similarly reduced. Asp-155, on the other hand, was crucial for the GDP-r ibosyl cyclase activity since its substitution with either Glu, Asn, or Gin stimulated the activity 3-15-fold, whereas other activities remained essen tially unchanged. In addition to these acidic residues, two tryptophans wer e also important, since all enzyme activities of W125F, W125Y, W189G and W1 89Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR h ydrolase activity. Homology modeling revealed all these critical residues a re clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.