The C-terminal cysteine-rich region dictates specific catalytic propertiesin chimeras of the ectonucleotidases NTPDase1 and NTPDase2

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) comprise a no vel family of ectonucleotidases that are important in the hydrolysis of ext racellular nucleotides. The related NTPDase1 (ecto-apyrase) and NTPDase2 (e cto-ATPase) share a common membrane topography with a transmembrane domain at both the N- and C-terminus, an extensive extracellular loop with five 'a pyrase conserved regions' (ACR1 to ACR5), and a cysteine-rich C-terminal re gion. Whereas NTPDase1 expressed in CHO cells hydrolyzes ATP and ADP equiva lently, NTPDase2 has a high preference for the hydrolysis of ATP over ADP. In addition recombinant NTPDase1 hydrolyzes ATP to AMP with the formation o f only minor amounts of free ADP. In contrast, ADP appears as the major fre e product when ATP is hydrolyzed by NTPDase2. In order to determine molecul ar domains responsible for these differences in catalytic properties, chime ric cDNAs were constructed in which N-terminal sequences of increasing leng th of NTPDase1 were substituted by the corresponding sequences of NTPDase2 and vice versa. The turnover points were contained within ACR1 to ACR5. Chi meric cDNAs were expressed in CHO cells and surface expression was verified by immunocytochemistry. ATP and ADP hydrolysis rates and ADP and AMP produ ct formation were determined using HPLC. Amino-acid residues between ACR3 a nd ACR5 and in particular the cysteine-rich region between ACR4 and ACR5 co nferred a phenotype to the chimeric enzymes that corresponded to the respec tive wild-type enzyme. Protein structure rather than the conserved ACRs may be of major relevance for determining differences in the catalytic propert ies between the related wild-type enzymes.