UC San Diego

Nucleoside triphosphate diphosphohydrolases (NTPDases) are ubiquitous enzymes hydrolyzing NTPs and/or NDPs. The human NTPDase 2 is a cell-surface NTPDase containing a large extracellular domain (ECD), two transmembrane domains (TMDs), short cytoplasmic tails at the N- and C-termini, and five apyrase conserved regions (ACRs). Human NTPDase 2, whether in the native tissue or expressed in tissue culture cells, displays several unique characteristics: inactivation by detergents, temperature, and substrate, and inhibition by pCMPS. These inhibitory effects are overcome by concanavalin A or glutaraldehyde that promote oligomerization. Since detergents and high temperatures perturb membranes, their inhibitory effects on human NTPDase 2 activity are likely mediated through the TMDs. To investigate this possibility, chimeric NTPDase 2 and a soluble human NTPDase 2 consisting of only its ECD were generated. The soluble human NTPDase 2 was no longer inactivated by membrane perturbation. These characteristics were also displayed by the hu-ck ACR1,5 chimera in which the ECD is anchored to the membrane by the two TMDs of the chicken NTPDase 8, which is not affected by membrane perturbation. These results indicate that the strength of interactions of the respective TMD pairs of the human NTPDase 2 and chicken NTPDase 8, which may be dictated by their amino acid sequences, determine their different responses to membrane perturbation. The target of pCMPS modification is a free cysteine residue at position 26 in the N-terminal TMD. A mutant in which C26 was replaced by a serine was no longer inhibited by pCMPS. Inhibition of pCMPS may result from the combined effects of disturbance of TMD interaction and active site conformation, since C26 is situated close to ACR1. While oligomer formation promoted by ConA or glutaraldehyde increases the human NTPDase 2 activity, oxidative cross- linking of wild-type enzyme and mutants containing a single cysteine residue in the C-terminal TMD is accompanied by reduction of ATPase activity. Similar reduction of activity is also obtained upon intramolecular disulfide formation of mutants containing a cysteine residue in both TMDs. These results indicate that mobility of TMDs is necessary for maximal catalysis