Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.
Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.
Musi
Elgilda
E
Thrombosis Research Laboratory, Research Service, Room 13026W, VA New York Harbor Healthcare System, 423 East 23rd Street, New York, NY 10010-5050, USA.
Islam
Naziba
N
Drosopoulos
Joan H F
JH
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001081:Apyrase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
D015703:Antigens, CD; D008565:Membrane Proteins; D001081:Apyrase; C067869:CD39 antigen