title: Substitution of His59 converts CD39 apyrase into an ADPase in a quaternary structure dependent manner.
abstract: The two transmembrane domains of CD39 ecto-apyrase regulate the formation of fully active homotetramers. We show that mutations in apyrase conserved region 1 (ACR1) have two dramatically different sets of effects determined by whether they occur in intact tetramers or in disrupted tetramers or monomers. In intact tetramers, substitution of H59 in the rat brain CD39 ACR1 with G or S abolishes more than 90% of the ATPase activity but less than 50% of the ADPase activity, converting the enzyme into an ADPase with relative ADP:ATP hydrolysis rates of 6:1 or 8:1, respectively. In contrast, the same substitutions in tetramers lacking either transmembrane domain, in monomers lacking both transmembrane domains, or in detergent-solubilized full-length monomers have no effect on ATPase activity and increase ADPase activity approximately 2-fold, resulting in equal ATPase and ADPase activities. N61R substitution has a much smaller effect on the ADPase:ATPase ratio in both cases. While the data for truncated and monomeric constructs are consistent with the proposed role of ACR1 as the beta-phosphate binding domain by analogy with the actin/hsp70/hexokinase superfamily, the finding that H59 substitutions in full-length CD39 primarily diminish the ATP hydrolysis rate suggests that ACR1 may play a different role in intact tetramers. We propose that CD39 uses different ATPase and ADPase mechanisms in different quaternary structure contexts, and that H59 in ACR1 plays a central role specifically in ATP hydrolysis in intact tetramers.
authors.affiliation: Department of Molecular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: A
authors.initials: A
authors.lastname: Grinthal
authors.forename: G
authors.initials: G
authors.lastname: Guidotti
mesh_terms: D000251:Adenosine Triphosphatases; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics*; D001081:Apyrase / Q000737:chemistry* / Q000235:genetics*; D001216:Asparagine / Q000235:genetics; D019556:COS Cells; D017124:Conserved Sequence; D003902:Detergents; D004789:Enzyme Activation / Q000235:genetics; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017384:Sequence Deletion; D012995:Solubility; D013329:Structure-Activity Relationship
chemical_list: D015703:Antigens, CD; D003902:Detergents; D008565:Membrane Proteins; D010446:Peptide Fragments; D006639:Histidine; D001216:Asparagine; D000251:Adenosine Triphosphatases; D001081:Apyrase; C067869:CD39 antigen