title: Site-directed mutagenesis of a human brain ecto-apyrase: evidence that the E-type ATPases are related to the actin/heat shock 70/sugar kinase superfamily.
abstract: On the basis of sequence homologies observed between members of the E-type ATPases and the phosphate binding motifs of the actin/heat shock protein 70/sugar kinase superfamily, a human ecto-apyrase was analyzed by site-directed mutagenesis of conserved amino acids in apyrase conserved regions (ACR) I and IV. The expressed proteins were analyzed to assess the significance of these amino acids. A conserved aspartic acid residue in ACR IV was mutated to alanine, asparagine, and glutamic acid, and the relative activity and Km for ATP and ADP were determined. Mutation of this Asp 219 to Ala or Asn yielded an enzyme severely reduced in ATP hydrolyzing activity (>90%) and completely devoid of ADPase activity, along with a similar extent of inhibition of hydrolysis of other nucleoside di- and triphosphates. Interestingly, mutation of Asp 219 to Glu completely restored the ability of the enzyme to hydrolyze nucleoside triphosphates at levels above that of the wild-type enzyme, while the ability to hydrolyze nucleoside diphosphates was slightly reduced. Mutation of a second conserved aspartic acid in ACR I (Asp 62) and two invariant glycine residues in both ACR I (Gly 64) and ACR IV (Gly 221) also severely disrupted nucleotidase activity. These results demonstrate that the E-type ATPases contain the nucleoside phosphate binding domains present in the actin/heat shock protein/sugar kinase superfamily. Together with analysis of computer-predicted secondary structures, the results suggest that the ecto-ATPases and ecto-apyrases are part of, or closely related to, the actin superfamily of proteins.
authors.affiliation: Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Ohio 45267-0575, USA.
authors.forename: T M
authors.initials: TM
authors.lastname: Smith
authors.forename: T L
authors.initials: TL
authors.lastname: Kirley
mesh_terms: D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D019556:COS Cells; D018076:DNA, Complementary / Q000096:biosynthesis; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D017433:Protein Structure, Secondary; D014162:Transfection
chemical_list: D000199:Actins; D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D000251:Adenosine Triphosphatases; C027201:ectoATPase