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

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 ectoapyrase 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 K-m 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.