A proposal for the Mg2+ binding site of P-type ion motive ATPases and the mechanism of phosphoryl group transfer.
Mutations of D586 in the DPPR sequence of sodium pump decrease the enzyme's affinity for inorganic phosphate [Farley R. A., Heart, E., Kabalin, M., Putnam, D., Wang, K., Kasho, V. N., and Faller, L. D. (1997) Biochemistry 36, 941-951]. Therefore, it was proposed that D586 coordinates the Mg2+ required for catalytic activity. This hypothesis is tested (1) by determining the substrate for catalysis of 18O exchange between inorganic phosphate and water and (2) by comparing conserved amino acid sequences in P-type pumps with the primary structures of enzymes of known tertiary structure that catalyze phosphoryl group transfer. From the isotope exchange data, it is concluded that the Mg2+-dependent and Na+- and K+-stimulated ATPase binds Mg2+ before inorganic phosphate. Sequence homology is demonstrated between the conserved DPPR and MV(I,L)TGD sequences of P-type pumps and two conserved adenylate kinase sequences that coordinate Mg2+ and/or bind nucleotide in the crystal structure of the yeast enzyme. A model for the Mg2+ site of P-type pumps and the mechanism of phosphoryl group transfer is proposed and tested by demonstrating that the conserved sequences are also structurally homologous.
Kasho
V N
VN
Department of Medicine, University of California at Los Angeles School of Medicine, USA.
Stengelin
M
M
Smirnova
I N
IN
Faller
L D
LD
D000249:Adenosine Monophosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009154:Mutation; D010104:Oxygen Radioisotopes; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013552:Swine; D014867:Water / Q000378:metabolism
D010104:Oxygen Radioisotopes; D010710:Phosphates; D014867:Water; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium