A PROPOSAL FOR THE MG2-SITE OF P-TYPE ION MOTIVE ATPASES AND THE MECHANISM OF PHOSPHORYL GROUP-TRANSFER( BINDING)

Mutations of D586 in the DPPR sequence of sodium pump decrease the enz yme's affinity for inorganic phosphate [Farley R. A., Heart, E., Kabal in, M., Putnam, D., Wang, K., Kasho, V. N., and Faller, L. D. (1997) B iochemistry 36, 941-951]. Therefore, it was proposed that D586 coordin ates the Mg2+ required for catalytic activity. This hypothesis is test ed (I)by determining the substrate for catalysis of O-18 exchange betw een 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+-dependen t and Na+- and K+-stimulated ATPase binds Mg2+ before inorganic phosph ate. Sequence homology is demonstrated between the conserved DPPR and MV(I,L)TGD sequences of P-type pumps and two conserved adenylate kinas e 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 pu mps and the mechanism of phosphoryl group transfer is proposed and tes ted by demonstrating that the conserved sequences are also structurall y homologous.