MUTANTS IN THE PUTATIVE NUCLEOTIDE-BINDING REGION OF THE PLASMA-MEMBRANE CA2-PUMP - A REDUCTION IN ACTIVITY DUE TO SLOW DEPHOSPHORYLATION()

Mutants of individual residues of the plasma membrane Ca2+-pump were m ade in the highly conserved region that (in related P-type ATPases) ha s been associated with nucleotide binding, Alteration of the strictly conserved Asp(672) to Glu nearly eliminated the ability of the pump to transport Ca2+, while alteration at Val(674), Arg(675), and Lys(686) reduced the activity, High levels of ATP (25 mhz) did not overcome the reduced activity, indicating that it could not be due to a reduction in the affinity for ATP, Effects not directly related to ATP binding s eemed to result from mutations in this area. For instance, the amount of phosphorylated intermediate in the most severely inhibited mutant, Asp(672)--> Glu, was nearly as high as that in the wild type, a much l arger amount of phosphorylated intermediate than was expected from its low activity, However, the rate of decomposition of this intermediate was much slower than that of the wild type, indicating that the inhib ition of this mutant resulted from an inhibition of the E similar to P --> E step in the enzyme cycle.