Hp. Adamo et al., MUTANTS IN THE PUTATIVE NUCLEOTIDE-BINDING REGION OF THE PLASMA-MEMBRANE CA2-PUMP - A REDUCTION IN ACTIVITY DUE TO SLOW DEPHOSPHORYLATION(), The Journal of biological chemistry, 270(50), 1995, pp. 30111-30114
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.