Cation-transporting
P-type ATPases comprise a major
membrane protein family, the members of which are found in eukaryotes, eubacteria, and archaea. A phylogenetically old branch of the
P-type ATPase family is involved in the transport of
heavy-metal ions such as
copper,
silver,
cadmium, and
zinc. In humans, two homologous
P-type ATPases transport
copper. Mutations in the human
proteins cause disorders of
copper metabolism known as Wilson and
Menkes diseases. E. coli possesses two genes for
heavy-metal translocating
P-type ATPases. We have constructed an expression system for one of them, ZntA, which encodes a 732
amino acid residue
protein capable of transporting Zn(2+). A
vanadate-sensitive, Zn(2+)-dependent
ATPase activity is present in the membrane fraction of our expression strain. In addition to Zn(2+), the
heavy-metal ions Cd(2+), Pb(2+), and Ag(+) activate the
ATPase. Incubation of membranes from the expression strain with [gamma-(33)P]
ATP in the presence of Zn(2+), Cd(2+), or Pb(2+) brings about phosphorylation of two
membrane proteins with molecular masses of approximately 90 and 190 kDa, most likely representing the ZntA monomer and dimer, respectively. Although Cu(2+) can stimulate phosphorylation by [gamma-(33)P]
ATP, it does not activate the
ATPase. Cu(2+) also prevents the Zn(2+) activation of the
ATPase when present in 2-fold excess over Zn(2+). Ag(+) and Cu(+) appear not to promote phosphorylation of the
enzyme. To study the effects of
Wilson disease mutations, we have constructed two site-directed mutants of ZntA, His475Gln and Glu470Ala, the human counterparts of which cause
Wilson disease. Both mutants show a reduced
metal ion stimulated
ATPase activity (about 30-40% of the wild-type activity) and are phosphorylated much less efficiently by [gamma-(33)P]
ATP than the wild type. In comparison to the wild type, the Glu470Ala mutant is phosphorylated more strongly by [(33)P]P(i), whereas the His475Gln mutant is phosphorylated more weakly. These results suggest that the mutation His475Gln affects the reaction with
ATP and P(i) and stabilizes the
enzyme in a dephosphorylated state. The Glu470Ala mutant seems to favor the E2 state. We conclude that His475 and Glu470 play important roles in the transport cycles of both the
Wilson disease ATPase and ZntA.