tailieunhanh - Báo cáo Y học: Introducing Wilson disease mutations into the zinc-transporting P-type ATPase ofEscherichia coli The mutation P634L in theÔhingeÕ motif (GDGXNDXP) perturbs the formation of the E2P state

ZntA, a bacterial zinc-transporting P-type ATPase, is homologous to two human ATPases mutated in Menkes and Wilson diseases. To explore the roles of the bacterial ATPase residues homologous to those involved in the humandiseases, we have introduced several pointmutations into ZntA. The mutants P401L, D628A and P634L corres-pond to the Wilson disease mutations P992L, D1267A and P1273L, respectively. | Eur. J. Biochem. 269 1579-1586 2002 FEBS 2002 Introducing Wilson disease mutations into the zinc-transporting P-type ATPase of Escherichia coli The mutation P634L in the hinge motif GDGXNDXP perturbs the formation of the E2P state Juha Okkeri Eija Bencomo Marja Pietila and Tuomas Haltia Institute of Biomedical Sciences Biochemistry University of Helsinki Finland ZntA a bacterial zinc-transporting P-type ATPase is homologous to two human ATPases mutated in Menkes and Wilson diseases. To explore the roles of the bacterial ATPase residues homologous to those involved in the human diseases we have introduced several point mutations into ZntA. The mutants P401L D628A and P634L correspond to the Wilson disease mutations P992L D1267A and P1273L respectively. The mutations D628A and P634L are located in the C-terminal part of the phosphorylation domain in the so-called hinge motif conserved in all P-type ATPases. P401L resides near the N-terminal portion of the phosphorylation domain whereas the mutations H475Q and P476L affect the heavy metal ATPase-speci c HP motif in the nucleotide binding domain. All mutants show reduced ATPase activity corresponding 0-37 of the wild-type activity. The mutants P401L H475Q and P476L are poorly phosphorylated by both ATP and Pi. Their dephosphorylation rates are slow. The D628A mutant is inactive and cannot be phosphorylated at all. In contrast the mutant P634L six residues apart in the same domain shows normal phosphorylation by ATP. However phosphorylation by Pi is almost absent. In the absence of added ADP the P634L mutant dephosphorylates much more slowly than the wildtype whereas in the presence of ADP the dephosphorylation rate is faster than that of the wild-type. We conclude that the mutation P634L affects the conversion between the states E P and E2P so that the mutant favors the E1 or E P state. Keywords P-type ATPase Wilson disease mutation heavy metal transport hinge motif ion translocation. P-Type ATPases form a large .

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