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Two serine residues control sequential steps during catalysis of the yeast Copper ATPase through different mechanisms that involve Kinase-mediatedPhosphorylations
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Edité par CCSD ; American Society for Biochemistry and Molecular Biology -
International audience. Ccc2, the yeast copper-transporting ATPase, pumps copper from the cytosol to the Golgi lumen. During its catalytic cycle, Ccc2 undergoes auto-phosphorylation on Asp$^{627}$ and uses the energy gained to transport copper across the cell membrane. We previously demonstrated that cAMP-dependent protein kinase (PKA) controls the energy interconversion CuE∼P → E-P + Cu when Ser$^{258}$ is phosphorylated. We now demonstrate that Ser$^{258}$ is essential in vivo for copper homeostasis in extremely low copper and iron concentrations. The S258A mutation abrogates all PKA-mediated phosphorylations of Ccc2, whereas the S971A mutation leads to a 100% increase in its global regulatory phosphorylation. With S258A, the first-order rate constant of catalytic phosphorylation by ATP decreases from 0.057 to 0.030 s−1, with an 8-fold decrease in the burst of initial phosphorylation. With the S971A mutant, the rate constant decreases to 0.007 s−1. PKAi5–24 decreases the amount of the aspartylphosphate intermediate (EP) in Ccc2 wt by 50% within 1 min, but not in S258A, S971A, or S258A/S971A. The increase of the initial burst and the extremely slow phosphorylation when the “phosphomimetic” mutant S258D was assayed (k = 0.0036 s−1), indicate that electrostatic and conformational (non-electrostatic) mechanisms are involved in the regulatory role of Ser$^{258}$. Accumulation of an ADP-insensitive form in S971A demonstrates that Ser971 is required to accelerate the hydrolysis of the E-P form during turnover. We propose that Ser$^{258}$ and Ser$^{971}$ are under long-range intramolecular, reciprocal and concerted control, in a sequential process that is crucial for catalysis and copper transport in the yeast copper ATPase.
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