Two serine residues control sequential steps during catalysis of the yeast copper ATPase through different mechanisms that involve kinase-mediated phosphorylations |
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Authors: | Valverde Rafael H F Britto-Borges Thiago Lowe Jennifer Einicker-Lamas Marcelo Mintz Elisabeth Cuillel Martine Vieyra Adalberto |
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Affiliation: | Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, Brazil. |
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Abstract: | 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 (Cu)E~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). PKAi(5-24) 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 Ser(971) 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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Keywords: | ATPases Copper Protein Kinases Protein Phosphorylation Yeast Metabolism Cell Signaling Yeast Copper ATPase |
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