Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate.
The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25 degrees C in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).
Carreira
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Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brasil.
Bastos
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Verjovski-Almeida
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D000818:Animals; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D009154:Mutation / Q000235:genetics*; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism
D010710:Phosphates; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases