Data/hora: 09/06/2024 02:59:07

Provedor de dados: | 56
País: | Brazil
Título: | Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate
Autores: | Carreira,A.C.O.; Bastos,C.M.V.; Verjovski-Almeida,S.
Data: | 2007-10-01
Ano: | 2007
Palavras-chave: | Phosphorylation site mutant; Catalytic site; Yeast expression system; Saccharomyces cerevisiae; P-type ATPase.
Resumo: | 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 25ºC 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).
Tipo: | Info:eu-repo/semantics/article
Idioma: | Inglês
Identificador: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2007001000004
Editor: | Associação Brasileira de Divulgação Científica
Relação: | 10.1590/S0100-879X2006005000160
Formato: | text/html
Fonte: | Brazilian Journal of Medical and Biological Research  v.40 n.10 2007
Fonte: | info:eu-repo/semantics/openAccess