Artículo
E2P-like states of plasma membrane Ca2+-ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues
Saffioti, Nicolas Andres
; de Sautu, Marilina
; Ferreira Gomes, Mariela Soledad
; Rossi, Rolando Carlos
; Berlin, Joshua; Rossi, Juan Pablo Francisco
; Mangialavori, Irene Cecilia
Fecha de publicación:
02/2019
Editorial:
Elsevier Science
Revista:
Biochimica et Biophysica Acta - Biomembranes
ISSN:
0005-2736
Idioma:
Inglés
Tipo de recurso:
Artículo publicado
Clasificación temática:
Resumen
The plasma membrane Ca2+‑ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeFx), aluminium fluoride (AlFx) and magnesium fluoride (MgFx) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·Pi product states. Our results show that the phosphate analogues BeFx, AlFx and MgFx inhibit PMCA Ca2+‑ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca2+‑ATPase inhibition by AlFx and BeFx depended on Mg2+ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlFx and BeFx but not MgFx apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E → E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA. In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.
Palabras clave:
METAL FLUORIDES
,
PHOSPHORYLATED STATE
,
PMCA
,
P‑ATPASES
,
REACTION CYCLE
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Articulos(IQUIFIB)
Articulos de INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Articulos de INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Citación
Saffioti, Nicolas Andres; de Sautu, Marilina; Ferreira Gomes, Mariela Soledad; Rossi, Rolando Carlos; Berlin, Joshua; et al.; E2P-like states of plasma membrane Ca2+-ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1861; 2; 2-2019; 366-379
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