Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca2+-ATPase with Occluded Ca2+ by Beryllium Fluoride: STRUCTURAL CHANGES DURING PHOSPHORYLATION AND ISOMERIZATION*

AUTOR(ES)

Danko, Stefania

FONTE

American Society for Biochemistry and Molecular Biology

RESUMO

As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca2+-ATPase E1PCa2·Mg, a complex of E1Ca2·BeFx, was successfully developed by addition of beryllium fluoride and Mg2+ to the Ca2+-bound state, E1Ca2. In E1Ca2·BeFx, most probably E1Ca2·BeF3−, two Ca2+ are occluded at high affinity transport sites, its formation required Mg2+ binding at the catalytic site, and ADP decomposed it to E1Ca2, as in E1PCa2·Mg. Organization of cytoplasmic domains in E1Ca2·BeFx was revealed to be intermediate between those in E1Ca2·AlF4− ADP (transition state of E1PCa2 formation) and E2·BeF3−·(ADP-insensitive phosphorylated intermediate E2P·Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca2·BeFx in contrast to no superfluorescence of TNP-AMP bound to E1Ca2·AlFx. E1Ca2·BeFx with bound TNP-AMP slowly decayed to E1Ca2, being distinct from the superfluorescent complex of TNP-AMP with E2·BeF3−, which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca2·BeFx mimics E1PCa2·Mg, and between those of E1Ca2·AlF4−·ADP and E2·BeF3−. E1Ca2·BeFx at low 50–100 μm Ca2+ was converted slowly to E2·BeF3− releasing Ca2+, mimicking E1PCa2·Mg → E2P·Mg + 2Ca2+. Ca2+ replacement of Mg2+ at the catalytic site at approximately millimolar high Ca2+ decomposed E1Ca2·BeFx to E1Ca2. Notably, E1Ca2·BeFx was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca2+ with 15 mm Mg2+. Also, stable E1Ca2·BeFx was produced from E2·BeF3− at 0.7 mm lumenal Ca2+ by binding two Ca2+ to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P· Mg + 2Ca2+ → E1PCa2·Mg.

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