Metal Fluoride Complexes of plasma membrane calcium pump: Characterization of fluoride-stabilized phosphoenzyme analogues

DE SAUTU, M.; FERREIRA GOMES, M. S.; SAFFIOTI, N. A.; ROSSI, R. C.; ROSSI, J. P. F. C.; MANGIALAVORI I. C.

Sierra de la Ventana

Congreso; SAB 2014; 2014

Sociedad Argentina de Biofísica

The plasma membrane calcium pump (PMCA) belongs to the P-type ATPase family of active cation pumps. According to the conventional E1?E2 theory, E1 and E2 are the high-affinity and low affinity states for Ca2+, respectively. Gating of the ion pathway is coupled to the phosphorylation and dephosphorylation of the ATPase. Phosphoryl transfer from ATP to an Asp residue in the cytoplasmic domain (i.e., E1 to E1P) closes the cytoplasmic gate, and the release of ADP triggers a change in the affinity of the Ca2+ binding sites (i.e., E1P to E2P) opening the luminal gate. Hydrolysis of the aspartylphosphate (E2P to E2) closes the gate. Metal fluorides like magnesium, beryllium, and aluminum fluorides, operate as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogues to the specific phosphoenzyme intermediates. Thus, MgFx, AlFx, and BeFx (MeF) stabilize analogues of the E2P product state (E2MgF4 2-), the E2P transition state (E2AlF4-), and theE2P ground state (E2BeF3-), respectively. Recently, we demonstrated that the inhibition of PMCA Ca2+-ATPase activity by these complexes have different characteristics [1]. Our results show that (1) the apparent velocity constant (kobs) for inhibition of PMCA activity by different fluoride complexes shows fluoride concentration dependence; (2) its dependence differs with each MeF; and (3) Ca2+ reverses PMCA inhibition bythese MeF.