Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 3 isoforms by steady-state and transient kinetic analyses

Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca2+-transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca2+ in activation of the overall reaction due to a decreased E-2 to E1Ca2 transition rate and an increased rate of Ca2+ dissociation from E1Ca2. At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca2+ level (either an enhanced efflux of lumenal Ca2+ through the pump in E-2 form or insensitivity to inhibition by lumenal Ca2+). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca2+ and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E2P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.