Mutational analysis of the conserved TGES loop of sarcoplasmic reticulum Ca2+-ATPase

Crystal structures have shown that the conserved TGES loop of the Ca2+-ATPase is isolated in the Ca(2)E1 state but becomes inserted in the catalytic site in E2 states. Here, we have examined the kinetics of the partial reaction steps of the transport cycle and the binding of the phosphoryl analogs BeF, AlF, MgF, and vanadate in mutants with alterations to the TGES residues. The mutations encompassed variation of size, polarity, and charge of the side chains. Differential effects on the Ca(2)E1P -> E2P, E2P -> E2, and E2 -> Ca(2)E1 reactions and the binding of the phosphoryl analogs were observed. In the E183D mutant, the E2P -> E2 dephosphorylation reaction proceeded at a rate as high as one-third that of the wild type, whereas it was very slow in the other Glu(183) mutants, including E183Q, thus demonstrating the need for a negatively charged carboxylate group to catalyze dephosphorylation. By contrast, the Ca(2)E1P -> E2P transition was accomplished at a reasonable rate with glutamine in place of Glu(183), but not with aspartate, indicating that the length of the Glu(183) side chain, in addition to its hydrogen bonding potential, is critical for Ca2E1P -> E2P. This transition was also slowed in mutants with alteration to other TGES residues. The data provide functional evidence in support of the proposed role of Glu(183) in activating the water molecule involved in the E2P -> E2 dephosphorylation and suggest a direct participation of the side chains of the TGES loop in the control and facilitation of the insertion of the loop in the catalytic site. The interactions of the TGES loop furthermore seem to facilitate its disengagement from the catalytic site during the E2 -> Ca(2)E1 transition.