Sarcoplasmic reticulum (SR) Ca²⁺-ATPase of rabbit fast twitch skeletal muscle is an ion-translocating ATPase of P-type, which mediates active uptake of Ca²⁺ from the cytoplasm to the endoplasmic reticulum lumen, thereby allowing rapid oscillation of the cytoplasmic Ca²⁺ concentration during cellular activation events. Site-specific mutagenesis was used to replace individually W107 or W967, located in transmembrane domains of SR Ca²⁺-ATPase, with either alanine (A) or phenylalanine (F). Mutant enzymes were expressed in Saccharomyces cerevisiae cells under the control of galactose promoter. Mutation W107F resulted in an increase in ATP hydrolysis (230%), whereas W107A showed only 20% of the wild-type turnover activity. However, both of them retained the capacity to form a phosphoenzyme intermediate from ATP (in the presence of Ca²⁺) and from Pi (in the absence of Ca²⁺) at wild-type levels. In W107A, apparent Ca²⁺ affinity measured in the phosphorylation assays with ATP was higher (pCa 5.3) than that of the wild-type (pCa 4). Analysis of the functional changes in W107 mutants in light of the high resolution structures of the Ca²⁺-ATPase, suggests that W107 (M2 helix) can be intimately related to residues in the possible pathway to the Ca²⁺ sites, in a negatively charged tunnel between M1, M2 and M3, as described for the E2 (TG complex) structure. Mutation to the other tryptophan analyzed in this work, W967, had a profound impact on the function of Ca²⁺-ATPase. Mutants were characterized by a reduced ATPase activity and a drastic reduction in the ability to form a phosphoenzyme intermediate from ATP and Pi. Trypsinolysis of W967 mutants identified a higher resistance to digestion than wild-type indicating they are in an overall correct folding. In the Ca²⁺-ATPase crystal structures, W967 residue is located in the M10 transmembrane helix and participates in a hydrogen bond network with Y949 in the M9 transmembrane segment. Interference with this hydrogen bond may constitute the reason for the observed effects of amino acid substitutions in W967 mutants on the ATPase activity and phosphoenzyme formation. This intramembrane interaction may be essential to interhelical packing in the Ca²⁺-ATPase, important for its stability and proper function. In conclusion, this work has identified two tryptophan residues that play a significant role in the catalytic cycle, providing new insights into the mechanism of Ca²⁺-ATPase.

    Supported  by  FAPESP and  CNPq.