Critical role of Val-304 in conformational transitions that allow Ca2+ occlusion and phosphoenzyme turnover in the Ca2+ transport ATPase.
Site-directed mutations were produced in the distal segments of the Ca(2+)-ATPase (SERCA) transmembrane region. Mutations of Arg-290 (M3-M4 loop), Lys-958, and Thr-960 (M9 - M10 loop) had minor effects on ATPase activity and Ca(2+) transport. On the other hand, Val-304 (M4) mutations to Ile, Thr, Lys, Ala, or Glu inhibited transport by 90-95% while reducing ATP hydrolysis by 83% (Ile, Thr, and Lys), 56% (Ala), or 45% (Glu). Val-304 participates in Ca(2+) coordination with its main-chain carbonyl oxygen, and this function is not expected to be altered by mutations of its side chain. In fact, despite turnover inhibition, the Ca(2+) concentration dependence of residual ATPase activity remained unchanged in Val-304 mutants. However, the rates (but not the final levels) of phosphoenzyme formation, as well the rates of its hydrolytic cleavage, were reduced in proportion to the ATPase activity. Furthermore, with the Val-304 --> Glu mutant, which retained the highest residual ATPase activity, it was possible to show that occlusion of bound Ca(2+) was also impaired, thereby explaining the stronger inhibition of Ca(2+) transport relative to ATPase activity. The effects of Val-304 mutations on phosphoenzyme turnover are attributed to interference with mechanical links that couple movements of transmembrane segments and headpiece domains. The effects of thermal activation energy on reaction rates are thereby reduced. Furthermore, inadequate occlusion of bound Ca(2+) following utilization of ATP in Val-304 side-chain mutations is attributed to inadequate stabilization of the Glu-309 side chain and consequent defect of its gating function.
California Pacific Medical Center Research Institute, San Francisco, California 94107.
Cheng
C
Xu
California Pacific Medical Center Research Institute, San Francisco, California 94107.
Anand Mohan
AM
Prasad
California Pacific Medical Center Research Institute, San Francisco, California 94107. Electronic address: ginesi@CPMCRI.com.
Giuseppe
G
Inesi
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.
Chikashi
C
Toyoshima
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D007532:Isoleucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D014633:Valine / Q000737:chemistry*
D007532:Isoleucine; D013912:Threonine; D000251:Adenosine Triphosphatases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506943:ATP2A1 protein, human; D014633:Valine; D008239:Lysine; D002118:Calcium