Phosphorylation and reaction intermediates of the prokaryotic Ca(2+)-ATPase.
A P-type Ca(2+)-ATPase activity has been identified previously in membranes of the Gram-negative bacteria Flavobacterium odoratum. The reaction mechanism of the prokaryotic Ca(2+)-ATPase was examined by isolation of reaction cycle intermediates formed during reversal of the normal cycle using inorganic P(i). The eukaryotic ATPase can be directly phosphorylated by P(i) only in the absence of calcium, and half-maximal inhibition is observed at 1-2 microM calcium. The F. odoratum Ca(2+)-ATPase is also phosphorylated by P(i) in the absence of calcium, but in contrast to the eukaryotic pump, a 30-35-fold increase in phosphoenzyme (E-P) formation is observed when the enzyme is preincubated in millimolar calcium. Furthermore, the half-maximal activation of E-P formation is observed at approximately 100 microM calcium, similar to the value for low-affinity calcium binding in the sarcoplasmic reticulum Ca(2+)-ATPase. The data suggest that the prokaryotic Ca(2+)-ATPase forward reaction is ordered and that the prokaryotic enzyme does not appear to bind calcium at the high-affinity site until ATP binds. Thus, calcium does not inhibit phosphoenzyme formation by P(i) because the high-affinity Ca2+ binding site is not exposed on the free enzyme (E1).
Department of Medicine, Medical University of South Carolina, Charleston 29425.
A M
AM
Gambel
D R
DR
Menick
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005417:Flavobacterium / Q000201:enzymology*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014461:Ultracentrifugation
D006898:Hydroxylamines; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D019811:Hydroxylamine; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium