Calcium dependence of Pi phosphorylation of sarcoplasmic reticulum Ca2+-ATPase at low water content: water dependence of the E2-->E1 conversion.
Enzymes entrapped in reverse micelles can be studied in low-water environments that have the potential of restricting conformational mobility in specific steps of the reaction cycle. Sarcoplasmic reticulum Ca2+-ATPase was incorporated into a reverse-micelle system (TPT) composed of toluene, phospholipids, Triton X-100 and varying amounts of water (0.5-7%, v/v). Phosphorylation of the Ca2+-ATPase by ATP required the presence of both water and Ca2+ in the micelles. No phosphoenzyme (EP) was detected in the presence of EGTA. Phosphorylation by Pi (inorganic phosphate) in the absence of Ca2+ was observed at water content below that necessary for phosphorylation by ATP. In contrast to what is observed in a totally aqueous medium, EP formed by Pi was partially resistant to dephosphorylation by Ca2+. However, the addition of non-radioactive Pi to the EP already formed caused a rapid decrease in radiolabelled enzymes, as expected for the isotopic dilution, indicating the existence of an equilibrium (E+Pi<-->EP). Phosphorylation by Pi also occurred in TPT containing millimolar Ca2+ concentrations in a range of water concentrations (2-5% v/v). The substrates p-nitrophenyl phosphate, acetyl phosphate, ATP and GTP increased the EP level under these conditions. These results suggest that: (1) the rate of conversion of the ATPase conformer E2 into E1 is greatly reduced at low water content, so that E2-->E1 becomes the rate-limiting step of the catalytic cycle; and (2) in media of low water content, Pi can phosphorylate both E1Ca and E2. Thus, the effect of enzyme hydration is complex and involves changes in the phosphorylation reaction at the catalytic site, in the equilibrium between E2 and E1 conformers, and in their specificity for substrates.
Sodré
C L
CL
Departamento de Bioquímica Medica, ICB/CCS, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro RJ, Brazil.
Scofano
H M
HM
Barrabin
H
H
D000255:Adenosine Triphosphate; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D006160:Guanosine Triphosphate; D008823:Micelles; D009596:Nitrophenols; D010755:Organophosphates; D009943:Organophosphorus Compounds; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014867:Water / Q000494:pharmacology*
D008823:Micelles; D009596:Nitrophenols; D010755:Organophosphates; D009943:Organophosphorus Compounds; D010710:Phosphates; D014867:Water; C008644:nitrophenylphosphate; C011632:acetyl phosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium