There is only one phosphoenzyme intermediate with bound calcium on the reaction pathway of the sarcoplasmic reticulum calcium ATPase.
Identical first-order rate constants for phosphorylation of the calcium ATPase of sarcoplasmic reticulum by bound inorganic phosphate (Pi) of 25 +/- 2 s-1 with empty vesicles and 25 +/- 1 s-1 with vesicles that were passively loaded with 40 mM Ca2+ were obtained by treating the reaction as an approach to equilibrium (4 mM [32P]Pi, 20 mM MgCl2, 10 mM EGTA, and 100 mM KCl at pH 7.0 and 25 degrees C). The formation of ADP-sensitive phosphoenzyme from Pi with Ca(2+)-loaded vesicles also proceeds with a first-order rate constant of 25 s-1 and no detectable induction period. These identical rate constants show that lumenal Ca2+ does not inhibit the rate of phosphorylation of the enzyme by bound Pi and that there is no significant kinetic barrier for the conformational change that converts an ADP-insensitive to an ADP-sensitive phosphoenzyme intermediate with bound Ca2+. We conclude that there is no evidence for the existence of two stable phosphoenzyme intermediates with bound Ca2+, such as E1 approximately P.Mg.Ca2 and Ca2.E2-P.Mg, that are included in the E1-E2 and related two-state models for calcium transport by this enzyme. In general, coupling of a physical reaction, such as muscle contraction or vectorial transport, to a chemical reaction, such as ATP hydrolysis, requires more than two states in the reaction cycle.(ABSTRACT TRUNCATED AT 250 WORDS)
Myung
J
J
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Jencks
W P
WP
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium