Manganese as a cosubstrate for the phosphorylation of the sarcoplasmic reticulum Ca-dependent adenosine triphosphatase with orthophosphate.
The phosphorylation of the sarcoplasmic reticulum Ca-ATPase (EC 3.6.1.38) with P(i) was characterized using Mn as a Mg analogue. Steady state and transient fluorescence and radioisotopic techniques were used; the affinities of Mn and P(i) for the enzyme and the rate constants of the phosphorylation and dephosphorylation reactions were determined, under several conditions. The reactions were carried out at pH 5.5 to minimize the binding of contaminant Ca to the transport sites, thus avoiding the use of Ca chelators. The apparent affinity of Mn binding at low [Mn] is larger in the absence of P(i) (35 microM) than in the presence of saturating P(i) (70 microM). On the contrary, the apparent affinity of Mn for the formation of the phosphoenzyme increases, from 1.5 mM to 0.15 mM, upon increasing [P(i)] in the millimolar range. The apparent affinty of P(i) for the formation of the phosphoenzyme also increases, from 2.2 mM to 0.2 mM, upon increasing [Mn] in the millimolar range. The equilibrium of the phosphoenzyme with the noncovalent Mn.P(i). Enzyme complex favors the covalent species. The simulation of a reaction model including the random binding of 2 Mn and I P(i) per mol of ATPase and a noncovalent complex in equilibrium with the phosphoenzyme, using a set of equilibrium constants deduced from the results, agree with the experimental data.
González
D A
DA
Cátedra de Biofísica, Facultad de Odontología, Universidad de Buenos Aires, Argentina. debora@biofis.odon.uba.ar
Alonso
G L
GL
Lacapère
J J
JJ
D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008345:Manganese / Q000378:metabolism* / Q000494:pharmacology; D008956:Models, Chemical; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D010710:Phosphates; D008345:Manganese; D000252:Calcium-Transporting ATPases