Effect of magnesium on the calcium-dependent transient kinetics of sarcoplasmic reticulum ATPase, studied by stopped flow fluorescence and phosphorylation.
At pH 7 and 20 degrees C and in the absence of potassium and magnesium, the intrinsic fluorescence rise after addition of calcium to a calcium-deprived enzyme was monoexponential. On the other hand, when the calcium-deprived enzyme was preincubated with magnesium, this fluorescence rise was clearly biphasic at high calcium concentrations. For a constant magnesium concentration (higher than millimolar), the rate constant of the slow phase and the amplitude of the fast phase rose for the same range of calcium concentrations, between pCa 5 and 4. At pH 6, fluorescence signals were monophasic even with 20 mM Mg2+. We also found that at pH 7, phosphorylation of the enzyme after simultaneous addition of calcium, magnesium, and ATP was faster when sarcoplasmic reticulum was originally calcium-deprived in the presence of magnesium. At pH 6, on the contrary, preincubation with magnesium did not influence the phosphorylation time course. It is concluded that (a) magnesium produces an enzyme conformation which is able to react with calcium and ATP as required by the kinetics of the ATPase reaction; (b) the magnesium-enzyme complex presents one readily accessible site of relatively low affinity (Kd congruent to 25 microM) for calcium; (c) occupancy of this site by calcium triggers a relatively slow (4-6 s-1) conformational change unmasking a second high affinity site; (d) interaction between and occupancy of these two calcium sites occur with positive cooperativity and yield enzyme activation; and (e) the kinetic difference between the low time constant of the second component of fluorescence rise following addition of calcium (in the absence of ATP) and the relatively fast phosphorylation obtained in the same conditions (but in the presence of ATP) is attributed to an Mg2+-dependent accelerating effect of ATP on enzyme isomerization.
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D000252:Calcium-Transporting ATPases; D008274:Magnesium