Cj. Herscher et al., THE DEPHOSPHORYLATION REACTION OF THE CA2-ATPASE FROM PLASMA-MEMBRANES(), The Journal of biological chemistry, 269(14), 1994, pp. 10400-10406
The breakdown of phosphoenzyme (EP) of the Ca2+-ATPase from pig red bl
ood cell membranes was studied at 37 degrees C by means of a rapid che
mical quenching technique. When the enzyme was phosphorylated with [ga
mma-P-32]ATP in media without added MgCl2, all the EP formed disappear
ed along two single exponential curves, a rapid one with k(app) = 90 /- 10 s(-1) and a slow one with k(app) = 0.7 +/- 0.3 s(-1). The amount
of EP involved in each reaction was close to 50% of the EP present at
the beginning. Only EP of rapid breakdown could account for the stead
y-state hydrolysis of ATP observed under the same experimental conditi
ons. ADP accelerated the slow reaction 45-fold (k(app) = 31 +/- 9 s(-1
)) with K-0.5 = 740 +/- 120 mu M as if this reaction represented the d
ecay of CaE(1)P, which donated its phosphate to water slowly in the fo
rward direction and rapidly to ADP in the reverse direction of the cyc
le. Combination of Mg2+ with K-0.5 = 26.3 +/- 5.0 mu M at a single cla
ss of site in E(1) before phosphorylation increased EP of rapid breakd
own at the expense of ADP-sensitive EP so that, at nonlimiting concent
rations of Mg2+ in the phosphorylation media, all EP decomposed at hig
h rate. Rapid decomposition was observed even with enough CDTA to chel
ate most of the Mg2+ remaining from phosphorylation, suggesting that t
he role of Mg2+ during dephosphorylation was to accelerate the transit
ion CaE(1)P --> CaE(2)P, preparing EP for hydrolysis. The combination
of ATP at a single class of site with K-m = 845 +/- 231 mu M accelerat
ed the hydrolysis of CaE(2)P. Calmodulin alone had no effects on depho
sphorylation but enhanced acceleration of hydrolysis of CaE(2)P by ATP
making the decay of EP under these conditions the fastest among those
measured. Comparison of the rates of dephosphorylation of EP made in
the presence of Mg2+ with those of steady-state Ca2+-ATPase activity w
ith and without calmodulin showed that the transition CaE(1)P --> CaE(
2)P and decomposition of CaE(2)P by hydrolysis are compatible with the
ir role as obligatory intermediate reactions in the cycle of hydrolysi
s of ATP by the Ca2+-ATPase.