Sequential dissociation of calcium from the calcium adenosinetriphosphatase of sarcoplasmic reticulum and the calcium requirement for its phosphorylation by ATP

Abstract

The kinetics for dissociation of the stable enzyme-calcium complex of the sarcoplasmic reticulum calcium ATPase, cE.Ca2, were followed by assay with simultaneous addition of [32P]ATP and EGTA, which gives 70% phosphorylation of cE.Ca2 with k = 300 s-1 (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). The binding of ATP to cE.Ca2 is described by kATP = 1.0 X 10(7) M-1 s-1, k-ATP = 120 s-1, and Kdiss = 12 microM; ATP binding is partially rate limiting for phosphorylation at less than 100 microM ATP. The sequential dissociation of Ca2+ from cE.Ca2 is described by k-2 = 55-60 s-1 for the first, "outer" Ca2+, k-1 = 25-30 s-1 for the second, "inner" Ca2+, and K0.5 = 3.4 microM, n = 1.9 (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). Dissociation of the inner Ca2+ is inhibited by external Ca2+, with K0.5 = k-1/k2 = 0.7 microM. This confirms the conclusion that dissociation of the two Ca2+ ions is sequential. The ability of cE.Ca2 to catalyze phosphorylation by ATP disappears in the presence of EGTA with k = 50-55 s-1, the same as k-2 for dissociation of the outer Ca2+ ion. This result, and the absence of the induction period that would occur if both cE.Ca2 and cE.Ca1 were catalytically competent, shows that both Ca2+ ions are required for phosphorylation. This conclusion is confirmed by the stoichiometry of 1.4/0.7 = 2.0 for the ratio of Ca2+ internalized to phosphoenzyme formed after simultaneous addition of ATP and EGTA. Phosphorylation of cE.Ca2 in the presence of 45Ca gives 0.15, not 0.3, 45Ca internalized, which corresponds to exchange of only 1 Ca2+ and is in agreement with this conclusion. The requirements for binding of two Ca2+ for catalytic specificity toward ATP and loss of two Ca2+ from E approximately P.Ca2 for specificity toward water account for the stoichiometry of Ca2+ transport and provide a possible reason for the two steps in the phosphorylation of cE.Ca2.ATP.

Molecular changes in the sarcoplasmic reticulum calcium ATPase during catalytic activity

“…We attribute these changes to ligand binding and conformational changes in the ATPase during the catalysis of Ca2 + -transport. This is supported by FTIR difference spectra of ATPase samples with caged ADP instead of caged ATP and of EGTA-inhibited [24] samples (data not shown). The transient absorbance changes of the 'normal' ATPase sample (Fig.…”

The Average Conformation at Micromolar [Ca2+] of Ca2+-ATPase with Bound Nucleotide Differs from That Adopted with the Transition State Analog ADP·AlFx or with AMPPCP under Crystallization Conditions at Millimolar [Ca2+]

“…Acceleration of the rate of Ca 2ϩ dissociation in fact seems to be a fairly general feature of the effect of nucleotides, because it is observed at all Mg 2ϩ concentrations with both ADP and ATP␥S, the latter nucleotide being a poorly hydrolyzable analog of ATP probably resembling ATP more than AMPPCP does (23). As concerns ATP itself, detailed time-resolved phosphorylation experiments previously suggested also that the rate of Ca 2ϩ dissociation from Ca 2ϩ -ATPase was slightly enhanced (but not much) in the presence of Mg⅐ATP: Ca 2ϩ dissociation from the non-covalent E⅐Ca 2 ⅐ATP complex was estimated to occur with a rate constant in the range of 45-80 s Ϫ1 at 25°C and pH 7 in the presence of 100 mM KCl and 5 mM Mg 2ϩ (13), compared with 25 or 50 s Ϫ1 in the absence of ATP (depending on whether overall dissociation of the two Ca 2ϩ ions or dissociation of the most accessible one is considered) (34). Thus, under ordinary conditions, Ca 2ϩ dissociation does not appear to be blocked by the mere binding of nucleotide.…”

Participation of a non‐covalent phosphointermediate in ATP hydrolysis by the sarcoplasmic reticulum Ca2+‐transport ATPase

“…All these models implicate that phosphorylation by ATP in the presence of Ca2+ proceeds through successive interdependent conformational changes [18,321, wereby the different binding domains shift relatively to each other. This further implicates possible changes of electrostatic interaction of the bound substrate with different amino acid residues during terminal Pi transfer and energy transduction.…”

“…Due to the demands of the filtration method we had to determine ATP binding (binding of the nucleoside moiety) with non-saturating 10 pM [I4C]ATP or [32P]ATP (for kATp and Kd at pH 7.0 see [18]; for pH dependence, as all our experiments were performed at pH 6.15, see [19]). We still observed that ATP binding was less sensitive to SDS than covalent phosphorylation (Fig.…”