Reactions of the sarcoplasmic reticulum calcium adenosine triphosphatase with adenosine 5'-triphosphate and calcium that are not satisfactorily described by an E1-E2 model

Abstract

Phosphorylation of the sarcoplasmic reticulum calcium ATPase, E, is first order with kb = 70 +/- 7 s-1 after free enzyme was mixed with saturating ATP and 50 microM Ca2+; this is one-third the rate constant of 220 s-1 for phosphorylation of enzyme preincubated with calcium, cE.Ca2, after being mixed with ATP under the same conditions (pH 7.0, Ca2+-loaded vesicles, 100 mM KCl, 5 mM Mg2+, 25 degrees C). Phosphorylation of E with ATP and Ca2+ in the presence of 0.25 mM ADP gives approximately 50% E approximately P.Ca2 with kobsd = 77 s-1, not the sum of the forward and reverse rate constants, kobsd = kf + kr = 140 s-1, that is expected for approach to equilibrium if phosphorylation were rate limiting. These results show that (1) kb represents a slow conformational change, rather than phosphoryl transfer, and (2) different pathways are followed for the phosphorylation of E and of cE.Ca2. The absence of a lag for phosphorylation of E with saturating ATP and Ca2+ indicates that all other steps, including the binding of Ca2+ ions and phosphoryl transfer, have rate constants of greater than 500 s-1. Chase experiments with unlabeled ATP or with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) show that the rate constants for dissociation of [gamma-32P]ATP and Ca2+ are comparable to kb. Dissociation of ATP occurs at 47 s-1 from E.ATP.Ca2+ and at 24 s-1 from E.ATP. Approximately 20% phosphorylation occurs following an EGTA chase 4.5 ms after the addition of 300 microM ATP and 50 microM Ca2+ to enzyme. This shows that Ca2+ binds rapidly to the free enzyme, from outside the vesicle, before the conformational change (kb). The fraction of Ca2+-free E.[gamma-32P]ATP that is trapped to give labeled phosphoenzyme after the addition of Ca2+ and a chase of unlabeled ATP is half-maximal at 6.8 microM Ca2+, with a Hill slope of n = 1.8. The calculated dissociation constant for Ca2+ from E.ATP.Ca2 is approximately 2.2 X 10(-10) M2 (K0.5 = 15 microM). The rate constant for the slow phase of the biphasic reaction of E approximately P.Ca2 with 1.1 mM ADP increases 2.5-fold when [Ca2+] is decreased from 50 microM to 10 nM, with half-maximal increase at 1.7 microM Ca2+. This shows that Ca2+ is dissociating from a different species, aE.ATP.Ca2, that is active for catalysis of phosphoryl transfer, has a high affinity for Ca2+, and dissociates Ca2+ with k less than or equal to 45 s-1.(ABSTRACT TRUNCATED AT 400 WORDS)

The reaction mechanism of Ca2+‐ATPase of sarcoplasmic reticulum

“…8 give an observed rate constant of 25 s-', a value similar to that deduced by Stahl and Jencks (1987) from dephosphorylation experiments. It is interesting to note that perfusion of either a magnesium-free or a magnesium-containing solution induces similar monoexponential ATP dissociation with a similar dissociation rate constant (data not shown).…”

“…ATP in the presence of calcium. This value is lower than the 9 -15 pM proposed by several authors (Pickart and Jencks, 1984;Fernandez-Belda and Inesi, 1986;Stahl and Jencks, 1987) (Fernandez-Belda and Inesi, 1986;Teruel et al, 1987). The best fit was obtained with the following values: 9 s-l and 0.36 s-l for the observed rate constants; and 2.6 nmol/mg and 1.8 nmol/mg for the ampliAddition of Mg ' ATP to SR vesicles in the presence of EGTA induces a fluorescence increase (Dupont et al, , 1988Lacapere et al, 1990a) and Fig.…”

“…ATP binding has not been directly measured. Equilibrium and rate constants for ATP binding have been deduced from kinetics of phosphorylation and dephosphorylation (Sumida et al, 1980;Pickart and Jencks, 1984;Fernandez-Belda et al, 1984, 1986Teruel et al, 1987;Stahl and Jencks, 1987;Petithory and Jencks, 1986). However, working at low temperature and in the absence of potassium, we have recently completed a detailed study of the reaction mechanism with Ca .…”

Crosslinking the active site of sarcoplasmic reticulum Ca(2+)-ATPase completely blocks Ca2+ release to the vesicle lumen.

“…The demonstration of a stable occluded form of the phosphoenzyme with no access for the Ca2l to the lumen shows that Ca2l release in this direction requires a further event (step 3). The difficulty experienced in detecting the E2-P.2Ca intermediate from kinetic studies of the native enzyme (22) suggests that it is more unstable than some of the other, more prominent intermediates. Its instability can also be deduced from the destabilization of the E2-vanadate complex, considered to be similar to E2-P, by Ca2' binding to luminal low-affinity sites (23,24).…”

Different classes of tryptophan residues involved in the conformational changes characteristic of the sarcoplasmic reticulum Ca2+‐ATPase cycle

“…Our finding here, that membrane tryptophan residues contribute to the phosphorylation-induced fluorescence changes as well as does a cytoplasmic tryptophan residue, implies that the consequences of this phosphorylation reaction are not restricted to the cytoplasmic catalytic center. The further conclusion that different classes of tryptophans are preferentially involved in the conformational changes occurring as a result of calcium binding or phosphorylation rather points to a different model of the catalytic cycle, in which calcium-induced fluorescence changes result from reorganization of membrane helices [20] after calcium binding to preexisting calcium sites present on the cytoplasmic side of the sarcoplasmic reticulum membrane, while phosphorylation by Pi of the Ca2 +-free ATPase would allow these sites to become accessible from the lumen of the reticulum [35] …”