Eur. J. Biochem. 211, 117-126 zyxwvutsr (1993) zyxwvutsr 0 FEBS 1993 zyxwvutsrqpo The reaction mechanism of zyxw Ca2 zyxwv -ATPase of sarcoplasmic reticulum Direct measurement of the Mg ATP dissociation constant gives similar values in the presence or absence of calcium Jean-Jacques LACAPERE and Florent GUILLAIN DCpartement de Biologie Cellulaire et MolCculaire et URA CNRS 1290, Centre d'Etudes NuclCaires, Gif sur Yvette, France (Received June 16/September 9,1992) - EJB 92 0843 Combining rapid filtration and rapid acid quenchmg, we have directly measured, at pH 7.0 and 5"C, the association and dissociationrate constants of Mg . ATP binding to the sarcoplasmicreticulum (SR) ATPase in the presence of 50 pM calcium and 5 mM MgC12 (3-4x lo6 M-' . s-' and 9 s-', respectively). Therefore, we have determined the true affinity for Mg . ATP zyxw (Kd = 3 pM) in the presence of calcium, which can not be measured at equilibrium because of spontaneous and fast phosphorylation. At low concentrations, Mg . ATP binding is the rate limiting step in the phosphoryla- tion process, and Mg . ATP dissociation is slower than dephosphorylation. The kinetics of Ca2+ binding measured by rapid filtration are biphasic, reflecting a two-step mechanism, both steps being accelerated by Mg . ATP. Combining rapid filtration and rapid monitor- ing of the intrinsic fluorescence of SR Ca2+-ATPase,we showed that rate constants for calcium binding are always lower than those of Mg . ATP binding to an EGTA-incubated enzyme. We measured dissociation and association rate constants of Mg . ATP binding in the absence of calcium (k-1 = 25 s-' and kl = 7.5 lo6 M-' . s-' ). This gives a Kd similar to that obtained by equilibrium measurements (3 - 4 pM). Both non-phosphorylated conformations of the enzyme have similar affinity for Mg . ATP. Therefore, activation of ATPase activity by an excess of ATP cannot be explained by a change in affinity of the non-phophorylated enzyme for Mg . ATP. In conjunction with previous results, these data are used to discuss the molecular mechanism for the Ca2+-ATPase cycle, in which ATP is sequentially substrate and activator on a multiple-function single site. Calcium transport by Ca2+-ATPase of skeletal-musclesar- coplasmic reticulum (SR) proceeds at the expense of ATP hydrolysis. The reaction mechanism is commonly represented by a scheme in which the enzyme resides in two main confor- mations: El in the presence of calcium, and E2 in the absence of calcium (reviews: de Meis, 1981; Inesi, 1985). It has already been observed that ATP concentrations higher than that required for phosphoenzyme formation accel- erate ATP hydrolysis (Weber et al., 1966). Several hypotheses have been proposed to explain t h s activation process. One proposes that, similar to Na/K-ATPase (for a review, see Skou, 1990) the affinity of the enzyme for Mg . ATP could change depending on the enzyme conformation (Reynolds et al., 1985). Two distinct sites have also been postulated, one cata- lytic and one regulatory (de Meis and de Mello, 1973; Coll and Murphy, 1985,1991). However, it has been recently dem- onstrated that only 1 mol ATP binds/mol Ca2+-ATPase under Correspondence to J.-J. Lacapere, Section de Biophysique des Prottines et des Membranes, Dtpartement de Biologie Cellulaire et Moleculaire, Centre d'Etudes NuclCaires de Saclay, F-91191 Gif sur Yvette, France Abbreviations. SR, sarcoplasmic reticulum; kobsr observed rate constant; k,,, association rate constant; koff, dissociation rate con- stant; Kp, equilibrium constant of phosphorylation; Vo, initial rate; Np,ATP, 2'(3')-0-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. Enzyme. Ca-transporting ATPase (EC 3.6.1.38). various conditions (Champeil et al., 1988; Oliveira et al., 1988; Lacapere et al., 1990a) and the most probable explanation for the activation is that a second ATP could bind following phosphorylation and ADP dissociation (Cable et al., 1985; Champeil and Guillain, 1986; Bishop et al., 1987; Lacapere, 1987; Champeil et al., 1988). In the present study, we measured and compared the affin- ity of the enzyme for Mg . ATP in the absence or presence of calcium. Mg . ATP promotes fast phosphorylation when calcium is bound to the transport sites (Froehlich and Taylor, 1975; Verjovski-Almeida and Inesi, 1979; Stahl and Jencks, 1984,1987; Petithory and Jencks, 1986; Fernandez-Beldaand Inesi, 1986), and, at room temperature in the presence of potassium, the reaction steps leading to phosphoenzyme for- mation are so fast that Mg . 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, 1986; Teruel 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 . ATP as a substrate (Lacapere and Guillain, 1990), in which we have been able to differentiate substrate binding, phosphorylation and ADP dissociation, thus measuring the true affinity of the enzyme for Ca . ATP.