Role of divalent cation bound to phosphoenzyme intermediate of sarcoplasmic reticulum ATPase

Effect of divalent cations bound to the phosphoenzyme intermediate of the ATPase of sarcoplasmic reticulum was investigated at 0 degree C and pH 7.0 using the purified ATPase preparations. Our previous study (Shigekawa, M., Wakabayashi, S., and Nakamura, H. (1983) J. Biol. Chem. 258, 14157-14161) indicated that 1 mol of the ADP-sensitive phosphoenzyme (E1P) formed from CaATP has 3 mol of high affinity binding sites for Ca2+, of which two are transport sites for calcium while the remainder is the acceptor site for calcium derived from the substrate, CaATP ("substrate site"). When incubated with a chelator of divalent cation, E1P formed from CaATP released all of its bound calcium to form a divalent cation-free phosphoenzyme. Evidence was presented that calcium dissociation from the substrate site was faster than that from the transport sites and primarily responsible for the ADP sensitivity loss of E1P induced by the chelator. Divalent cation-free phosphoenzyme was kinetically stable but when treated with divalent cations, it behaved similarly to the ADP-insensitive phosphoenzyme (E2P) which is the normal reaction intermediate of ATP hydrolysis. 45Ca bound at the substrate site on E1P formed from 45CaATP exchanged readily with nonradioactive ionized Ca2+ in the reaction medium whereas 45Ca at the transport sites on E1P was displaced only at a very slow rate which was almost the same as that for the phosphoenzyme hydrolysis. It was suggested that calcium at the transport sites on E1P formed from CaATP is released only after the rate-limiting conformational transition of the phosphoenzyme from E1P to E2P and that removal of calcium by a chelator from the substrate site facilitates this conformational transition, thereby allowing calcium bound at the transport sites to be released readily from the phosphoenzyme.     

Effect of divalent cation bound to the ATPase of sarcoplasmic reticulum. Activation of phosphoenzyme hydrolysis by Mg2+

In order to study the mechanism for activation of ATP hydrolysis by Mg2+, the stoichiometry of the high affinity calcium-binding sites with respect to each form of reaction intermediate of sarcoplasmic reticulum ATPase was determined at 0 degrees C and pH 7.0 in the presence and absence of added Mg2+ using the purified ATPase preparation. High affinity calcium binding to the enzyme-ATP complex and to ADP-sensitive (E1P) and ADP-insensitive (E2P) phosphoenzymes occurred with stoichiometric ratios of 2, 2, and 0, and 3, 3, and 1 in the presence and absence of added Mg2+, respectively. The results were interpreted to indicate that in addition to 2 mol of calcium bound to the transport sites of the ATPase, 1 mol of divalent cation, which is derived from the metal component of the substrate, the metal-ATP complex, remains bound to each mole of the enzyme at least until E2P is hydrolyzed. As activation of phosphoenzyme hydrolysis by Mg2+ was blocked by the low concentrations of Ca2+ used in the calcium binding experiments, it was concluded that it is the magnesium derived from MgATP that is responsible for rapid hydrolysis of the phosphoenzyme intermediate.     

Lanthanum inhibits steady-state turnover of the sarcoplasmic reticulum calcium ATPase by replacing magnesium as the catalytic ion

LaATP is shown to be an effective inhibitor of the calcium ATPase of sarcoplasmic reticulum because the binding of LaATP to cE.Ca2 results in the formation of lanthanum phosphoenzyme, which decays slowly. Steady-state activity of the calcium ATPase in leaky sarcoplasmic reticulum vesicles is inhibited 50% by 0.16 microM LaCl3 (15 nM free La3+, 21 nM LaATP) in the presence of 25 microM Ca2+ and 49 microM MgATP (5 mM MgSO4, 100 mM KCl, 40 mM 4-morpholinepropanesulfonic acid, pH 7.0, 25 degrees C). However, 50% inhibition of the uptake of 45Ca and phosphorylation by [gamma-32P]ATP in a single turnover experiment requires 100 microM LaCl3 (28 microM free La3+) in the presence of 25 microM Ca2+; this inhibition is reversed by calcium but inhibition of steady-state turnover is not. Therefore, binding of La3+ to the cytoplasmic calcium transport site is not responsible for the inhibition of steady-state ATPase activity. The addition of 6.7 microM LaCl3 (1.1 microM free La3+) has no effect on the rate of dephosphorylation of phosphoenzyme formed from MgATP and enzyme in leaky vesicles, while 6.7 mM CaCl2 slows the rate of phosphoenzyme hydrolysis as expected; 6.7 microM LaCl3 and 6.7 mM CaCl2 cause 95 and 98% inhibition of steady-state ATPase activity, respectively. This shows that inhibition of ATPase activity in the steady state is not caused by binding of La3+ to the intravesicular calcium transport site of the phosphoenzyme. Inhibition of ATPase activity by 2 microM LaCl3 (0.16 microM free La3+, 0.31 microM LaATP) requires greater than 5 s, which corresponds to approximately 50 turnovers, to reach a steady-state level of greater than or equal to 80% inhibition. Inhibition by La3+ is fully reversed by the addition of 0.55 mM CaCl2 and 0.50 mM EGTA; this reactivation is slow with t1/2 approximately 9 s. Two forms of phosphoenzyme are present in reactions that are partially inhibited by La3+: phosphoenzyme with Mg2+ at the catalytic site and phosphoenzyme with La3+ at the catalytic site, which undergo hydrolysis with observed rate constants of greater than 4 and 0.05 s-1, respectively. We conclude, therefore, that La3+ inhibits steady-state ATPase activity under these conditions by replacing Mg2+ as the catalytic ion for phosphoryl transfer. The slow development of inhibition corresponds to the accumulation of lanthanum phosphoenzyme. Initially, most of the enzyme catalyzes MgATP hydrolysis, but the fraction of enzyme with La3+ bound to the catalytic site gradually increases because lanthanum phosphoenzyme undergoes hydrolysis much more slowly than does magnesium phosphoenzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sarcoplasmic reticulum Ca-ATPase: distinction of phosphoenzymes formed from MgATP and CaATP as substrates and interconversion of the phosphoenzymes by Mg2+ and Ca2+

In order to characterize the phosphoenzymes (EPs) formed from MgATP and CaATP as substrates, the effects of Mg2+ and Ca2+ outside SR vesicles on the hydrolysis rates of EPs were examined by using purified and unpurified Ca-ATPases of sarcoplasmic reticulum (SR) at low [gamma-32P]ATP (4-10 microM), 0.1 M KCl, pH 7.0, and 0 degrees C. When the phosphorylation reaction was stopped by adding an excess of EDTA over Ca and Mg, two components of EP, EPfast (rate constant, kfast = 15-20 min-1), and EPslow (kslow = 0.3-0.4 min-1), were recognized in the time course of EP decomposition. These two rates did not depend on the Ca2+ or Mg2+ concentration in the medium during the phosphorylation reaction, although the proportions of EPfast and EPslow essentially depended on the concentrations of MgATP and CaATP in the phosphorylation reaction medium. The proportion of EPfast increased with increasing [MgATP]/[CaATP] in the medium, whereas that of EPslow decreased. The rate of EPslow hydrolysis in the presence of excess EDTA was basically the same as that of EP formed from CaATP. These results suggest that EPfast and EPslow are derived from MgATP and CaATP, respectively, and EPfast is a reaction intermediate with Mg bound at the substrate site (MgEP), while the main EPslow is a reaction intermediate with Ca bound at the substrate site (CaEP) which is readily converted to metal-free EP by EDTA addition (Shigekawa et al., (1983) J. Biol. Chem. 258, 8698-8707). Mg2+ added outside SR vesicles stimulated the conversion of CaEP to MgEP and inhibited the hydrolysis of MgEP in the relatively high concentration range (K(Mg) = 7.9 mM). Ca2+ added outside SR vesicles stimulated the conversion of MgEP to CaEP and inhibited the conversion of CaEP to MgEP by Mg2+ addition. The Ca2+ outside SR vesicles did not essentially affect the hydrolysis of MgEP. These results suggest that the interconversion between MgEP and CaEP takes place during the reaction by exchange of the divalent cation on the substrate site. The following scheme is proposed. (formula: see text)     

The role of tightly bound ADP on chloroplast ATPase

Isolated chloroplast coupling factor 1 ATPase is known to retain about 1 mol of tightly bound ADP/mol of enzyme. Some experimental results have given evidence that the bound ADP is at catalytic sites, but this view has not been supported by observations of a slow replacement of the bound ADP when CaATP or MgATP is added. The experiments reported in this paper show why a slow replacement of ADP bound at a catalytic site can occur. When coupling factor 1, labeled with tightly bound [3H]ADP, is exposed to Mg2+ or Ca2+ prior to the addition of MgATP or CaATP, a pronounced lag in the onset of ATP hydrolysis is observed, and only slow replacement of the [3H]ADP occurs. Mg2+ or Ca2+ can induce inhibition very rapidly, as if an inhibited form of the enzyme results whenever the enzyme with tightly bound ADP encounters Mg2+ or Ca2+ prior to ATP. The inhibited form can be slowly reactivated by incubation with EDTA, although some irreversible loss in activity is encountered. In contrast, when MgATP or CaATP is added to enzyme depleted of Mg2+ and Ca2+ by incubation with EDTA, a rapid onset of ATP hydrolysis occurs and most of the tightly bound [3H]ADP is released within a few seconds, as expected for binding at a catalytic site. The Mg2+-induced inhibition of both the ATPase activity and the lack of replacement of tightly bound [3H] ADP can be largely prevented by incubation with Pi under conditions favoring Pi addition to the site containing the tightly bound ADP. Our and other results can be explained if enzyme catalysis is greatly hindered when MgADP or CaADP without accompanying Pi is tightly bound at one of the three catalytic sites on the enzyme in a high affinity conformation.     

Reaction mechanism of the calcium-transport ATPase in endoplasmic reticulum of rat liver. Demonstration of different reactive forms of the phosphorylated intermediate

A calcium-transport ATPase is inserted into the endoplasmic reticulum of rat liver. Catalysis of calcium translocation involves transient covalent binding of the terminal phosphate residue of ATP by the enzyme, resulting in the formation of an alkali- and hydroxylamine-labile phosphorylprotein intermediate. Both MgATP as well as CaATP can be utilized in the phosphorylation reaction which requires calcium as a cofactor. Magnesium accelerates the turnover of the phosphorylprotein intermediate. An ADP-reactive and ADP-unreactive state of the phosphoenzyme could be distinguished. In the ADP-reactive state with tightly bound calcium, the phosphoenzyme can transphosphorylate its phosphate residue to ADP, giving rise to synthesis of ATP. The ADP-reactive phosphoenzyme can be converted into an ADP-unreactive state by prolonged incubation with excess EGTA (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid). It is suggested that this conversion is brought about by the removal of tightly bound calcium from the phosphoenzyme. A strong homology of the sequential reaction steps during calcium translocation by the calcium-transport ATPases in endoplasmic reticulum from rat liver and sarcoplasmic reticulum from skeletal muscle is suggested.     

Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase

The yeast plasma membrane proton-pumping ATPase forms a phosphorylated intermediate during the hydrolysis of ATP. The fraction of enzyme phosphorylated during steady-state ATP hydrolysis was studied as a function of substrate concentration (MgATP), Mg2+ concentration, and pH. The dependence of the fraction of enzyme phosphorylated on the concentration of MgATP is sigmoidal, and the isotherms can be fit with parameters and mechanisms similar to those used to describe ATP hydrolysis. The isotherm is significantly more sigmoidal at pH 5.5 than at pH 6.0, with the limiting percentage (100.mol of phosphate/mol of enzyme) of enzyme phosphorylated being 70% and 6%, respectively, at the two pH values. The maxima in the steady-state rate of ATP hydrolysis occur at higher concentrations of Mg2+ and higher pH than the maxima in the fraction of enzyme phosphorylated. This suggests that the rate-determining step for ATP hydrolysis is different from that for enzyme phosphorylation and the hydrolysis of phosphoenzyme is enhanced by Mg2+ and high pH. The rate of phosphoenzyme formation was investigated with the quenched-flow method, but only a lower bound of 140 s-1 could be obtained for the rate constant at MgATP concentrations greater than 2.5 mM. Since the turnover number for ATP hydrolysis under similar conditions is 14 s-1, the rate-determining step in ATP hydrolysis occurs after enzyme phosphorylation.     

Phosphorylation and dephosphorylation kinetics of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa.

Partial reactions of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa were studied by means of a rapid-mixing apparatus. At 21 degrees C, in the presence of 2 mM MgCl2 and 5 microM [gamma-32P]ATP there was a rapid phosphorylation of the enzyme with a pseudofirst order rate constant of 1400 min-1. Addition of the ATP about 120 ms before the MgCl2 increased this rate constant to 4400 min-1. In the absence of MgCl2 there was no phosphorylation. Addition of 4 or 10 mM KCl to the phosphoenzyme which had been formed in the absence of KCl produced a rapid initial rate of dephosphorylation (k = 2600 and 3200 min-1 respectively). An additional slow component of dephosphorylation was observed when unlabeled ATP was added together with the KCl (k = 700 to 900 min-1). At a 4 mM concentration, KCl stimulated the ATPase activity about 9-fold. At higher concentrations, the activity was reduced in parallel with a reduction of the steady state level of phosphoenzyme. Addition of KCl to the enzyme before the addition of ATP plus MgCl2 resulted in a low rate and extent of phosphorylation. KCl appeared to inhibit the phosphorylation at a level preceeding the E.ATP complex.     

Intermediate reaction states of the red beet plasma membrane ATPase

The phosphorylation reaction for the plasma membrane ATPase of red beet (Beta vulgaris L.) was examined in order to further understand the mechanism of this enzyme. The level of steady-state phosphorylation had a pH optimum of about 6.0 while ATPase activity (32Pi production) measured under identical conditions had a pH optimum of 7.0. Phosphoenzyme decomposition was accelerated as both the pH and temperature were increased. The former effect may account for the observed difference between the pH optimum for phosphorylation and ATPase. Although the kinetics of K+ stimulation of ATP hydrolysis have been observed to be complex, the kinetics of K+ stimulation of phosphoenzyme turnover were observed to be simple Michaelis-Menten. An antagonism was observed between MgATP and K+ for the stimulation of phosphoenzyme turnover. Increased MgATP concentration reduced the degree of K+ stimulation of phosphoenzyme turnover and ATPase activity. These effects could be explained by the observation that two forms of phosphoenzyme occur during ATP hydrolysis. One form is discharged by ADP while the other form is ADP insensitive. Potassium stimulation of phosphoenzyme breakdown occurs primarily because of effects on the ADP-insensitive phosphoenzyme form. These results are consistent with a mechanism of ATP hydrolysis involving interconversions of conformational states.     

Bovine brain Na+, K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. Detection of a transient [32P]phosphoenzyme formed in the presence of potassium ions.

1. Conditions for binding of [gamma-32P]ATP to bovine brain Na+,K+-stimulated ATPase were investigated by the indirect technique of measuring the initial rate of 32P-labelling of the active site of the enzyme. 2. At 100 muM [gamma-32P]ATP in the presence of 3 mM MgCl2, approximately the same very high rate of formation of [32P]phosphoenzyme was obtained irrespective of whether [gamma-32P]ATP was added to the enzyme simultaneously with, or 70 ms in advance of the addition of NaCl. A comparatively slow rate of phosphorylation was obtained at 5 muM[gamma-32P]ATP without preincubation. However, on preincubation of the enzyme with 5 muM[gamma-32P]ATP a rate of formation of [32P]phosphoenzyme almost as rapid as at 100 muM[gamma-32P]ATP was observed. 3. A transient [32P]phosphoenzyme was discovered. It appeared in the presence of K+, under conditions which allowed extensive binding of [gamma-32P]-ATP. The amount of [gamma-32P]ATP that could be bound to the enzyme seemed to equal the amount of [32P] phosphorylatable sites. 4. The formation of the transient [32P] phosphoenzyme was inhibited by ADP. The transient [32P] phosphoenzyme was concluded mainly to represent the K+-insensitive and ADP-sensitive E1-32P. 5. When KCl was present in the enzyme solution before the addition of NaCl only a comparatively slow rate of phosphorylation was observed. On preincubation of the enzyme with [gamma-32]ATP an increase in the rate of formation of [32P] phosphoenzyme was obtained, but there was no transient [32P]-phosphoenzyme. The transient [32P]phosphoenzyme was, however, detected when the enzyme solution contained NaCl in addition to KCl and the phosphorylation was started by the addition of [gamma-32P]ATP.     

Phosphoenzyme formation from ATP in the ATPase of sarcoplasmic reticulum. Effect of KCl or ATP and slow dissociation of ATP from precursor enzyme-ATP complex

The ATP-dependent phosphoenzyme formation and its reversal were studied at 0 degrees C and pH 7.0 in the ATPase of sarcoplasmic reticulum. Addition of KCl or several other salts (approximately 100 mM) decreased the maximum rate of ADP-induced dephosphorylation of phosphoenzyme as well as the apparent affinity of the phosphoenzyme toward ADP. High ATP had a similar effect on the latter, whereas it had little effect on the former. In contrast, high KCl or a considerable change in the ionic strength had little effect on the initial rate of phosphoenzyme formation at saturating ATP concentrations. During steady state phosphorylation at 1.0 mM MgCl2 and 5.0 mM CaCl2 in the absence of added KCl, a significant amount of [gamma-32P]ATP remained bound to the enzyme even when the enzyme concentration was much in excess over that of [gamma-32P]ATP. Evidence is presented that this enzyme-ATP complex represents a precursor to the phosphoenzyme. ATP dissociated slowly (0.20 s-1) from this enzyme-ATP complex and addition of high KCl or other salts accelerated its dissociation. In contrast, when the enzyme was complexed with adenyl-5'-yl (beta, gamma-methylene)diphosphonate in the absence of added KCl under these conditions, dissociation of the nucleotide from the complex as estimated in the displacement experiment with [gamma-32P]ATP, was found to be much faster than that of ATP.     

Slow dissociation of ATP from the calcium ATPase

The acyl-phosphate intermediate of the sarcoplasmic reticulum calcium ATPase reaction, formed in a brief incubation of vesicular enzyme with 5 microM [gamma-32P]ATP and calcium, reacts biphasically with added ADP (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4). Both the burst size and the rate constant for the slow phase increase with increasing ADP concentration in the way that is expected if the burst represents very rapid formation of an equilibrium amount of enzyme-bound ATP and the slow phase represents rate-limiting dissociation of ATP. Also consistent with this interpretation are the slow labeling of phosphoenzyme under conditions in which unlabeled ATP must dissociate first and the observation of a burst of ATP formation on ADP addition to phosphoenzyme. Values of the equilibrium constants for ADP dissociation from phosphoenzyme (0.75 mM), for ATP formation on the enzyme (2.3), and for the ATP dissociation rate constant (37 s-1) were obtained from a quantitative analysis of the data.     

Na+ATPase of the mammalian erythrocyte membrane. Reversibility of phosphorylation at 0 degrees.

When human erythrocyte membranes are phosphorylated with a very low concentration of [gamma-32P]ATP (0.02 muM) at 0 degrees, and then EDTA is added, rapid disappearance of the phosphoenzyme intermediate of Na+ATPase is observed. The initial rapid phase of phosphoenzyme disappearance is, for the most part, not associated with P1 release and its rate constant, kD, is severalfold greater than the ratio of Na+ATPase activity to phosphoenzyme intermediate, v:EP, at steady state. It is concluded that this rapid disappearance of phosphoenzyme is due to resynthesis of ATP via reversal of phosphorylation. In contrast, rapid reversal is not observed when excess nonradioactive ATP is added to reduce E32P formation, provided Mg2+ is present; however, K+ added with the ATP stimulates reversal. Rapid reversal following EDTA addition is unlikely also when higher ATP concentrations (greater than or equal to 10(-6) M) are used to phosphorylate the enzyme since, at higher ATP, kD congruent to v:EP. The results are compatible with the concept that the Na+ATPase enzyme is composed of two or more catalytic subunits, in which ATP at one catalytic site can regulate the reactivity at another site.     

Bovine brain Na+,K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. K+-stimulated liberation of [32P] orthophosphate from [32P] phosphoenzyme and resolution of the dephosphorylation into two phases.

Dephosphorylation of [32P]phosphoenzyme of bovine brain Na+,K+-stimulated ATP phosphohydrolase (EC 3.6.1.3), labelled by [gamma-32P]ATP, was investigated at 21 degrees C by means of a rapid-mixing technique. On addition of a high concentration of KCl (10 mM) to [32P]phosphoenzyme at steady state in the presence of Mg2+ and Na+, very rapid dephosphorylation was obtained. Simultaneously, the amount of [32P]orthophosphate increased at about the same rate. It was concluded that this K+-stimulated dephosphorylation and liberation of [32P]orthophosphate from the [32P]phosphoenzyme was rapid enough to participate in the Na+,K+-stimulated hydrolysis of ATP. In order to study the dephosphorylation in absence of continuing 32P-labelling, excess unlabelled ATP or a chelator of Mg2+ was added. Simultaneous addition of a high concentration of KCl to the [32P]phosphoenzyme formed in the presence of Mg2+ and Na+ but in the absence of K+, resulted in an initial very rapid phase and a subsequent slower phase of dephosphorylation. With KCl also initially present in the incubation medium, only the slow phase was observed. The slow phase of dephosphorylation also seemed to be sufficiently rapid to participate in the Na+, K+-stimulated ATPase reaction. On addition of a high concentration of ADP (5 mM) to [32P]phosphoenzyme formed in the presence of Mg2+ and Na+, an initial comparatively rapid, and later slow phase of dephosphorylation were detected. This gave further support for different forms of phosphoenzyme. Approximate concentrations of these forms, in the absence and presence of KCl, were estimated by extrapolation and the turnover of these forms was calculated. The nature of the kinetically different components of phosphoenzyme and their role in the Na+, K+-stimulated ATPase reaction is discussed.     

Sarcoplasmic reticulum ATPase phosphorylation from inorganic phosphate in the absence of a calcium gradient. Steady state and kinetic fluorescence studies

The intrinsic fluorescence of sarcoplasmic reticulum vesicles was measured under conditions allowing ATPase phosphorylation from inorganic phosphate. Significant fluorescence enhancement of up to 4% resulted from gradient-independent enzyme phosphorylation at pH 6, in the absence of KCl. The equilibrium fluorescence data obtained at various magnesium and phosphate concentrations agree with a reaction scheme in which Mg2+, as direct activator, and free phosphate, as the true substrate, bind to the enzyme in random order to give a noncovalent ternary complex (Mg.*E.Pi), in equilibrium with the covalent phosphoenzyme (Mg.*E-P). The transient kinetics of the fluorescence rise was also studied, and the resulting data were generally consistent with the above scheme, assuming that binding reactions are fast compared to covalent phosphoenzyme formation. This, however, might be valid only as a first approximation. At 20 degrees C and pH 6, the phosphate concentration for half-maximum phosphorylation rate constant, at 20 mM magnesium, was higher than 20 mM. Similarly, the magnesium concentration for half-maximum phosphorylation rate constant, at 20 mM phosphate, was also higher than 20 mM. The maximum phosphorylation rate was faster than 25 s-1, and the phosphoenzyme hydrolysis rate constant was 1.5-2 s-1 under these conditions, so that the equilibrium constant between Mg.*E.Pi and Mg.*E-P largely favors the phosphoenzyme.     

Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional roles of 18 amino acid residues located at or near the "hinge-domain," a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to arginine, alanine, histidine, and glutamine resulted in complete loss of calcium transport function and ATPase activity. For the Lys684----Ala, histidine, and glutamine mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi. The Lys684----Arg mutant retained the ability to phorphorylate from ATP with normal apparent affinity, demonstrating the importance of the positive charge. On the other hand, no phosphorylation was observed with Pi as substrate in this mutant. Examination of the partial reactions after phosphorylation from ATP in the Lys684----Arg mutant demonstrated a reduction of the rate of transformation of the ADP-sensitive phosphoenzyme intermediate (E1P) to the ADP-insensitive phosphoenzyme intermediate (E2P), which could account for the loss of transport function. Once accumulated, the E2P intermediate was able to decompose rapidly in the presence of K+ at neutral pH. These results may be interpreted in terms of a preferential destabilization of protein phosphate interactions in the E2P form of this mutant. The Asp703----Ala and Asn-Asp707----Ala-Ala mutants were completely inactive and unable to form phosphoenzyme intermediates from ATP or Pi. In these mutants as well as in the Lys684----Ala mutant, nucleotides were found to protect with normal affinity against intramolecular cross-linking induced with glutaraldehyde, indicating that the nucleotide binding site was intact. Mutation of Glu646, Glu647, Asp659, Asp660, Glu689, Asp695, Glu696, Glu715, and Glu732 to alanine did not affect the maximum rates of calcium transport and ATP hydrolysis or the apparent affinities for calcium and ATP. Mutation of the 2 highly conserved proline residues, Pro681 and Pro709, as well as Lys728, to alanine resulted in partially inhibited Ca(2+)-ATPase enzymes with retention of the ability to form a phosphoenzyme intermediate from ATP or Pi and with normal apparent affinities for ATP and calcium. The proline mutants retained the biphasic ATP concentration dependence of ATPase activity, characteristic of the wild-type, and therefore the partial inhibition of turnover could not be ascribed to a disruption of the low affinity modulatory ATP site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in the profile structure of the sarcoplasmic reticulum membrane induced by phosphorylation of the Ca2+ ATPase enzyme in the presence of terbium: a time-resolved x-ray diffraction study.

The design of the time-resolved x-ray diffraction experiments reported in this and an accompanying paper was based on direct measurements of enzyme phosphorylation using [gamma-32P]ATP that were employed to determine the extent to which the lanthanides La3+ and Tb3+ activate phosphorylation of the Ca2+ATPase and their effect on the kinetics of phosphoenzyme formation and decay. We found that, under the conditions of our experiments, the two lanthanides are capable of activating phosphorylation of the ATPase, resulting in substantial levels of phosphoenzyme formation and they slow the formation and dramatically extend the lifetime of the phosphorylated enzyme conformation, as compared with calcium activation. The results from the time-resolved, nonresonance x-ray diffraction work reported in this paper are consistent with the enzyme phosphorylation experiments; they indicate that the changes in the profile structure of the SR membrane induced by terbium-activated phosphorylation of the ATPase enzyme are persistent over the much longer lifetime of the phosphorylated enzyme and are qualitatively similar to the changes induced by calcium-activated phosphorylation, but smaller in magnitude. These results made possible the time-resolved, resonance x-ray diffraction studies reported in an accompanying paper utilizing the resonance x-ray scattering from terbium, replacing calcium, to determine not only the location of high-affinity metal-binding sites in the SR membrane profile, but also the redistribution of metal density among those sites upon phosphorylation of the Ca2+ATPase protein, as facilitated by the greatly extended lifetime of the phosphoenzyme.     

Phosphoenzymes formed from Mg.ATP and Ca.ATP during pre-steady state kinetics of sarcoplasmic reticulum ATPase

We have investigated here the pre-steady state kinetics of sarcoplasmic reticulum ATPase incubated under conditions where significant amounts of Mg.ATP and Ca.ATP coexist, both of them being substrates for the ATPase. We confirmed that these two substrates are independently hydrolyzed by the ATPase, which thus apparently catalyzes Pi production by two simultaneous and separate pathways. External calcium (or the Ca2+/Mg2+ ratio) determines the extent to which Ca2+ or Mg2+ is bound at the phosphorylation site, while internal calcium controls the rate of processing of both the slow, calcium-containing and the fast, magnesium-containing phosphoenzyme. Time-dependent binding of calcium at the catalytic site is correlated with the observed burst of Pi liberation, which therefore results from reequilibration during pre-steady state of magnesium- and calcium-containing phosphoenzyme pools. Independently of direct exchange of metal at the catalytic site, ADP produced by the hydrolysis reaction contributes to reequilibration of these pools through reversal of phosphorylation by the ATP-ADP exchange pathway.     

The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase

The coupling of Ca2+ movements and phosphate fluxes as well as the time-dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg-dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 microM does not allow more than one reaction cycle to occur. The respective fractions of ADP-sensitive and ADP-insensitive phosphoenzyme have been determined. The chosen experimental conditions (0-1 degree C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively. It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P----E2P transition, E2P hydrolysis, and E2----E1 transformation). Mg2+ represents the sole ionic co-factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P----E2P transition rate (including Ca2+ translocation and Ca2+ release from low-affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded. We find Ca2+ release to occur time-coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low-affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].     

The partial reactions in the catalytic cycle of the calcium-dependent adenosine triphosphatase purified from erythrocyte membranes

A preparation of purified erythrocyte membrane ATPase whose activation by Ca2+ is or is not dependent on calmodulin depending on the enzyme dilution was used in the low dilution state for these studies. In appropriate conditions, the purified ATPase in the absence of calmodulin exhibited a Ca2+ concentration dependence identical to that of the native enzyme in the erythrocyte membrane ghost in the presence of calmodulin. Accordingly, an apparent Kd approximately equal to 1 X 10(-7) M was derived for cooperative calcium binding to the activating and transport sites of the nonphosphorylated enzyme. The kinetics of enzyme phosphorylation in the transient state following addition of ATP to enzyme activated with calcium were then resolved by rapid kinetic methods, demonstrating directly that phosphoenzyme formation precedes Pi production, consistent with the phosphoenzyme role as an intermediate in the catalytic cycle. Titration of a low affinity site (Kd approximately equal to 2 X 10(-3) M) with calcium produced inhibition of phosphoenzyme cleavage and favored reversal of the catalytic cycle, indicating that calcium dissociation from the transport sites precedes hydrolytic cleavage of the phosphoenzyme. The two different calcium dissociation constants of the nonphosphorylated and phosphorylated enzyme demonstrate that a phosphorylation-induced reduction of calcium affinity is the basic coupling mechanism of catalysis and active transport, with an energy expenditure of approximately 6 kcal/mol of calcium in standard conditions. From the kinetic point of view, a rate-limiting step is identified with the slow dissociation of calcium from the phosphoenzyme; another relatively slow step following hydrolytic cleavage and preceding recycling of the enzyme is suggested by the occurrence of a presteady state phosphoenzyme overshoot.