From chemistry to biochemistry to catalysis to movement.

Mechanisms of chemical reaction can often be predicted by determining the dependence of the lifetime of reaction intermediates on the structure of the reactants. When there is no lifetime in the presence of another reactant or catalyst the reaction proceeds through an enforced concerted mechanism. Noncovalent binding interactions between enzymes and their substrates provide a major contribution to catalysis by decreasing entropy and by destabilizing the ground state relative to the transition state, as well as by covalent and noncovalent chemical interactions with the substrate. Movement in biological systems, such as muscle contraction and the active transport of ions, is generally brought about through a series of alternating chemical and vectorial steps that involve a series of changes in the specificity for catalysis of the chemical and vectorial reactions. These changes divide the overall reaction into segments so that neither the chemical nor the vectorial reaction will be completed unless the other is also completed.

Phosphorylation of the sodium-potassium adenosinetriphosphatase with adenosine triphosphate and sodium ion that requires subconformations in addition to principal E1 and E2 conformations of the enzyme.

Phosphorylation of the free sodium-potassium adenosinetriphosphatase of sheep kidney upon the addition of 0.02-2.0 mM ATP and a saturating concentration of Na+ (125 mM) follows pseudo-first-order kinetics. The first-order rate constant increases with increasing [ATP] and levels off at high [ATP] with a limiting rate constant of 180 s-1 at 25 degrees C, pH 7.4, 125 mM NaCl, and 3.0 mM MgCl2. This rate constant is about 1/3 of the maximum rate constant of 460 s-1 for phosphorylation of enzyme that had been preincubated with Na+ under identical conditions [Keillor, J. W., & Jencks, W. P., (1996) Biochemistry 35, 2750-2753]; this rate ratio is similar to that for phosphorylation of the calcium ATPase with and without initial incubation with Ca2+ [Petithory, J. R., & Jencks, W. P. (1986) Biochemistry 25, 4493-4497]. The K0.5 for ATP is 18 +/- 3 microM for the free enzyme, which is about 1/4 of K0.5 = 75 microM for enzyme that was preincubated with Na+. Addition of ADP to ATP and Na+ decreases the yield of E approximately P progressively with increasing ADP concentration. Upon an increase of [ADP] from 0 to 2.0 mM, the rate constant for phosphorylation decreases 4-fold (167 to 41 s-1) at low [ATP] (0.25 mM) and about 2.7-fold (174 to 64 s-1) at high [ATP] (2.0 mM). The absence of an induction period for phosphorylation of E upon the addition of saturating concentrations of ATP and Na+ indicates that all the prior reaction steps are much faster than the rate-limiting step. These results are consistent with a rate-determining conformational change of the E.ATP.Na3 intermediate. The decrease of the rate constant with increasing [ADP] is attributed to competition between ATP and ADP for the free enzyme.

Phosphorylation of the sodium--potassium adenosinetriphosphatase proceeds through a rate-limiting conformational change followed by rapid phosphoryl transfer.

The sodium-potassium adenosinetriphosphatase of sheep kidney, preincubated with sodium and magnesium (E.Nae), reacts with 0.01-2.00 mM ATP to form covalent phosphoenzyme (E-P). The first order rate constant for phosphorylation increases hyperbolically with ATP concentration with a maximum value of (4.6 +/- 0.9) x 10(2) s-1 and K0.5 = 75 +/- 25 microM (ph 7.4, 25 degrees C, 120 mM NaCl, and 3 mM MgCl2). If the phosphoryl-transfer step were rate-limiting, the approach to equilibrium to give 50% E-P in the presence of ADP would follow kobsd=Kf+Kr+9.2 x 10(2) s-1. However, the formation of phosphoenzyme from E.Na3 with 1.0 mM ATP plus 2.0 mM ADP proceeds to 50% completion with kobsd=(4.2 +/- 0.8) x 10(2) s-1. This result show that phosphoryl transfer from bound ATP to the enzyme is not the rate limiting step for phosphoenzyme formation from E.Na3. The result is consistent with a rate-limiting conformational change of the E.Na3.ATP intermediate that is followed by rapid phosphoryl transfer, with kcat > or = 3000 s-1.

The mechanism of coupling chemical and physical reactions by the calcium ATPase of sarcoplasmic reticulum and other coupled vectorial systems.

The coupling of the chemical reaction of ATP hydrolysis to the transport of calcium from the cytoplasm into the lumen of sarcoplasmic reticulum vesicles can be defined by a set of rules that define alternating changes in the specificities of the enzyme for catalysis of chemical and physical reactions.

Role of binding energy with coenzyme A in catalysis by 3-oxoacid coenzyme A transferase.

Succinyl-CoA:3-oxoacid coenzyme A transferase (EC 2.8.3.5), which catalyzes the reversible conversion of succinyl-CoA and acetoacetate into acetoacetyl-CoA and succinate through a covalent enzyme thiol ester intermediate, E-CoA, utilizes binding energy from noncovalent interactions with CoA to bring about an increase in kcat/KM of approximately 10(10)-fold. The approximately 40-fold stronger binding of desulfo-CoA (KI = 2.7 +/- 0.7 mM) compared to desulfopantetheine (KI = 110 +/- 15 mM), both of which inhibit competitively with respect to acetoacetyl-CoA, shows that binding to the nucleotide domain of CoA at the active site provides ca. -2.2 kcal/mol of binding energy to stabilize noncovalent complexes with the enzyme. This is much smaller than the ca. -8.9 kcal/mol that the nucleotide domain contributes to the stabilization of the transition state and the ca. -7.2 kcal/mol that it contributes to stabilizing the E-CoA intermediate [Fierke, C. A., & Jencks, W. P. (1986) J. Biol. Chem. 261, 7603-7606]. This shows that most of the approximately 10(6)-fold increase in kcat/KM that is brought about by binding to this domain is in kcat, which is increased by a factor of about 10(5). Binding to the central pantoic acid domain of CoA is stronger in the transition state than in the Michaelis complex by ca. -3.4 kcal/mol; this corresponds to an additional increase in kcat of approximately 350-fold. Covalent enzyme thiol esters analogous to E-CoA but containing the short-chain CoA analogues N-acetylaletheine (NAA) and N-acetylcysteamine (NAC) are more stable than the enzyme thiol ester containing pantetheine (E-Pant) by approximately 3.5 and approximately 4.8 kcal/mol, respectively. Thus, interactions between the pantoic acid domain of CoA and the active site destabilize E-CoA by approximately 4.8 kcal/mol, approximately 1.3 kcal/mol of which arises from interaction with the amide group of the pantoic acid domain and approximately 3.5 kcal/mol of which arises from interaction with other portions of the pantoic acid domain. E-Pant is more reactive toward acetoacetate and succinate by a factor of approximately 10(7) than E-NAA and E-NAC. This shows that the destabilization caused by these interactions in E-CoA is relieved in the transition state, in which binding to the pantoic acid moiety is strongly favorable with delta delta G approximately -5.2 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS)

There is only one phosphoenzyme intermediate with bound calcium on the reaction pathway of the sarcoplasmic reticulum calcium ATPase.

Identical first-order rate constants for phosphorylation of the calcium ATPase of sarcoplasmic reticulum by bound inorganic phosphate (Pi) of 25 +/- 2 s-1 with empty vesicles and 25 +/- 1 s-1 with vesicles that were passively loaded with 40 mM Ca2+ were obtained by treating the reaction as an approach to equilibrium (4 mM [32P]Pi, 20 mM MgCl2, 10 mM EGTA, and 100 mM KCl at pH 7.0 and 25 degrees C). The formation of ADP-sensitive phosphoenzyme from Pi with Ca(2+)-loaded vesicles also proceeds with a first-order rate constant of 25 s-1 and no detectable induction period. These identical rate constants show that lumenal Ca2+ does not inhibit the rate of phosphorylation of the enzyme by bound Pi and that there is no significant kinetic barrier for the conformational change that converts an ADP-insensitive to an ADP-sensitive phosphoenzyme intermediate with bound Ca2+. We conclude that there is no evidence for the existence of two stable phosphoenzyme intermediates with bound Ca2+, such as E1 approximately P.Mg.Ca2 and Ca2.E2-P.Mg, that are included in the E1-E2 and related two-state models for calcium transport by this enzyme. In general, coupling of a physical reaction, such as muscle contraction or vectorial transport, to a chemical reaction, such as ATP hydrolysis, requires more than two states in the reaction cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate specificity for catalysis of phosphoryl transfer by the calcium ATPase of sarcoplasmic reticulum.

When alpha,beta-methylene ADP (alpha,beta-CH2-ADP) is added to the phosphorylated calcium ATPase of sarcoplasmic reticulum with Ca(2+)-bound, Ca2.E approximately P.Mg, alpha,beta-methylene ATP is not synthesized (5 mM MgCl2, 100 mM KCl, pH 7.0, 25 degrees C). Similarly, adenosine 5'-O-(2-thiotriphosphate) is not synthesized from reaction of the phosphoenzyme with adenosine 5'-O-(2-thiodiphosphate), ADP beta S. In contrast, ATP is formed rapidly and reversibly from the reaction of the phosphoenzyme with ADP. Both ADP analogs are competitive inhibitors for the binding of ADP to the phosphoenzyme with KADPS = 0.45 mM: alpha,beta-CH2-ADP and ADP beta S bind to the phosphoenzyme with K alpha,beta-CH2-ADPS = 0.92 mM and KADP beta SS = 0.05 mM, respectively. We conclude that phosphoryl transfer from the phosphoenzyme to alpha,beta-CH2-ADP is kinetically blocked, although it is thermodynamically favorable. The rate acceleration of > 10(5) for phosphoryl transfer from Ca2.E approximately P.Mg to ADP compared to alpha,beta-CH2-ADP can be attributed to the differences in both the structure and the net charge of ADP compared with alpha,beta-CH2-ADP at pH 7.0. Phosphoryl transfer from the phosphoenzyme to ADP beta S is thermodynamically so unfavorable that we cannot determine whether the transition state is also unfavorable.

Alkaline phosphatase is an almost perfect enzyme.

The second-order rate constant, kcat/km, for catalysis of the hydrolysis of 4-nitrophenyl phosphate by alkaline phosphatase decreases with increasing viscosity in the presence of sucrose or arabinose, with a slope of delta[kcat/Km)0/(kcat/Km)]/delta(eta/eta 0) = 1.4 at pH 8.0, 25 degrees C. This is consistent with rate-limiting diffusional encounter of the substrate with active enzyme and indicates that alkaline phosphatase is a "perfect enzyme". However, the reported second-order rate constants of kcat/Km = 6.6 x 10(6) to 4.6 x 10(7) M-1 s-1 are smaller than the diffusional limit; this shows that only approximately 0.1-1% of the diffusional encounters are productive. The first-order rate constant, kcat, for rate-limiting hydrolysis of the phosphoenzyme intermediate at pH = 6 with saturating substrate concentration is independent of viscosity in aqueous sucrose solutions. This shows that sucrose does not destabilize the transition state for phosphoenzyme hydrolysis. However, at pH 8.0 product dissociation is rate limiting and kcat decreases with increasing viscosity in the presence of sucrose, with slopes of delta(k0/kobsd)/delta(eta/eta 0) = 1.2 in 0.04 M Mops buffer, 1.0 in 0.1 M Tris, and 1.2 in 0.67 M Tris buffer. This is consistent with rate-limiting diffusional separation of inorganic phosphate and of Tris phosphate from the enzyme. In contrast, glycerol causes a large decrease in kcat/Km at pH 8.0 and also decreases kcat at pH 6. This shows that glycerol decreases the rate by a solvent effect on the catalytic activity of the enzyme, as well as by increasing the viscosity.

Lumenal and cytoplasmic binding sites for calcium on the calcium ATPase of sarcoplasmic reticulum are different and independent.

The calcium ATPase of sarcoplasmic reticulum reacts with inorganic phosphate (Pi) to form phosphoenzyme that can bind two Ca2+ ions from the lumen of intact vesicles. Therefore, as the concentration of lumenal Ca2+ is increased, the concentration of phosphoenzyme at equilibrium increases; however, it levels off at lower maximal concentrations with decreasing concentrations of Pi. This requires that two Ca2+ ions can bind to lumenal binding sites of both the phosphoenzyme and the unphosphorylated enzyme. If lumenal Ca2+ could bind only to the phosphoenzyme, saturating concentrations of lumenal Ca2+ would drive phosphoenzyme formation to completion even at low concentrations of Pi. Phosphorylation is inhibited by cytoplasmic Ca2+ with K0.5 = 2.1 and 4 microM in the absence and in the presence of 40 mM lumenal Ca2+, respectively. K0.5 = 4 microM is much lower than K0.5 = 70 microM, which is expected if lumenal Ca2+ could bind only to the phosphoenzyme. Occupancy of the lumenal sites on the unphosphorylated enzyme by Ca2+ does not significantly change the rate constants of kp = 220 s-1 for phosphorylation by ATP, kCa = 90 s-1 for dissociation of Ca2+, and kMg = 50 s-1 for dissociation of Mg2+. We conclude that the calcium ATPase has two low-affinity lumenal Ca(2+)-binding sites that are independent of the high-affinity cytoplasmic Ca(2+)-binding sites. The results are consistent with a mechanism of Ca2+ transport in which phosphorylation of the enzyme by ATP drives the translocation of two Ca2+ ions from the high-affinity to the low-affinity sites.

Catalysis of the hydrolysis of phosphorylated pyridines by alkaline phosphatase has little or no dependence on the pKa of the leaving group.

Bacterial alkaline phosphatase is an active catalyst for the hydrolysis of N-phosphorylated pyridines, with values of the second-order rate constant kcat/Km in the range 0.4-1.2 x 10(6) M-1 s-1 at pH 8.0, 25 degrees C. There is little or no dependence of the rate on the pKa of the leaving group; the value of beta 1g is 0 +/- 0.05, which may be compared with beta 1g = -1.0 for the nonenzymic reaction. Phosphorylated pyridines do not have a free electron pair available for protonation or coordination of the leaving group. Therefore, this result means that the similar, small dependence on leaving group structure for the enzyme-catalyzed hydrolysis of phosphate esters [Hall, A. D., & Williams, A. (1986) Biochemistry 25, 4784-4790) does not provide evidence for general acid catalysis or electrophilic assistance of leaving group expulsion. The results are consistent with the hypothesis that productive binding of the substrate, which may involve a conformational change, is largely rate limiting for turnover of the enzyme at low substrate concentrations.

Calcium ATPase of sarcoplasmic reticulum has four binding sites for calcium.

The calcium-transporting ATPase of sarcoplasmic reticulum is known to bind two Ca2+ ions from the cytoplasm to the free enzyme and two Ca2+ ions from the lumen to the phosphoenzyme. The concentration of phosphoenzyme formed at equilibrium from Pi and Mg2+ increases with increasing concentration of calcium in the lumen, which binds to the phosphoenzyme to form Ca2.E approximately P.Mg. However, at subsaturating concentrations of Mg2+ increasing the concentration of lumenal Ca2+ does not drive phosphoenzyme formation to completion. The maximal levels of phosphoenzyme that are formed at saturating concentrations of lumenal Ca2+ increase with increasing concentrations of Mg2+. This result requires that Ca2+ can bind to low-affinity lumenal sites on both the free enzyme and the phosphoenzyme, as well as to the high-affinity cytoplasmic calcium-binding sites. If there were no lumenal binding sites for Ca2+ on the free enzyme, high concentrations of lumenal Ca2+ would convert all of the enzyme to the same maximal concentration of Ca2.E approximately P.Mg at subsaturating concentrations of Mg2+ and Pi. We conclude that there are two low-affinity lumenal sites as well as two high-affinity cytoplasmic sites for Ca2+ on the free enzyme. Phosphorylation by ATP results in translocation of Ca2+ from the high-affinity to the low-affinity sites.

The binding of ATP and Mg2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum follows a random mechanism.

The enzyme form of the calcium adenosinetriphosphatase of sarcoplasmic reticulum (CaATPase) that is stable in the presence of calcium, cE.Ca2, has a binding site for the catalytic Mg2+ ion with a dissociation constant of 0.94 +/- 0.15 mM at 25 degrees C, pH 7.0, and 100 mM KCl. This is approximately 10 times smaller than that reported for the free enzyme, E, (8.8 mM) under similar conditions [Punzengruber, C., Prager, R., Kolassa, N., Winkler, F., & Suko, J. (1978) Eur. J. Biochem. 92, 349-359]. This difference shows that the sites for the catalytic and the transported ions interact in the absence of ATP. The addition of ATP and EDTA to enzyme that had been incubated with Ca2+ and Mg2+ resulted in the formation of 61% phosphoenzyme. The addition of unlabeled ATP and Mg2+ to enzyme that had been incubated with 3.5 microM free Ca2+ and labeled ATP gave 39% labeled phosphoenzyme. This shows that the binding of ATP and Mg2+ to cE.Ca2 follows a random mechanism. The rate constants for dissociation of ATP and Mg2+ from cE.Ca2.ATP.Mg are different: kdiss(ATP) = 120 s-1 and kdiss(Mg2+) = 60 s-1. This shows that Mg2+ and ATP can bind and dissociate independently; they do not have to associate or dissociate from cE as a Mg.ATP complex. Calcium-free enzyme binds metal-free ATP at the active site with a dissociation constant of 44 +/- 4 microM, kdiss = 130 +/- 7 s-1, and a calculated association rate constant of 3 x 10(6) M-1 s-1. Calcium-free enzyme that was incubated with [gamma-32P]ATP gave 38% labeled phosphoenzyme when chased with unlabeled ATP, Mg2+, and Ca2+. An increase of the Mg2+ concentration did not increase the amount of E32P formed. This shows that the binding of Mg2+ and ATP to free E also follows a random mechanism. The Mg2+ ion is not buried under ATP, and ATP is not under a Mg2+ ion. Incubation of free E with Mg2+ and ATP causes a conformational change that activates the enzyme for phosphorylation and decreases the rate constant for the dissociation of ATP from kdiss = 120 s-1 to kdiss = 47 s-1.

The kinetics for the phosphoryl transfer steps of the sarcoplasmic reticulum calcium ATPase are the same with strontium and with calcium bound to the transport sites.

Rate constants for most of the steps of the reaction cycle of the sarcoplasmic reticulum calcium-ATPase are similar or identical with Ca2+ or Sr2+ as the transported ions in spite of the large differences in the size and affinity of Ca2+ and Sr2+ (5 mM MgCl2, 100 mM KCl, pH 7.0, 25 degrees C). Phosphorylation of cE.Sr2 and cE.Ca2 by ATP occurs with kp = 220-235 s-1, whereas phosphorylation of E.ATP+Ca2+ or Sr2+ is consistent with kb = 50-70 s-1. Hydrolysis of E approximately P.Sr2 and E approximately P.Ca2 occurs with kt = 20 s-1, and the addition of 7 mM ADP to E approximately P.Sr2 or to E approximately P.Ca2 gives a burst of approximately 43% dephosphorylation, followed by dephosphorylation with k = 46 s-1. However, one Sr2+ ion dissociates from cE.Sr2 and from cE.ATP.Sr2 with k congruent to 120 s-1, whereas one Ca2+ ion dissociates from cE.Ca2 with k = 38 s-1 and from cE.ATP.Ca2 with k = 80 s-1.

Binding of two Sr2+ ions changes the chemical specificities for phosphorylation of the sarcoplasmic reticulum calcium ATPase through a stepwise mechanism.

The sequential binding of Sr2+ and Ca2+ to the cytoplasmic transport sites of the sarcoplasmic reticulum calcium ATPase allows the formation of two different mixed complexes: cE.Sr.Ca, with Sr2+ bound to the "inner" site and Ca2+ bound to the "outer" site, and cE. Ca.Sr, with Ca2+ bound to the inner site and Sr2+ bound to the outer site (pH 7.0, 25 degrees C, 10 mM MgCl2, 100 mM KCl). Both cE.Sr.45Ca and cE.45Ca.Sr react with ATP to internalize one 45Ca/phosphoenzyme. The value of K0.5 = 83 microM Sr2+ for activation of the enzyme for phosphorylation by ATP is much larger than K0.5 = 28 microM Sr2+ for inhibition of phosphoenzyme formation from inorganic phosphate (eta H = 1.0-1.3). These results are consistent with the sequential binding of two strontium ions with negative cooperativity and dissociation constants of KSr1 = 35 microM and KSr2 = 55 microM. The species cE.Sr2 and cE.Ca2 react rapidly with ATP but not inorganic phosphate. However, enzyme with one strontium bound, cE.Sr, does not react with either inorganic phosphate or ATP. Therefore, the conformational changes in the enzyme that alter the chemical specificity for phosphorylation by ATP and by inorganic phosphate are different. This requires the existence of at least three forms of the unphosphorylated enzyme with three different chemical specificities for catalysis.

Dissociation of calcium from the phosphorylated calcium-transporting adenosine triphosphatase of sarcoplasmic reticulum: kinetic equivalence of the calcium ions bound to the phosphorylated enzyme.

The internalization of 45Ca by the calcium-transporting ATPase into sarcoplasmic reticulum vesicles from rabbit muscle was measured during a single turnover of the enzyme by using a quench of 7 mM ADP and EGTA (25 degrees C, 5 mM MgCl2, 100 mM KCl, 40 mM MOPS.Tris, pH 7.0). Intact vesicles containing either 10-20 microM or 20 mM Ca2+ were preincubated with 45Ca for approximately 20 s and then mixed with 0.20-0.25 mM ATP and excess EGTA to give 70% phosphorylation of Etot with the rate constant k = 300 s-1. The two 45Ca ions bound to the phosphoenzyme (EP) become insensitive to the quench with ADP as they are internalized in a first-order reaction with a rate constant of k = approximately 30 s-1. The first and second Ca2+ ions that bind to the free enzyme were selectively labeled by mixing the enzyme and 45Ca with excess 40Ca, or by mixing the enzyme and 40Ca with 45Ca, for 50 ms prior to the addition of ATP and EGTA. The internalization of each ion into loaded or empty vesicles follows first-order kinetics with k = approximately 30 s-1; there is no indication of biphasic kinetics or an induction period for the internalization of either Ca2+ ion. The presence of 20 mM Ca2+ inside the vesicles has no effect on the kinetics or the extent of internalization of either or both of the individual ions. The Ca2+ ions bound to the phosphoenzyme are kinetically equivalent. A first-order reaction for the internalization of the individual Ca2+ ions is consistent with a rate-limiting conformational change of the phosphoenzyme with kc = 30 s-1, followed by rapid dissociation of the Ca2+ ions from separate independent binding sites on E approximately P.Ca2; lumenal calcium does not inhibit the dissociation of calcium from these sites. Alternatively, the Ca2+ ions may dissociate sequentially from E approximately P.Ca2 following a rate-limiting conformational change. However, the order of dissociation of the individual ions can not be distinguished. An ordered-sequential mechanism for dissociation requires that the ions dissociate much faster (k greater than or equal to 10(5) s-1) than the forward and reverse reactions for the conformational change (k-c = approximately 3000 s-1). Finally, the Ca2+ ions may exchange their positions rapidly on the phosphoenzyme (kmix greater than or equal to 10(5) s-1) before dissociating. A Hill slope of nH = 1.0-1.2, with K0.5 = 0.8-0.9 mM, for the inhibition of turnover by binding of Ca2+ to the low-affinity transport sites of the phosphoenzyme was obtained from rate measurements at six different concentrations of Mg2+.

The vectorial specificity for calcium binding to the CaATPase of sarcoplasmic reticulum is controlled by phosphorylation, not by an E-E* conformational change.

The E-E* model for calcium pumping by the CaATPase of sarcoplasmic reticulum includes two distinct conformational states of the enzyme, E and E*. Exterior Ca2+ binds only to E and interior Ca2+ binds only to E*. Therefore, it is expected that there will be competition between the binding of calcium to the unphosphorylated enzyme from the two sides of the membrane. The equilibrium concentration of cECa2, the enzyme with Ca2+ bound at the exterior site, was measured at different Ca2+ concentrations with empty sarcoplasmic reticulum vesicles (SRV) and with SRV loaded with 40 mM Ca2+ by reaction with 0.5 mM [gamma-32P]ATP plus 20 mM EGTA for 13 ms (100 mM KCl, 5 mM MgSO4, 40 mM Mops/KOH, pH 7.0, 25 degrees C). The sigmoidal dependence on free exterior calcium concentration of the concentration of cECa2, measured as [32P]phosphoenzyme, is identical with empty and loaded SRV, within experimental error. The value of K0.5 is 2.8 microM, and the Hill coefficient is 2. This result shows that there is no competition between binding of Ca2+ to the outside and the inside of the membrane. This is consistent with a model in which the vectorial specificity for calcium binding is controlled by the chemical state of the enzyme, rather than a simple conformational change. It is concluded that there are not two interconverting forms of the free enzyme, E and E*, instead the vectorial specificity for binding and dissociation of Ca2+ is determined by the state of phosphorylation of the CaATPase.