Modulation of the binding characteristics of a fluorescent nucleotide derivative to the sarcoplasmic reticulum adenosinetriphosphatase

Trinitrophenyladenosine monophosphate (TNP-AMP) binding to the phosphorylated Ca2+-ATPase of sarcoplasmic reticulum results in manyfold higher fluorescence intensity and longer lifetimes of the nucleotide analogue, as compared to TNP-AMP binding to the nonphosphorylated enzyme. This is observed when the phosphoenzyme intermediate is formed either from ATP or from inorganic phosphate (Pi). An important question is whether the TNP-AMP fluorescence properties can also reflect the kinetically defined interconversions of different phosphoenzyme species during catalysis. We have approached this question by manipulating the phosphorylation conditions in a manner which is known to result in accumulation of different species of the phosphoenzyme, i.e., by variations in pH, substrates, and K+ and Ca2+ concentrations. Decreasing pH or increasing [K+] caused large decreases in fluorescence intensity at a given concentration of TNP-AMP under conditions of phosphorylation with either ATP or Pi. In contrast, low to high intravesicular Ca2+ concentrations had no effect on fluorescence during steady-state turnover. TNP-AMP titrations of the phosphorylated enzyme stabilized in different states revealed that H+ and K+ caused a shift in TNP-AMP binding affinity to the site responsible for high fluorescence enhancement, while maintaining approximately the same maximal fluorescence yield at saturation. The fluorescence lifetimes of TNP-AMP bound to phosphoenzyme did not change with variations in pH, [K+], and substrates. We conclude that the environment of that part of the TNP-AMP binding site which binds the trinitrophenyl moiety undergoes a change upon enzyme phosphorylation resulting in enhanced fluorescence yield; this change is invariant between different phosphoenzyme species.(ABSTRACT TRUNCATED AT 250 WORDS)     

2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine mono-, di-, and triphosphates as photoaffinity probes of the Ca2+-ATPase of sarcoplasmic reticulum. Regulatory/superfluorescent nucleotides label the catalytic site with high efficiency

We have synthesized a new class of ATP photo-affinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido (TNP-8N3)-ATP, -ADP, and -AMP, and their radiolabeled derivatives, and characterized their interaction with sarcoplasmic reticulum vesicles. The nucleotides bind with high affinity (Kd = 0.04-0.4 microM) to the catalytic site of the Ca2+-ATPase. TNP-8N3-ATP and TNP-8N3-ADP, at low concentrations (less than 10 microM), accelerate ATPase activity 1.5- and 1.4-fold, respectively, indicating that they bind to a regulatory site. In the same concentration range, they all undergo a large increase in fluorescence ("superfluorescence") during enzyme turnover in the presence of ATP and Ca2+, or on phosphorylation from Pi in a Ca2+-depleted medium. Irradiation at alkaline pH results in specific covalent incorporation of the nucleotide at the catalytic site on the A1 tryptic subfragment. The efficiency of catalytic site labeling is greatest (up to 80% of available sites/irradiation period) in the presence of ATP, Ca2+, and Mg2+, conditions in which the probe binds only to the regulatory and superfluorescent sites. The covalently attached nucleotide exhibits fluorescence enhancement on enzyme turnover in the presence of acetyl phosphate plus Ca2+ or on phosphorylation from Pi in a Ca2+-depleted medium, but not in the presence of ATP plus Ca2+. The results suggest that the catalytic, regulatory, and superfluorescent nucleotide sites are at the same locus and that the binding domain includes portions of the A1 subfragment. The high efficiency with which the site is photolabeled during turnover is ascribed to water exclusion and possibly cleft closure in E2-P.     

A conformational change of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled sarcoplasmic reticulum Ca2+-ATPase upon ATP binding to the catalytic site

Sarcoplasmic reticulum vesicles were modified with a fluorescent thiol reagent, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. One mol of readily reactive thiols per mol of the Ca2+-ATPase was labeled without a loss of the catalytic activity. The fluorescence of the label increased by 8% upon binding of Ca2+ to the high affinity sites of the enzyme. This fluorescence enhancement probably reflects a conformational change responsible for Ca2+-induced enzyme activation. Upon addition of ATP to the Ca2+-activated enzyme, the fluorescence decreased by 15%. This fluorescence drop and formation of the phosphoenzyme intermediate were determined under the same conditions with a stopped-flow apparatus and a rapid quenching system. The amplitude of the fluorescence drop thus determined was saturated with 3 microM ATP. This shows that the fluorescence drop was caused by ATP binding to the catalytic site. In contrast, the rate of the fluorescence drop was not saturated even with 50 microM ATP. The fluorescence drop coincided with phosphoenzyme formation at 0.5 or 3 microM ATP, but it became much faster than phosphoenzyme formation when the ATP concentration was raised to 100 microM. These results indicate that the ATP-induced fluorescence drop reflects a conformational change in the enzyme.ATP complex. The fluorescence drop was accompanied by a red spectrum shift, which suggests that the label was exposed to a more hydrophilic environment. The electrophoretic analysis of the tryptic digest of the labeled enzyme (10.9 kDa) showed that almost all of the label was located on the 5.2-kDa fragment which includes the carboxyl terminus and the putative ATP-binding domain. The sequencing of the two major labeled peptides, which were isolated from the thermolytic digest of the labeled enzyme, revealed that the labeled site in either of these peptides was Cys674. It seems likely that the label bound to this Cys674 could be involved in the observed fluorescence changes.     

Functional role of "N" (nucleotide) and "P" (phosphorylation) domain interactions in the sarcoplasmic reticulum (SERCA) ATPase

Experimental perturbations of the nucleotide site in the N domain of the SR Ca2+ ATPase were produced by chemical derivatization of Lys492 or/and Lys515, mutation of Arg560 to Ala, or addition of inactive nucleotide analogue (TNP-AMP). Selective labeling of either Lys492 or Lys515 produces strong inhibition of ATPase activity and phosphoenzyme intermediate formation by utilization of ATP, while AcP utilization and reverse ATPase phosphorylation by Pi are much less affected. Cross-linking of the two residues with DIDS, however, drastically inhibits utilization of both ATP and AcP, as well as of formation of phosphoenzyme intermediate by utilization of ATP, or reverse phosphorylation by Pi. Mutation of Arg560 to Ala produces strong inhibition of ATPase activity and enzyme phosphorylation by ATP but has a much lower effect on enzyme phosphorylation by Pi. TNP-AMP increases the ATPase activity at low concentrations (0.1-0.3 microM), but inhibits ATP, AcP, and Pi utilization at higher concentration (1-10 microM). Cross-linking with DIDS and TNP-AMP binding inhibits formation of the transition state analogue with orthovanadate. It is concluded that in addition to the binding pocket delimited by Lys 492 and Lys515, Arg560 sustains an important and direct role in nucleotide substrate stabilization. Furthermore, the effects of DIDS and TNP-AMP suggest that approximation of N (nucleotide) and P (phosphorylation) domains is required not only for delivery of nucleotide substrate, but also to favor enzyme phosphorylation by nucleotide and nonnucleotide substrates, in the presence and in the absence of Ca2+. Domain separation is then enhanced by secondary nucleotide binding to the phosphoenzyme, thereby favoring its hydrolytic cleavage.     

Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphoenzyme conformational states and nucleotide-binding site hydrophobicity following thiol modification of the Ca2+-ATPase of sarcoplasmic reticulum from skeletal muscle

Enhanced fluorescence of the ATP analogue 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)adenosine 5'-triphosphate (TNP-ATP), bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, is closely related to phosphoenzyme levels (Bishop, J. E., Johnson, J. D., and Berman, M. C. (1984) J. Biol. Chem. 259, 15163-15171) and has an emission maximum consistent with decreased polarity of the TNP-ATP-binding site. The phosphoenzyme conformation responsible for increased nucleotide-binding site hydrophobicity has been studied by redistribution of phosphoenzyme intermediates following specific thiol group modification. N-Ethylmaleimide, in the presence of 50 microM Ca2+, 1 mM adenyl-5'-yl imidodiphosphate, pH 7.0, at 25 degrees C for 30 min, selectively modified the SH group essential for phosphoenzyme decomposition, which resulted in decreased ATPase activity, Ca2+ uptake, and a decrease in ATP-induced TNP-ATP fluorescence. Phosphorylated (Ca2+, Mg2+)-ATPase levels from [gamma-32P] ATP remained relatively unaffected (3.1 nmol/mg), but the ADP-insensitive fraction decreased from 56 to 15%. Phosphoenzyme levels from 32Pi were also decreased to the same extent as turnover, with equivalent loss of Pi-induced TNP-ATP fluorescence. The E1 to E2 transition, as monitored by the change in intrinsic tryptophan fluorescence, was unaffected. Modification of thiol groups of unknown function did not modify turnover-induced TNP-ATP fluorescence. It is concluded that the ADP-insensitive phosphoenzyme, E2-P, is responsible for enhanced TNP-ATP fluorescence. This suggests that the conformational transition, 2Ca2+outE1 approximately P----2Ca2+inE2-P, is associated with altered properties of the noncatalytic, or regulatory, nucleotide-binding site.     

Characterization of the substrate-induced conformational change of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled sarcoplasmic reticulum Ca2(+)-ATPase by using different kinds of substrate

Cys-674 of the sarcoplasmic reticulum Ca2(+)-ATPase was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity, and changes in the fluorescence intensity upon addition of seven kinds of substrate were followed by the stopped-flow method. The steady-state fluorescence intensity and anisotropy were also determined. When Ca2+ was present, the fluorescence intensity and anisotropy decreased greatly upon addition of any substrate used. The observed affinity for each substrate agreed with the previously observed affinity of the catalytic site. The fluorescence drop induced by the adenine nucleotides, ATP and adenosine 5'-(beta, gamma-methylene)triphosphate (a nonhydrolyzable ATP analog), was much faster than that induced by other substrates. The ATP-induced fluorescence drop preceded phosphoenzyme formation when the ATP concentration was high, but the fluorescence drop coincided with phosphoenzyme formation when it was slowed by reducing ATP concentrations. The fluorescence drop induced by ITP or acetyl phosphate was slow even at high concentrations of the substrate, and it coincided with phosphoenzyme formation. When Ca2+ was absent, the fluorescence intensity and anisotropy decreased only slightly upon addition of any substrate other than the adenine nucleotides. They decreased substantially upon addition of the adenine nucleotides, but the kinetics of this fluorescence drop were quite different from that of the fluorescence drop induced by any substrate in the presence of Ca2+. These results show that the conformational change, which makes the bound label less constrained, is induced by substrate binding to the catalytic site of the Ca2(+)-activated enzyme. This change precedes phosphoenzyme formation in the catalytic cycle and is greatly accelerated by the adenine moiety of the substrate.     

ATP regulation of sarcoplasmic reticulum Ca2+-ATPase. Metal-free ATP and 8-bromo-ATP bind with high affinity to the catalytic site of phosphorylated ATPase and accelerate dephosphorylation

To localize and characterize the regulatory nucleotide site of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase, we have investigated the effects of ADP, ATP, and analogues of these nucleotides on the rate of dephosphorylation of both native ATPase and ATPase modified with fluorescein 5'-isothiocyanate (FITC), a reagent which hinders access of nucleotides to the ATPase catalytic site without affecting phosphorylation from Pi. Dephosphorylation of the phosphoenzyme formed from Pi was monitored by rapid filtration or stopped-flow fluorescence, mostly at 20 degrees C, pH 6.0, and in the absence of potassium. Fluorescence measurements were made possible through the use of 8-bromo-ATP, which selectively quenched certain tryptophan residues of the ATPase, thereby allowing the intrinsic fluorescence changes associated with dephosphorylation to be measured in the presence of bound nucleotide. ATP, 8-bromo-ATP, and trinitrophenyladenosine diand triphosphate, but not ADP, enhanced the rate of dephosphorylation of native ATPase 2-3-fold when added in the absence of divalent cations. Millimolar concentrations of Mg2+ eliminated the accelerating effects. Acceleration in the absence of Mg2+ was observed at relatively low concentrations of ATP and 8-bromo-ATP (0.01-0.1 mM) and binding of metal-free ATP and ADP, but not Mg.ATP, to the phosphoenzyme in this concentration range was demonstrated directly. Modification of the ATPase with FITC blocked nucleotide binding in the submillimolar concentration range and eliminated the nucleotide-induced acceleration of dephosphorylation. These results show that dephosphorylation, under these conditions, is regulated by ATP but not by Mg.ATP or ADP, and that the catalytic site is the locus of this "regulatory" ATP binding site.     

Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.     

Evaluation of H2O activity in the free or phosphorylated catalytic site of Ca2+-ATPase

The sarcoplasmic reticulum Ca2+-ATPase catalyses a reversible calcium transport coupled to phosphate transfer between ATP and water. It has been proposed [Biochemistry (1980) 19, 4252-4261] that the reactivity of the acyl-phosphate bond is dependent on the water activity within the catalytic site. We have tested this hypothesis and found that the polarity in the free catalytic site is lower than that of water, a further and large decrease is observed when the enzyme is phosphorylated by Pi. Phosphorylation by ATP indicates that this polarity change is specifically associated with the formation of the ADP-insensitive phosphoenzyme.     

Vanadate inhibition of the Ca2+-ATPase from human red cell membranes

(1) VO3(-) combines with high affinity to the Ca2+-ATPase and fully inhibits Ca2+-ATPase and Ca2+-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state of the Ca2+-dependent phosphoenzyme. (2) VO3(-) blocks hydrolysis of ATP at the catalytic site. The sites for VO3(-) also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca2+-ATPase. (3) The sites for VO39-) show positive interaactions in affinity with sites for Mg2+ and K+. This accounts for the dependence on Mg2+ and K+ of the inhibition by VO3(-). Although, with less effectiveness, Na2+ and K+ substitutes for K+ whereas Li+ does not. The apparent affinites for Mg24 and K+ for inhibiton by VO3(-) seem to be less than those for activation of the Ca2+-ATPase. (4) Inhibition by VO3(-) is independent of Ca2+ at concentrations up to 50 microM. Higher concentrations of Ca2+ lead to a progressive release of the inhibitiory effect of VO3(-).     

Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum. The role of Mg2+ that activates hydrolysis of the phosphoenzyme

The role of Mg2+ in the activation of phosphoenzyme hydrolysis has been investigated with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. The enzyme of the native and solubilized vesicles was phosphorylated with ATP at 0 degrees C, pH 7.0, in the presence of Ca2+ and Mg2+. When Ca2+ and Mg2+ in the medium were chelated, phosphoenzyme hydrolysis continued for about 15 s and then ceased. The extent of this hydrolysis increased with increasing concentrations of Mg2+ added before the start of phosphorylation. This shows that the hydrolysis was activated by the Mg2+ added. The Mg2+ which activated phosphoenzyme hydrolysis was distinct from Mg2+ derived from MgATP bound to the substrate site. The Mg2+ site at which Mg2+ combined to activate phosphoenzyme hydrolysis was located on the outer surface of the vesicular membranes. During the catalytic cycle, Mg2+ combined with the Mg2+ site before Ca2+ dissociated from the Ca2+ transport site of the ADP-sensitive phosphoenzyme with bound Ca2+. This Mg2+ did not activate hydrolysis of the ADP-sensitive phosphoenzyme with bound Ca2+, but markedly activated hydrolysis of the ADP-insensitive phosphoenzyme without bound Ca2+. It is concluded that during the catalytic cycle, Mg2+ activates phosphoenzyme hydrolysis only after Ca2+ has dissociated from the Ca2+ transport site of phosphoenzyme.     

ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.     

ATP/ADP exchange activity of gastric (H+ +K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the (H+ +K+)-ATPase by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to P1, P5-di(adenosine-5') pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration (K0.5=116 microM) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+.     

Compound 48/80 and calmodulin modify the interaction of ATP with the (Ca2+ + Mg2+)-ATPase of red cell membranes

Compound 48/80, an anti-calmodulin agent, reduces the maximum effect of ATP and does not affect the apparent affinity for ATP of the high-affinity site of the Ca2+-ATPase from calmodulin-bound membranes of human red cells. In the same preparation, 48/80 reduces more than 50-times the apparent affinity for ATP of the low-affinity site with little change in the maximum effect of the nucleotide at this site of the Ca2+-ATPase. The effects of compound 48/80 are independent of the concentration of Ca2+ between 30 and 200 microM. The apparent affinity of the low-affinity site of the Ca2+-ATPase for ATP is almost 100-fold less in calmodulin-stripped membranes than in calmodulin-bound membranes. In calmodulin-stripped membranes, exogenous calmodulin increases the apparent affinity for ATP up to the control values. These results indicate that apart from increasing the apparent affinity of the transport site for Ca2+, calmodulin also increases the apparent affinity of the regulatory site of the Ca2+-ATPase for ATP. Since this effect is exerted within the physiological ranges of ATP concentrations, it may participate in the physiological regulation of Ca2+ pumping by calmodulin.     

Transient kinetic analysis of turnover-dependent fluorescence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP bound to Ca2+-ATPase of sarcoplasmic reticulum

The fluorescence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum is greatly enhanced during turnover induced by ATP plus Ca2+ (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). We have studied the kinetics of induction of TNP-ATP fluorescence and of its decay and have found a close correlation with levels of phosphorylated intermediate of the enzyme, E-P. Steady-state kinetic studies suggested competitive binding of ATP and TNP-ATP to the catalytic site, with Km and Ki values of 2.4 and 1.0 microM, respectively. Rate constants for fluorescence enhancement and for E-P formation in the presteady state were 1.2 s-1 or 97-130 s-1 under conditions resulting in TNP-ATP or ATP saturation respectively, of the enzyme at inception of reaction. The slow process was concluded to be the koff for dissociation of TNP-ATP from the catalytic site. Following this dissociation, a second TNP-ATP site was detected, which both formed (97-130 s-1) and decayed (0.22 s-1) synchronously with E-P. TNP-ATP binding to this noncatalytic site was rapid (5 X 10(7) M-1 s-1) and resulted in high fluorescence during steady-state turnover. Fluorescence was found to be dissociated from E-P by KCl (100 mM). KCl had little effect on E-P levels, but decreased fluorescence by 68%. These studies provide independent kinetic evidence for the existence of both catalytic and noncatalytic, or "regulatory," nucleotide-binding sites, but cannot distinguish whether the two sites exist independently or whether the catalytic site is transformed into a regulatory site on phosphorylation. The latter site, which shows relatively high selectivity for TNP-ATP over ATP, and which is simultaneously hydrophobic and freely accessible to the medium, may play a role during energy transduction. The changes occurring at this site during catalysis are conveniently monitored with TNP-ATP fluorescence.     

Functional characterization of lanthanide binding sites in the sarcoplasmic reticulum Ca(2+)-ATPase: do lanthanide ions bind to the calcium transport site?

Gd3+ binding sites on the purified Ca(2+)-ATPase of sarcoplasmic reticulum were characterized at 2 and 6 degrees C and pH 7.0 under conditions in which 45Ca2+ and 54Mn2+ specifically labeled the calcium transport site and the catalytic site of the enzyme, respectively. We detected several classes of Gd3+ binding sites that affected enzyme function: (a) Gd3+ exchanged with 54Mn2+ of the 54MnATP complex bound at the catalytic site. This permitted slow phosphorylation of the enzyme when two Ca2+ ions were bound at the transport site. The Gd3+ ion bound at the catalytic site inhibited decomposition of the ADP-sensitive phosphoenzyme. (b) High-affinity binding of Gd3+ to site(s) distinct from both the transport site and the catalytic site inhibited the decomposition of the ADP-sensitive phosphoenzyme. (c) Gd3+ enhanced 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence in NBD-modified enzyme by probably binding to the Mg2+ site that is distinct from both the transport site and the catalytic site. (d) Gd3+ inhibited high-affinity binding of 45Ca2+ to the transport site not by directly competing with Ca2+ for the transport site but by occupying site(s) other than the transport site. This conclusion was based mainly on the result of kinetic analysis of displacement of the enzyme-bound 45Ca2+ ions by Gd3+ and vice versa, and the inability of Gd3+ to phosphorylate the enzyme under conditions in which GdATP served as a substrate. These results strongly suggest that Ln3+ ions cannot be used as probes to structurally and functionally characterize the calcium transport site on the Ca(2+)-ATPase.     

Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex

The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H2O by D2O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex.     

Detection of the conformational change in the catalytic site of adenosine triphosphatase from beef liver mitochondria by affinity labeling with the dialdehyde derivative of ethenoadenosine triphosphate

Beef liver mitochondrial F1ATPase was inactivated by the 2',3'-dialdehyde derivative of ethenoATP (epsilon ATP) in a pseudo-first order reaction. The kinetics of protection of the enzyme against inactivation by various nucleoside triphosphates (NTPs) revealed that the dial-epsilon ATP was bound to the catalytic site as an affinity label. Certain anions (sulfate or bicarbonate) were ineffective for protection. In the early phase of the reaction, inactivation was due to the binding of 1 mol dial-epsilon ATP per mol enzyme. In this phase, dial-epsilon ATP bound exclusively to the subunit beta of the enzyme, indicating that the catalytic site is in this subunit. The fluorescence of the ethenoadenosine moiety, bound exclusively to the subunit beta of the enzyme, was measured as a conformational probe of the catalytic site region. Addition of ATP or CTP to the labeled enzyme resulted in a decrease in the fluorescence intensity. GTP and other NTPs were less effective than ATP or CTP. The anions (sulfate of bicarbonate) suppressed the ability of ATP to decrease the fluorescence in a competitive manner. Quantitative analysis of these fluorescence changes suggested that they might originate from the binding of the NTP to the regulatory site of the enzyme. These findings are in good agreement with the two-site model proposed by us (Wakagi, T. & Ohta, T. (1981) J. Biochem. 89, 1205) which was deduced from the steady state kinetics of the NTPase reactions catalyzed by the F1ATPase.     

Effects of calmodulin on the phosphoenzyme of the Ca2+-ATPase of human red cell membranes

Comparison of the effects of calmodulin on the Ca2+-ATPase activity and on the steady-state level of the phosphoenzyme, indicates that activation of the Ca2+-ATPase is mainly due to an increase in the turnover of the phosphoenzyme and does not require occupation of the regulatory site of the Ca2+-ATPase by ATP.