Phosphoenzyme conversion of the sarcoplasmic reticulum Ca(2+)-ATPase. Molecular interpretation of infrared difference spectra.

Time-resolved Fourier transform infrared difference spectra of the phosphoenzyme conversion and Ca(2+) release reaction (Ca(2)E(1)-P --> E(2)-P) of the sarcoplasmic reticulum Ca(2+)-ATPase were recorded at pH 7 and 1 degrees C in H(2)O and (2)H(2)O. In the amide I spectral region, the spectra indicate backbone conformational changes preserving conformational changes of the preceding phosphorylation step. beta-sheet or turn structures (band at 1685 cm(-1)) and alpha-helical structures (band at 1653 cm(-1)) seem to be involved. Spectra of the model compound EDTA for Ca(2+) chelation indicate the assignment of bands at 1570, 1554, 1411 and 1399 cm(-1) to Ca(2+) chelating Asp and Glu carboxylate groups partially shielded from the aqueous environment. In addition, an E(2)-P band at 1638 cm(-1) has been tentatively assigned to a carboxylate group in a special environment. A Tyr residue seems to be involved in the reaction (band at 1517 cm(-1) in H(2)O and 1515 cm(-1) in (2)H(2)O). A band at 1192 cm(-1) was shown by isotopic replacement in the gamma-phosphate of ATP to originate from the E(2)-P phosphate group. This is a clear indication that the immediate environment of the phosphoenzyme phosphate group changes in the conversion reaction, altering phosphate geometry and/or electron distribution.     

Phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase from ATP and ATP analogs studied by infrared spectroscopy

Phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) was studied with time-resolved Fourier transform infrared spectroscopy. ATP and ATP analogs (ITP, 2'- and 3'-dATP) were used to study the effect of the adenine ring and the ribose hydroxyl groups on ATPase phosphorylation. All modifications of ATP altered conformational changes and phosphorylation kinetics. The differences compared with ATP increased in the following order: 3'-dATP > ITP > 2'-dATP. Enzyme phosphorylation with ITP results in larger absorbance changes in the amide I region, indicating larger conformational changes of the Ca(2+)-ATPase. The respective absorbance changes obtained with 3'-dATP are significantly different from the others with different band positions and amplitudes in the amide I region, indicating different conformational changes of the protein backbone. ATPase phosphorylation with 3'-dATP is also much ( approximately 30 times) slower than with ATP. Our results indicate that modifications to functional groups of ATP (the ribose 2'- and 3'-OH and the amino group in the adenine ring) affect gamma-phosphate transfer to the phosphorylation site of the Ca(2+)-ATPase by changing the extent of conformational change and the phosphorylation rate. ADP binding to the ADP-sensitive phosphoenzyme (Ca(2)E1P) stabilizes the closed conformation of Ca(2)E1P.     

Toward a general method to observe the phosphate groups of phosphoenzymes with infrared spectroscopy

A general method to study the phosphate group of phosphoenzymes with infrared difference spectroscopy by helper enzyme-induced isotope exchange was developed. This allows the selective monitoring of the phosphate P-O vibrations in large proteins, which provides detailed information on several band parameters. Here, isotopic exchange was achieved at the oxygen atoms of the catalytically important phosphate group that transiently binds to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a). [gamma-(18)O(3)]ATP phosphorylated the ATPase, which produced phosphoenzyme that was initially isotopically labeled. The helper enzyme adenylate kinase regenerated the substrate ATP from ADP (added or generated upon ATP hydrolysis) with different isotopic composition than used initially. With time this produced the unlabeled phosphoenzyme. The method was tested on the ADP-insensitive phosphoenzyme state of the Ca(2+)-ATPase for which the vibrational frequencies of the phosphate group are known, and it was established that the helper enzyme is effective in mediating the isotope exchange process.     

P-O bond destabilization accelerates phosphoenzyme hydrolysis of sarcoplasmic reticulum Ca2+ -ATPase

The phosphate group of the ADP-insensitive phosphoenzyme (E2-P) of sarcoplasmic reticulum Ca2+ -ATPase (SERCA1a) was studied with infrared spectroscopy to understand the high hydrolysis rate of E2-P. By monitoring an autocatalyzed isotope exchange reaction, three stretching vibrations of the transiently bound phosphate group were selectively observed against a background of 50,000 protein vibrations. They were found at 1194, 1137, and 1115 cm(-1). This information was evaluated using the bond valence model and empirical correlations. Compared with the model compound acetyl phosphate, structure and charge distribution of the E2-P aspartyl phosphate resemble somewhat the transition state in a dissociative phosphate transfer reaction; the aspartyl phosphate of E2-P has 0.02 A shorter terminal P-O bonds and a 0.09 A longer bridging P-O bond that is approximately 20% weaker, the angle between the terminal P-O bonds is wider, and -0.2 formal charges are shifted from the phosphate group to the aspartyl moiety. The weaker bridging P-O bond of E2-P accounts for a 10(11)-10(15)-fold hydrolysis rate enhancement, implying that P-O bond destabilization facilitates phosphoenzyme hydrolysis. P-O bond destabilization is caused by a shift of noncovalent interactions from the phosphate oxygens to the aspartyl oxygens. We suggest that the relative positioning of Mg2+ and Lys684 between phosphate and aspartyl oxygens controls the hydrolysis rate of the ATPase phosphoenzymes and related phosphoproteins.     

Phosphorus-31 nuclear magnetic resonance of phosphoenzymes of sodium- and potassium-activated and of calcium-activated adenosinetriphosphatase

Prior studies identified phosphoenzyme intermediates in the turnover of sodium- and potassium-activated adenosinetriphosphatase [(Na,K)ATPase] from several sources and of the calcium-activated adenosinetriphosphatase [(Ca)-ATPase] of skeletal muscle sarcoplasmic reticulum. In both cases, the transphosphorylation is to a beta-aspartyl carboxyl group at the active site. We now report observation of a K+-sensitive phosphorylated intermediate of purified (Na,-K)ATPase from the salt gland of the duck using high-field 31P nuclear magnetic resonance. Addition of ATP to a suspension of this enzyme in the presence of Mg2+ and Na+ produced a resonance at about +17 ppm relative to 85% phosphoric acid. Addition of inorganic phosphate and Mg2+ to (Na,K)ATPase also produced a resonance at about +17 ppm which was enhanced in the presence of a saturating concentration of the inhibitor, ouabain; again, addition of K+ made this resonance disappear. These findings are consistent with earlier kinetic characterization of an acid-stable (Na,K)ATPase phosphoenzyme intermediate by 32P-labeled phosphate incorporation into a denatured precipitate of the enzyme. We attribute the +17-ppm resonance to formation of an acyl phosphate at an aspartyl residue of the catalytic site of (Na,K)ATPase. This is supported by our finding of a similar resonance at +17 ppm after phosphorylation of another membrane-bound cation transport enzyme, sarcoplasmic reticulum (Ca)ATPase, as well as by a similar resonance at about +17 ppm after phosphorylation of the model dipeptide L-seryl-L-aspartate.     

Infrared spectroscopic signals arising from ligand binding and conformational changes in the catalytic cycle of sarcoplasmic reticulum calcium ATPase.

Fourier transform infrared spectroscopy was used to investigate ligand binding and conformational changes in the Ca2(+)-ATPase of sarcoplasmic reticulum during the catalytic cycle. The ATPase reaction was started in the infrared sample by release of ATP from the inactive, photolabile ATP derivative P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP). Absorption spectroscopy in the visible spectral region using the Ca2(+)-sensitive dye Antipyrylazo III ensured that the infrared samples were able to transport Ca2+ in spite of their low water content, which is required for mid-infrared measurements (1800-950 cm-1). Small, but characteristic and highly reproducible infrared absorbance changes were observed upon ATP release. These infrared absorbance changes exhibit different kinetic properties. Comparison with model compound infrared spectra indicates that they are related to photolysis of caged ATP, hydrolysis of ATP in consequence of ATPase activity and to molecular changes in the active ATPase. The absorbance changes due to alterations in the ATPase were observed mainly in the region of Amide I and Amide II protein absorbance and presumably reflect the molecular processes upon phosphoenzyme formation. Since the absorbance changes were small compared to the overall ATPase absorbance, no major rearrangement of ATPase conformation as the result of catalysis could be detected.     

Structural changes of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding studied by fourier transform infrared spectroscopy

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding were recorded in H(2)O and (2)H(2)O at -7 degrees C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P(3)-1-(2-nitrophenyl) adenosine 5'-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca(2+)-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release (k(app) congruent with 3 s(-1) in H(2)O and k(app) congruent with 7 s(-1) in (2)H(2)O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca(2+)-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of alpha-helical and beta-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the gamma-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the gamma-phosphate group in the interactions between the Ca(2+)-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716 cm(-1) (H(2)O) and 1706 cm(-1) ((2)H(2)O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm(-1) (H(2)O) and 1514 cm(-1) ((2)H(2)O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21.     

The stereochemical course of phosphoric residue transfer catalyzed by sarcoplasmic reticulum ATPase

The stereochemical course of the phosphoric residue transfer from ADP to water catalyzed by the (Mg2+ + Ca2+)-dependent ATPase of sarcoplasmic reticulum has been determined. For this determination, the preparation is described of ATP gamma S, stereospecifically labeled in the gamma-position with both 17O and 18O. After hydrolysis of this nucleotide, the analysis of the product inorganic [16O,17O,18O]thiophosphate showed that the reaction proceeded with retention of configuration at the gamma-phosphorus atom. This result is expected since a phosphoenzyme is well characterized for this ATPase and provides support for the hypothesis that each phosphate transfer step occurs with inversion. In this case, the formation and breakdown of the phosphoenzyme occur each with inversion leading to the retention observed for the whole reaction.     

Phosphorylation and dephosphorylation reactions of the red beet plasma membrane ATPase studied in the transient state

The reaction mechanism of the solubilized red beet (Beta vulgaris L.) plasma membrane ATPase was studied with a rapid quenching apparatus. Using a dual-labeled substrate ([γ-³²P]ATP and [5′,8-³H]ATP), the presteady-state time course of phosphoenzyme formation, phosphate liberation and ADP liberation was examined. The time course for both phosphoenzyme formation and ADP liberation showed a rapid, initial rise while the timecourse for phosphate liberation showed an initial lag. This indicated that ADP was released with formation of the phosphoenzyme while phosphate was released with phosphoenzyme breakdown. Phosphoenzyme formation was Mg²⁺-dependent and preincubation of the enzyme with free ATP followed by the addition of Mg²⁺ increased the rate of phosphoenzyme formation 2.3-fold. This implied that phosphoenzyme formation could result from a slow reaction of ATP binding followed by a more rapid reaction of phosphate group transfer. Phosphoenzyme formation was accelerated as the pH was decreased, and the relationship between pH and the apparent first-order rate constants for phosphoenzyme formation suggested the role of a histidyl residue in this process. Transient kinetics of phosphoenzyme breakdown confirmed the presence of two phosphoenzyme forms, and the discharge of the ADP-sensitive form by ADP correlated with ATP synthesis. Potassium chloride increased the rate of phosphoenzyme turnover and shifted the steady-state distribution of phosphoenzyme forms. From these results, a minimal catalytic mechanism is proposed for the red beet plasma membrane ATPase, and rate constants for several reaction steps are estimated.     

Acetyl phosphate as a substrate for the calcium ATPase of sarcoplasmic reticulum

Acetyl phosphate is hydrolyzed by the calcium ATPase of leaky sarcoplasmic reticulum vesicles from rabbit skeletal muscle with Km = 6.5 mM and kcat = 7.9 s-1 in the presence of 100 microM calcium (180 mM K+, 5 mM MgSO4, pH 7.0, 25 degrees C). In the absence of calcium, hydrolysis is 6% of the calcium-dependent rate at low and 24% at saturating concentrations of acetyl phosphate. Values of K0.5 for calcium are 3.5 and 2.2 microM (n = 1.6) in the presence of 1 and 50 mM acetyl phosphate, respectively; inhibition by calcium follows K0.5 = 1.6 mM (n approximately 1.1) with 50 mM acetyl phosphate and K0.5 = 0.5 mM (n approximately 1.3) with 1.5 mM ATP. The calcium-dependent rate of phosphoenzyme formation from acetyl phosphate is consistent with Km = 43 mM and kf = 32 s-1 at saturation; decomposition of the phosphoenzyme occurs with kt = 16 s-1. The maximum fraction of phosphoenzyme formed in the steady state at saturating acetyl phosphate concentrations is 43-46%. These results are consistent with kc congruent to 30 s-1 for binding of Ca2+ to E at saturating [Ca2+], to give cE.Ca2, in the absence of activation by ATP. Phosphoenzyme formed from ATP and from acetyl phosphate shows the same biphasic reaction with ADP, rate constants for decomposition that are the same within experimental error, and similar or identical activation of decomposition by ATP. It is concluded that the reaction pathways for acetyl phosphate and ATP in the presence of Ca2+ are the same, with the exception of calcium binding and phosphorylation; an alternative, faster route that avoids the kc step is available in the presence of ATP. The existence of three different regions of dependence on ATP concentration for steady state turnover is confirmed; activation of hydrolysis at high ATP concentrations involves an ATP-induced increase in kt.     

ATP synthesis catalyzed by the purified erythrocyte Ca-ATPase in the absence of calcium gradients

The Ca2+-transporting ATPase of erythrocytes was isolated by calmodulin affinity chromatography. The backward reaction of the ATPase was investigated. The phosphorylation of the solubilized enzyme by Pi required Mg and was inhibited by Ca and vanadate in the micromolar concentration range. Significant amounts of phosphoenzyme could be obtained only in a medium containing high dimethyl sulfoxide concentrations (greater than 25%) in order to diminish water activity at the phosphorylation site. The phosphoenzyme formed in this way could not phosphorylate ADP. However, upon addition of Ca2+ ions and dilution of dimethyl sulfoxide in the phosphorylated preparation (water activity jump), a highly reactive phosphoenzyme species was obtained which could transfer phosphate in nearly stoichiometric amounts to ADP to form ATP.     

The significance of the 2' OH group and the influence of cations on the secondary structure of the RNA backbone.

In the IR spectra, the coupling of vibrations leads to band splitting and/or bands shifting in opposite directions which provides information on the mutual orientation of groupings. From such band shifts in the range 1800 to 1500 cm-1 one can draw conclusions on the double helix formation of polynucleotides. These band shifts are caused either by vibrational coupling of stretching vibrations within pairs of base residues or by coupling of stretching vibrations with the bending (scissor) vibration of the -NH2 groups; the latter is indicated by band shifts after deuterium substitution within the amino groups. Couplings of phosphate and 1 ibose vibrations in the range 1300 to 1000 cm-1 provide information on the secondary structure of the backbone. In order to obtain information of the structure of the RNA backbone, the IR spectra of poly(ribonucleotides) were studied in neutral media in which they were single-stranded. The shift due to coupling of the band of the 2'OD bending vibration and that of the antisymmetric stretching vibration of the ether group of the ribose residue proves that ribose residues of the backbone are cross-linked via hydrogen bonds. These are formed between the 2'OD or 2'OH groups, respectively, and the O atoms of the ether group of the neighboring ribose residues. This is the reason for the difference between DNA and RNA as regards the 2'OH group. The structure formation caused by these hydrogen bonds results in a stiffening of the RNA backbone. The tendency to form these hydrogen bonds increases in the order poly (U), poly(C), poly (A). This order of secondary structure stabilization is due to an interplay between the influences of (1) the 2'OH hydrogen bonds and (2) the base residues' stacking. Furthermore, the coupling of the antisymmetric stretching vibration of the greater than PO2- groups with a vibration involving the 2'OH group can result in a doublet structure of the band at about 1240 cm-1 if cations with strong fields are present. This probably shows that these cations can turn the greater than PO2-groups-which are usually turned outward at the backbone, as shown by construction of molecular models- towards the basic residues. Thus they cause stiff monohelices which are right-handed screws.     

Pre-steady-state charge translocation in NaK-ATPase from eel electric organ

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-- >E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s- 1.     

Reversal of the sarcoplasmic reticulum ATPase cycle by substituting various cations for magnesium. Phosphorylation and ATP synthesis when Ca2+ replaces Mg2+

Reversal of the cycle of sarcoplasmic reticulum ATPase starts from ATPase phosphorylation by Pi, in the presence of Mg2+, and leads to ATP synthesis. We show here that ATP can also be synthesized when Ca2+ replaces Mg2+. In the absence of a calcium gradient and in the presence of dimethyl sulfoxide, ATPase phosphorylation from Pi and Ca2+ led to the formation of an unstable phosphoenzyme. This instability was due to a competition between the phosphorylation reaction induced by Pi and Ca2+ and the transition induced by Ca2+ binding to the transport sites, which led to a conformation that could not be phosphorylated from Pi. Dimethyl sulfoxide and low temperature stabilized the calcium phosphoenzyme, which under appropriate conditions, subsequently reacted with ADP to synthesize ATP. Substitution of Co2+, Mn2+, Cd2+, or Ni2+ for Mg2+ induced ATPase phosphorylation from Pi, giving phosphoenzymes of various stabilities. However, substitution of Ba2+, Sr2+, or Cr3+ produced no detectable phosphoenzymes, under the same experimental conditions. Our results show that ATPase phosphorylation from Pi, like its phosphorylation from ATP, does not have a strict specificity for magnesium.     

Structures of the Ca2+-ATPase complexes with ATP, AMPPCP and AMPPNP. An FTIR study

We studied binding of ATP and of the ATP analogs adenosine 5'-(beta,gamma-methylene)triphosphate (AMPCP) and beta,gamma-imidoadenosine 5'-triphosphate (AMPPNP) to the Ca(2+)-ATPase of the sarcoplasmic reticulum membrane (SERCA1a) with time-resolved infrared spectroscopy. In our experiments, ATP reacted with ATPase which had AMPPCP or AMPPNP bound. These experiments monitored exchange of ATP analog by ATP and phosphorylation to the first phosphoenzyme intermediate Ca(2)E1P. These reactions were triggered by the release of ATP from caged ATP. Only small differences in infrared absorption were observed between the ATP complex and the complexes with AMPPCP and AMPPNP indicating that overall the interactions between nucleotide and ATPase are similar and that all complexes adopt a closed conformation. The spectral differences between ATP and AMPPCP complex were more pronounced at high Ca(2+) concentration (10 mM). They are likely due to a different position of the gamma-phosphate which affects the beta-sheet in the P domain.     

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.     

Intermediate phosphorylation reactions in the mechanism of ATP utilization by the copper ATPase (CopA) of Thermotoga maritima

Recombinant and purified Thermotoga maritima CopA sustains ATPase velocity of 1.78-2.73 micromol/mg/min in the presence of Cu+ (pH 6, 60 degrees C) and 0.03-0.08 micromol/mg/min in the absence of Cu+. High levels of enzyme phosphorylation are obtained by utilization of [gamma-32P]ATP in the absence of Cu+. This phosphoenzyme decays at a much slower rate than observed with Cu.E1 approximately P. In fact, the phosphoenzyme is reduced to much lower steady state levels upon addition of Cu+, due to rapid hydrolytic cleavage. Negligible ATPase turnover is sustained by CopA following deletion of its N-metal binding domain (DeltaNMBD) or mutation of NMBD cysteines (CXXC). Nevertheless, high levels of phosphoenzyme are obtained by utilization of [gamma-3)P]ATP by the DeltaNMBD and CXXC mutants, with no effect of Cu+ either on its formation or hydrolytic cleavage. Phosphoenzyme formation (E2P) can also be obtained by utilization of Pi, and this reaction is inhibited by Cu+ (E2 to E1 transition) even in the DeltaNMBD mutant, evidently due to Cu+ binding at a (transport) site other than the NMBD. E2P undergoes hydrolytic cleavage faster in DeltaNMBD and slower in CXXC mutant. We propose that Cu+ binding to the NMBD is required to produce an "active" conformation of CopA, whereby additional Cu+ bound to an alternate (transmembrane transport) site initiates faster cycles including formation of Cu.E1 approximately P, followed by the E1 approximately P to E2-P conformational transition and hydrolytic cleavage of phosphate. An H479Q mutation (analogous to one found in Wilson disease) renders CopA unable to utilize ATP, whereas phosphorylation by Pi is retained.     

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.     

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.     

Structural studies of a stabilized phosphoenzyme intermediate of Ca2+-ATPase

Ca(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species.