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J Biol Chem, Vol. 274, Issue 32, 22170-22175, August 6, 1999

Phosphoenzyme Conversion of the Sarcoplasmic Reticulum Ca²⁺-ATPase
MOLECULAR INTERPRETATION OF INFRARED DIFFERENCE SPECTRA^*

Andreas Barth

From the Institut für Biophysik, Johann Wolfgang Goethe Universität, Theodor Stern Kai 7, Haus 74, D-60590 Frankfurt am Main, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

Time-resolved Fourier transform infrared difference spectra of the phosphoenzyme conversion and Ca²⁺ release reaction (Ca₂E₁-P  E₂-P) of the sarcoplasmic reticulum Ca²⁺-ATPase were recorded at pH 7 and 1 °C in H₂O and ²H₂O. In the amide I spectral region, the spectra indicate backbone conformational changes preserving conformational changes of the preceding phosphorylation step. -sheet or turn structures (band at 1685 cm¹) and -helical structures (band at 1653 cm¹) seem to be involved. Spectra of the model compound EDTA for Ca²⁺ chelation indicate the assignment of bands at 1570, 1554, 1411 and 1399 cm¹ to Ca²⁺ chelating Asp and Glu carboxylate groups partially shielded from the aqueous environment. In addition, an E₂-P band at 1638 cm¹ 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 cm¹ in H₂O and 1515 cm¹ in ²H₂O). A band at 1192 cm¹ was shown by isotopic replacement in the -phosphate of ATP to originate from the E₂-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

Muscle relaxation is mediated by the removal of cytosolic Ca²⁺ by the Ca²⁺-ATPase of the sarcoplasmic reticulum membrane. The ATPase couples active Ca²⁺ transport to the hydrolysis of ATP (1-4) in a reaction cycle that is shown in Scheme 1. In an essential reaction step, ATP reacts with Asp-351 to form a phosphoenzyme intermediate (Ca₂E₁  Ca₂E₁-P), which then converts from an ADP-sensitive form (Ca₂E₁-P) to an ADP-insensitive form (E₂-P) that is more rapidly hydrolyzed. This phosphoenzyme conversion is associated with Ca²⁺ release toward the sarcoplasmic reticulum lumen against the concentration gradient (1, 3, 5). The structural origin of the change of accessibility of the Ca²⁺ sites and of the essential reduction of Ca²⁺ affinity upon phosphoenzyme conversion Ca₂E₁-P  E₂-P have yet to be elucidated.

View larger version (10K): [in this window] [in a new window]   Scheme 1.

The potentially large body of structural information regarding the ATPase reaction cycle provided by infrared spectroscopy has only recently begun to be exploited using the approach of effector molecule-induced infrared difference spectroscopy (6-13). The method uses the release of effector molecules from biologically "silent" photolabile derivatives, termed caged compounds (14-17), to generate high quality infrared difference spectra. (The reaction products and infrared difference spectra of caged ATP photolysis and of side reactions have been characterized (18-20).) The absorbance changes seen in these difference spectra give evidence for conformational changes of the polypeptide backbone and for alterations to the environment of amino acid side chains that take place in the reaction investigated.

Spectra of the phosphoenzyme conversion reaction in the 1800 to 1000 cm¹ spectral range have previously been described (8) and in that work were calculated by subtracting two difference spectra obtained with two different types of samples; a normalized difference spectrum of the Ca₂E₁  Ca₂E₁-P reaction was subtracted from a normalized difference spectrum of the Ca₂E₁  E₂-P reaction. The use of two different samples in the subtraction and the normalization to identical protein content limit the reliability of these spectra and make it desirable to obtain the phosphoenzyme conversion spectrum more directly. This is possible using time-resolved rapid scan Fourier transform infrared (FTIR)¹ spectroscopy (10). Using this approach, we present here the spectra of the phosphoenzyme conversion reaction obtained in H₂O and ²H₂O after the release of unlabeled ATP or [-¹⁸O₃]ATP. In particular, bands of the putative Ca²⁺ chelating carboxylate groups and of the phosphoenzyme phosphate group are discussed.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

Sample Preparation-- Samples for time-resolved infrared spectroscopy of the Ca₂E₁ P  E₂-P reaction were prepared as described previously (8, 10) by removal of free water from a sarcoplasmic reticulum suspension equilibrated in H₂O or ²H₂O buffer. Samples were immediately rehydrated with H₂O or ²H₂O with or without 20% Me₂SO. This method resulted in active ATPase samples (6). Approximate concentrations were 0.7 mM ATPase, 300 mM imidazole, pH 7.0, 1 mM CaCl₂, 20 mM glutathione, 20 mM caged ATP, 0.5 mg/ml A23187, 2 mg/ml adenylate kinase, and 20% Me₂SO in approximately 1 µl of sample volume. Approximately 2-3 mM ATP was released per flash. Caged [-¹⁸O₃]ATP at 91% isotopic enrichment per oxygen atom was a gift of M. R. Webb and J. E. T. Corrie (National Institute for Medical Research, London).

FTIR Measurements-- Time-resolved FTIR measurements of the Ca₂E₁-P  E₂-P reaction were performed at 1 °C with a modified Bruker IFS 66 spectrometer as described previously (10). Difference spectra for the reaction were obtained by subtracting a spectrum representing predominantly Ca₂E₁-P, and to a small extent E₂-P (recorded 3.3-11 s after photolysis of caged ATP in H₂O or after 11-19 s in ²H₂O), from a spectrum representing E₂-P (recorded after 88 and 146 s in H₂O and ²H₂O, respectively). The resulting spectrum was normalized as described (10) to the full amplitude of the absorbance changes associated with the phosphoenzyme conversion reaction.

As the difference spectra were obtained directly from the time-resolved measurements, a normalization of spectra to an identical protein concentration is in principle not necessary. However, for a better comparison of samples in H₂O and ²H₂O and to prevent the possible predominance of individual samples with high protein content in the averaged spectra, spectra were normalized to an identical protein concentration before averaging, as described (21).

Absorbance spectra in H₂O and ²H₂O of the model compound for Ca²⁺ release EDTA were recorded with and without Ca²⁺ at 20 °C and pH 12.8. Small variations in the path length between different samples were corrected by normalizing the water absorption of every sample to a single water spectrum that served as a standard for the path length. Sample concentrations were 84 mM.

Band-narrowing Procedures-- Apart from second-derivative spectra, the following procedure was applied. The original spectrum was smoothed over 24 cm¹ and multiplied by 0.95. This spectrum was then subtracted from the original spectrum, which eliminates broad features of the original spectrum. The resulting spectrum is dominated by the fine-structure in the original spectrum, and thus we term the method "fine-structure enhancement" in the following text. When tested with protein absorbance spectra, this method gives results similar to Fourier self-deconvolution (data not shown). For a clearer presentation, fine- structure enhanced spectra were multiplied by 10 and second-derivative spectra by 15.

To assess whether peaks in the fine-structure enhanced spectra correspond to "true" component bands or are the result of artifacts in the band-narrowing procedure (see Fig. 3), the original difference spectra were fitted, and the resulting fit was fine-structure enhanced and compared with the fine-structure enhanced original spectra (data not shown). Interestingly, it was found that the spectral region of 1700-1670 cm¹ can accurately be fitted by only three bands at 1693 (), 1686 (), and 1670 cm¹ (+) despite the plateau at 1678/1676 cm¹ in the fine-structure enhanced H₂O spectrum (solid line in Fig. 3B). It is not necessary to introduce an additional band in the fitting model to reproduce that plateau.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

Model Spectra of Ca2+ Release-- From site-directed mutagenesis studies it is thought that Glu-309, Glu-771, and Asp-800 form part of the high affinity Ca²⁺ binding sites of Ca₂E₁ and Ca₂E₁-P (4). Thus, we studied the effect of Ca²⁺ binding on the infrared spectrum of carboxylate groups using the Ca²⁺ chelator EDTA.

Fig. 1A shows absorbance spectra in ²H₂O of free EDTA (solid line) and of the EDTA complex with Ca²⁺ (dotted line). The bands near 1590 and 1410 cm¹ can be assigned to the COO antisymmetric stretching vibration (_as) and the symmetric stretching vibration (_s), respectively. The shoulder at 1612 cm¹ indicates some heterogeneity of the COO groups, resulting from a small proportion of EDTA with protonated nitrogen atoms (22, 23). Upon Ca²⁺ release there is a downshift of 4-8 cm¹ for both of the main bands (at 1590 and 1414 cm¹), which translates in the difference spectrum of Ca²⁺ release (absorbance of free EDTA minus absorbance of the Ca²⁺ complex) into the two minimum/maximum features at 1590/1568 and 1418/1400 cm¹.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Model spectra for Ca2+ release from carboxylate groups. A, absorbance spectra in 2H2O of EDTA (solid line) and the Ca2+ complex of EDTA (dotted line). B, Ca2+ release spectrum: absorbance of EDTA minus absorbance of the Ca2+ complex of EDTA.

When ²H₂O is replaced by H₂O, the bands of the antisymmetric stretching vibration are downshifted by 6-8 cm¹ for free EDTA and for the complex (data not shown). The band of the symmetric stretching vibration is less sensitive and shifts downwards only for free EDTA (by 2 cm¹). A similar behavior has been observed for sodium acetate (24) and for Asp and Glu (25, 26).

Thus, the model spectra have identified marker band pairs for Ca²⁺ release from carboxylate groups near 1575 and 1410 cm¹. The former is expected to be sensitive to H₂O  ²H₂O replacement with a higher frequency in ²H₂O.

The Phosphoenzyme Conversion Spectrum-- Fig. 2A shows the difference spectrum of the phosphoenzyme conversion and Ca²⁺ release reaction, Ca₂E₁-P  E₂-P (solid line), and the respective spectrum in ²H₂O buffer (dashed line). Positive bands are characteristic for E₂-P, negative bands for Ca₂E₁-P. Fig. 3A shows the same spectra on an expanded scale.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Spectra of phosphoenzyme conversion and Ca2+ release at pH 7.0 and 1 °C. The labels refer to the solid line spectra. A, full line, H2O; dotted line, 2H2O. B, spectra in H2O after the release of unlabeled ATP (solid line) and [-18O3]ATP (dotted line) from caged ATP.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Phosphoenzyme conversion spectra processed with band-narrowing techniques. A, original spectra: solid line, H2O; dotted line, 2H2O. The labels refer to the solid line spectrum. B, fine structure-enhanced spectra multiplied by 10 (see "Materials and Methods"): solid line, H2O; dotted line, 2H2O. The labels refer to the closest peak. C, second derivative spectra multiplied by 15: full line, H2O; dotted line, 2H2O.

Ca²⁺ Release from Carboxylate Groups-- In the phosphoenzyme conversion spectrum, similar band profiles are observed as in the model difference spectrum for Ca²⁺ release (Fig. 1B) at 1570/1554 and 1411/1399 cm¹ (numbers for H₂O). The slight upshift upon H₂O  ²H₂O exchange characteristic for the _as vibration of carboxylate groups (see above) is also notable (1570  1572 cm¹, 1554  1555 cm¹) and is best seen in Fig. 3A. However, the shift is less than that observed above for completely exposed carboxylate groups. The lower wavenumber of the 1570/1554 cm¹ band pair as compared with the model spectra (1586/1556 cm¹ in H₂O and 1590/1568 cm¹ in ²H₂O) is expected because the _as(COO) band position of free Asp and Glu residues (25, 26) is 2-4 cm¹ (Asp) and 18 cm¹ (Glu) lower than that of EDTA (in H₂O and ²H₂O). These observations support and substantiate the tentative assignment of the band pairs at 1570/1554 and 1411/1399 cm¹ (numbers for H₂O) to Ca²⁺ chelating residues of the ATPase (8). The relatively small shift observed for the 1570/1554 cm¹ band pair in ²H₂O may indicate that these groups are not completely exposed to water.

The band at 1638 cm¹ also shows the upshift characteristic of a _as(COO) band upon H₂O  ²H₂O replacement (best seen in Fig. 3A). Other bands absorbing in that region, i.e. the amide I mode of -sheet structures, and side chain modes of His, Arg, and Lys should either show downshifts of about 10 cm¹ (backbone and His) (26-30), 50 cm¹ (Arg) (25, 26), and 400 cm¹ (Lys) (31) or should remain unchanged if the respective groups are inaccessible to ¹H  ²H exchange. The position of the 1638 cm¹ band is rather unusual for a _as band of a COO group, which absorbs for model compounds in aqueous solution at 1574 cm¹ (Asp) or at 1560 cm¹ (Glu) (25). However, a strong salt bridge or hydrogen bond to one of the oxygens of a carboxylate group could shift the band well above 1600 cm¹ (24, 32), and a reasonable scenario for the band at 1638 cm¹ could be that the parting Ca²⁺ is replaced by a strong hydrogen bond donor or a positive charge.

Bands at 1758 and 1710 cm¹ have tentatively been assigned to the protonation of at least two chelating carboxylate groups (33), which is in line with the small downshift observed upon H₂O  ²H₂O replacement.

Protein Backbone: the Amide I Region-- The largest absorbance changes (up to 0.5% of the total protein absorbance) are observed in the amide I region of the spectrum (1700-1610 cm¹) as are the strongest effects of protein deuteration. In this region, the amide I mode of the polypeptide backbone as well as the side chains of Asn, Gln, Arg, HisH⁺, and Lys absorb strongly (25, 26, 30). In the lower wavenumber part of that region, Asp and Glu COO groups with strong interactions may contribute as discussed above.

Large downshifts (30 cm¹) of bands upon H₂O  ²H₂O replacement are characteristic for the side chain absorptions of Asn, Gln, Lys, and Arg, as are small upshifts as discussed above for COO bands and downshifts of up to 10 cm¹ for amide I and HisH⁺ bands. No band shifts are expected for groups that are located in parts of the protein that are not accessible to deuteration.

The large number of alterations in this spectral region, when H₂O is replaced by ²H₂O, makes it difficult to arrive at a unique explanation for the band shifts, and additional experiments as well as data processing were necessary, as described below.

The recording of spectra as soon as possible after H₂O  ²H₂O replacement (30 min to 1 h at 1 °C) shows that most of the observed effects take place in protein regions readily accessible to deuteration. The spectrum shortly after deuteration (data not shown) is very similar to the one after prolonged incubation. The only clear exception is the minimum at ~1630 cm¹ that develops over a few hours of incubation. This position is characteristic of amide I modes of -sheet structures and of the C=O group of deuterated Asn or Gln residues (26).

Band-narrowing procedures (see "Materials and Methods") were applied to identify band shifts upon ¹H  ²H exchange. Fig. 3A shows the original difference spectra and Fig. 3, B and C, the resulting spectra after band narrowing. These reveal essentially the same peak positions in H₂O and ²H₂O (see Fig. 3, B and C), with one exception. The negative band at 1689 cm¹ in the unprocessed original H₂O spectrum (solid line in Fig. 3A) is composed of at least two bands, giving minima in the processed spectrum at 1693 and 1685 cm¹ (solid line in Fig. 3, B and C). The highest wavenumber component of the negative band seems to be nearly unaffected by protein deuteration and is observed in ²H₂O at 1692 cm¹. It could be caused by a conformational change of -sheet or turn structures or an Asn, Gln, or Arg side chain that is located in the core of the protein and is inaccessible to deuteration.

The other component of the 1689 cm¹ band seems to shift from its position in the processed spectrum (Fig. 3B) at 1685 (H₂O) to 1677 cm¹ (²H₂O) and to cancel part of the adjacent positive band observed at 1671 cm¹ in H₂O (Fig. 3A). This shift is characteristic of amide I modes of the polypeptide backbone, and the position then indicates a conformational change of -sheet or turn structures. These structures are predicted to occur only in the extramembraneous domains of the protein (34, 35), and thus the observed conformational change is likely to take place in these protein domains.

As the positions of the other bands in the amide I region are hardly affected by deuteration, the isotope effects are the result either of intensity changes or of bands that are not evident after applying the band-narrowing procedures because they are broader than the ones detected. Narrow bands tend to dominate the processed spectra.

The bands hardly affected by ¹H  ²H exchange are found at 1653, 1638, 1621, and 1607 cm¹. A band at 1653 cm¹ is often observed for -helical structures and is often hardly affected by H₂O²H₂O replacement as observed here. The band at 1638 cm¹, which shifts slightly upward, was tentatively assigned above to a COO group. The bands at 1621 and 1607 cm¹ seem to retain their position in ²H₂O with a possible overlap of additional bands in H₂O or ²H₂O. The band at 1621 cm¹ may be assigned to a -sheet structure or to the imide group of a Pro residue in a helical or unordered conformation. The imide band is found approximately 20 cm¹ lower than an amide band (36). Thus, the band at 1607 cm¹, the position of which seems to be too low for an amide I band, could also be caused by a Pro imide group. Interestingly, there are three Pro residues in the putative Ca²⁺ binding transmembrane helices M4 and M6, mutation of which affects Ca²⁺ affinity and phosphoenzyme conversion (4). Alternatively, both bands may be caused by COO side chain groups not interacting with bulk water. This assumption relies on the fact that the bands do not show the upshift upon H₂O  ²H₂O replacement characteristic for carboxylate groups in water.

Protein Backbone: the Amide II Region-- None of the three bands in the amide II region (1570-1530 cm¹) shows the strong sensitivity toward ¹H  ²H exchange expected for the amide II mode of the protein backbone. The amide II mode of backbone elements accessible to deuteration therefore does not seem to be affected by phosphoenzyme conversion and Ca²⁺ release. Two of the bands (at 1570 and 1554 cm¹) have been tentatively attributed above to the _as vibration of COO groups.

Protein Backbone: the Amide III Region-- The amide III mode absorbs between 1400 and 1200 cm¹ and is sensitive to deuteration (37). This property is observed in the spectra for the bands at 1337 and 1318 cm¹ (see Fig. 2A), which therefore might be attributed to amide III modes. The position of these bands is characteristic for turn structures (37), and they are probably related to the amide I band at 1687 cm¹, which has tentatively been assigned to turn or -sheet structures. Alternatively, the bands at 1337 and 1318 cm¹ may be caused by the (COH) mode of Ser, Asp, or Glu with a weakly bonded OH group.

Side Chain Modes Other than Carboxyl Modes-- As mentioned above, bands of the side chains of Asn, Gln, Arg, and Lys in the amide I region show relatively large shifts upon deuteration. The extinction coefficient of the former three residues is relatively high (25, 26), whereas that of Lys is smaller. Thus, Lys bands may be masked by stronger bands. The HisH⁺ mode near 1631 cm¹ (30) shows a 10 cm¹ downshift in ²H₂O (26) and absorbs relatively strongly in H₂O (30). These characteristic shifts have not been observed in the spectra, and thus there is no clear evidence for the participation of Asn, Gln, Arg, and HisH⁺ in the phosphoenzyme conversion and Ca²⁺ release reaction. This statement holds only for those residues that are accessible to ¹H  ²H exchange, which should include residues in the ATP binding site, the catalytic site, and at least part of the Ca²⁺ binding sites, because several bands that were tentatively assigned to the Ca²⁺ binding sites show an effect upon H₂O  ²H₂O replacement (see above).

The position of the band at 1517 cm¹ and its slight downshift of 2 cm¹ in ²H₂O is characteristic for a ring mode of protonated Tyr (25, 26, 30). Also, the band pairs at 1283 and 1264 cm¹ may be attributed to the (C-O) mode of Tyr but also to a Trp mode, which is observed for indole at 1276 cm¹ (38, 39). It has been suggested that Tyr-763 is involved in the cytoplasmic gate to the Ca²⁺ binding sites (4). The very small band at 1365 cm¹ might be caused by a _s(CH₃) mode of aliphatic side chains.

Absorption of the Phosphate Group-- Fig. 2B shows a comparison between the conversion spectrum obtained with unlabeled ATP and that obtained with [-¹⁸O₃]ATP. With the heavier isotope, phosphate bands are expected to be down-shifted, thereby enabling the identification in the spectrum of alterations to the phosphate group. As the -phosphate is transferred to Asp-351 before phosphoenzyme conversion, differences between the spectra with the two isotopes will identify the absorbance of the phosphoenzyme phosphate group. As expected, the two spectra superimpose very well above 1250 cm¹, where phosphate groups do not absorb. However, the band at 1192 cm¹ is reduced upon isotopic substitution. Instead, the intensity is higher for [-¹⁸O₃]ATP between 1180 and 1150 cm¹. A difference spectrum between labeled and unlabeled phosphate group (data not shown) shows that the band at 1192 cm¹ for the unlabeled phosphate seems to shift to 1157 cm¹ for the labeled phosphate, in agreement with the expected isotopic shift of 20-30 cm¹ for phosphate groups (18, 40). This identification of a phosphate band in the difference spectrum clearly shows that the conversion reaction considerably changes the environment of the phosphoenzyme phosphate group, thus affecting its electron density distribution and/or binding geometry. The band position at 1192 cm¹ is rather unusual for a phosphate group and could be explained in two ways: (i) a widening of the P-O angles leading to a stronger coupling between the P-O vibrations and thus to a stronger splitting between the _as and _s modes; or (ii) an increase in electron density of some or all of the P-O bonds, either by breaking of the hydrogen bonds to all phosphate oxygens or by strong hydrogen binding to one or two of the phosphate oxygen atoms, thus increasing the electron density in the other P-O bond(s). The band is also affected by H₂O  ²H₂O replacement, which seems to indicate that the phosphate oxygen interacts with either a water molecule or deuterated protein residues.

The active site of E₂-P was shown to be in a closed conformation (41) shielded from bulk water with a hydrophobic environment detected close to the ribose OH groups of a fluorescent ATP analogue (42, 43). This finding is in contrast to the properties of Ca₂E₁-P, and it is thought that the decrease in the active site water activity upon the Ca₂E₁-P  E₂-P conversion is responsible for the higher hydrolysis rate of E₂-P as compared with Ca₂E₁-P (44). The infrared spectra presented here show that this conformational change has a direct effect on phosphate conformation and/or electron density distribution. Creation of a hydrophobic environment alone however, may not be sufficient to explain the phosphate band at 1192 cm¹, because dehydration of the model compound acetyl phosphate shifts the _as PO₃² band only from 1132 to 1177 cm¹ at most (data not shown).

Comparison with Spectra of ATPase Phosphorylation-- Fig. 4 shows spectra in ²H₂O of ATPase phosphorylation Ca₂E₁·ATP  Ca₂E₁-P (solid line) (21) and of the overall reaction of phosphorylation and phosphoenzyme conversion Ca₂E₁·ATP  E₂-P (dotted line). These spectra were obtained from time-resolved measurements of the same set of samples. Negative bands are characteristic for Ca₂E₁·ATP, positive bands either for Ca₂E₁-P (solid line) or E₂-P (dotted line). (The spectrum of Ca₂E₁·ATP  Ca₂E₁-P also shows a small contribution because of the E₂-P that is already formed, as indicated by the E₂-P marker band near 1750 cm¹.) The spectral region shown includes the amide I region (1700-1610 cm¹) that is sensitive to conformational changes of the protein backbone. The comparison indicates that bands characteristic for Ca₂E₁-P are still present in E₂-P, which is especially obvious for the bands at 1682, 1630, and 1593 cm¹. This finding indicates that at least some of the alterations to the protein conformation induced by ATPase phosphorylation seem to be preserved or even "enhanced" in the subsequent transition to E₂-P. It seems as if the enzyme conformation on the way from Ca₂E₁-P to E₂-P goes further away from the pre-phosphorylation conformation of Ca₂E₁·ATP instead of returning to it.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Difference spectra of the Ca2E1·ATP  Ca2E1-P (solid line) and the Ca2E1·ATP  E2-P (dotted line) reaction at pH 7 and 1 °C.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

Infrared difference spectra show that the protein conformation changes in the phosphoenzyme conversion reaction, preserving conformational changes of the preceding step of enzyme phosphorylation. -sheet or turn structures of the extramembraneous domains and most likely -helical structures seem to be affected. The net change of secondary structures, however, is small (10).

The release of Ca²⁺ proceeds from carboxylate groups that seem to be partly shielded from the aqueous environment. It is associated with the protonation of at least two carboxylate groups presumably involved in Ca²⁺ chelation. A change of environment of a Tyr residue was detected as well as a direct effect of phosphoenzyme conversion on the geometry and/or electron density distribution of the phosphoenzyme phosphate group. The latter is likely to be a prerequisite for the higher susceptibility toward hydrolytic attack of E₂-P as compared with Ca₂E₁-P. Interestingly, the currently assigned bands of the phosphate group and of Ca²⁺ release from carboxylate groups appear with the same reaction rate (10), showing that Ca²⁺ release and the change of phosphate environment proceed at the same time. This finding rules out a significant population of Ca₂E₂-P, a postulated state (45) with phosphate properties of E₂-P that still binds Ca²⁺.

	ACKNOWLEDGEMENTS

Valuable discussions with Prof. W. Mäntele and the opportunity to use his laboratory are gratefully acknowledged. The author thanks Prof. W. Hasselbach (Max-Planck-Institut, Heidelberg) for the gift of Ca²⁺-ATPase, Dr. M. R. Webb (National Institute for Medical Research, London) for the gift of [-¹⁸O₃]ATP, and Dr. J. E. T. Corrie (National Institute for Medical Research, London) for caging ATP and [-¹⁸O₃]ATP.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-69-6301-6087; Fax: 49-69-6301-5838; E-mail: barth@biophysik.uni-frankfurt.de.

	ABBREVIATIONS

The abbreviations used are: FTIR, Fourier transform infrared; Ca²⁺-ATPase, Ca²⁺ transporting ATPase (EC 3.6.1.38); caged ATP, P³-1-(2-nitrophenyl)ethyladenosine 5'-triphosphate; Ca₂E₁-P, ADP-sensitive phosphoenzyme; E₂-P, ADP-insensitive phosphoenzyme; _s, symmetric stretching vibration; _as, antisymmetric stretching vibration.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

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