Reduction in water activity greatly retards the phosphoryl transfer from ATP to enzyme protein in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase.

Cys-674 of the sarcoplasmic reticulum Ca-ATPase was labeled with N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The ATP-induced drop in the fluorescence of the label, which was shown in our previous studies to reflect the conformational change upon formation of the calcium•enzyme•ATP complex, was followed by the stopped-flow method. The subsequent phosphoenzyme formation was followed by the rapid quenching method. Effects of a partial substitution of organic solvents for water in the medium on the conformational change and phosphoenzyme formation were investigated in the presence of 100 μM CaCl at pH 7.5, 0°C. The rate of the conformational change increased with increasing ATP concentration (0.1-100 μM) and was unaffected by 30% (v/v) dimethyl sulfoxide. In contrast, the rate of phosphoenzyme formation decreased sharply with increasing concentration of dimethyl sulfoxide (20-40% (v/v)), even when phosphoenzyme formation was saturated with ATP. N,N-Dimethylformamide and glycerol had essentially the same effects as dimethyl sulfoxide. These results show that the reduction in water activity does not affect the rate of the conformational change upon formation of the calcium•enzyme•ATP complex, but greatly retards the subsequent phosphoryl transfer from ATP to the enzyme protein. This strongly suggests that in this early stage of the catalytic cycle water plays a critical role in ensuring the rapid turnover of the enzyme.

Finally, ADP-insensitive EP is hydrolyzed to liberate P i . This catalytic cycle is fully reversible.
The and Hasselbach (10) showed previously that the rate of EP hydrolysis is markedly reduced by Me 2 SO. Later, de Meis et al. (11)(12)(13) found that EP formation from P i in the reverse reaction is greatly favored by substituting organic solvents such as Me 2 SO, DMF, and glycerol for water in the medium. They further suggested that, in contrast to the catalytic site of ADP-insensitive EP, which is thought to be hydrophobic (11), the catalytic site of ADP-sensitive EP is hydrophilic. According to this proposal, a change in water activity within the catalytic site may be essential for the conformational transition from ADP-sensitive EP to ADP-insensitive EP (11,14,15). However, the role of water in the formation of ADP-sensitive EP from ATP in the early stage of the catalytic cycle has not yet been explored.
Previously, we labeled Cys-674 of the enzyme by EDANS selectively without a loss of the catalytic activity and found that the fluorescence of bound EDANS decreases greatly upon formation of the calcium-enzyme-substrate complex in the initial step of the catalytic cycle (16 -18). This fluorescence drop reflects a conformational change in the vicinity of the ATPbinding site, because Cys-674 is surrounded by amino acid residues which contribute the conformation of the ATP-binding site (19 -25). This conformational change is immediately followed by the phosphoryl transfer from ATP to the enzyme protein (16 -18).
In this study, we have investigated the effects of organic solvents (Me 2 SO, DMF, and glycerol) on the conformational change in the calcium-enzyme-substrate complex and EP formation from ATP by using EDANS-labeled SR vesicles. The conformational change has been followed by the stopped-flow measurements of the fluorescence of bound EDANS and EP formation followed by the continuous-flow rapid quenching method. The results demonstrate that the reduction in water activity by addition of the organic solvents does not appreciably affect the rate of the conformational change, but greatly retards the subsequent phosphoryl transfer. These findings suggest that in this early stage of the catalytic cycle water plays a critical role in ensuring the rapid turnover of the enzyme.
Labeling of SR Vesicles with I-EDANS-SR vesicles were labeled with I-EDANS as described previously (16). The content of bound EDANS was about 1 mol per mole of phosphorylation site. The Ca 2ϩ -* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Steady-state Measurements of Fluorescence-
The steady-state fluorescence intensity of bound EDANS was measured on a computerinterfaced spectrofluorometer as described previously (17). The excitation and emission wavelengths were set to 380 and 475 nm, respectively.
Stopped-flow Measurements of Fluorescence-Rapid kinetic measurements of the fluorescence of bound EDANS were made by using a stopped-flow spectrofluorometer interfaced with a personal computer which was programmed to accumulate the digitized data, as described  Fig. 1 in the presence of 30% (v/v) Me 2 SO is shown after subtraction of the base-line level, which was determined by mixing the vesicles with the ATP-free medium.
previously (17). The excitation wavelength was 370 nm. The emitted light was passed through a filter which cut off the light below 450 nm. The reaction was started by mixing equal volumes of solutions from two syringes, one containing Ca 2ϩ -loaded, EDANS-labeled SR vesicles in a medium described in the figure legends and the other containing ATP in the same medium. The measurement was repeated 30 -80 times, and the data were accumulated.
Determination of EP-Rapid kinetic measurements of EP formation were made by using a continuous-flow rapid quenching system as described previously (17). The reaction was started by mixing equal volumes of solutions from two syringes, one containing Ca 2ϩ -loaded, EDANS-labeled SR vesicles and the other containing [␥-32 P]ATP, and quenched with trichloroacetic acid containing carrier ATP and P i . When the reaction was long enough, the above procedures were carried out by manual pipetting. After the reaction was quenched, bovine serum albumin was added. The sample was washed four times by centrifugation with perchloric acid containing carrier P i and PP i , dissolved in 0.5 N NaOH containing 1% SDS, and the radioactivity was measured.
Miscellaneous Methods-Protein concentrations were determined by the method of Lowry et al. (28) with bovine serum albumin as a standard. Data were analyzed by the nonlinear least squares method as described previously (26). ATP and ADP were from Yamasa Biochemicals (Choshi, Japan). AMP-PCP was from Sigma. I-EDANS was from Aldrich. [␥-32 P]ATP was purchased from Amersham Corp. EDANS-labeled SR vesicles in the absence of organic solvents or in the presence of 30% (v/v) Me 2 SO, 30% (v/v) DMF, or 30% (v/v) glycerol. When organic solvents were absent, the fluorescence decreased rapidly after addition of ATP (Fig. 1A). The initial rate of the fluorescence drop increased with increasing concentration of ATP. The maximum amplitude of this drop was 25%. These results are in agreement with our previous findings (16,18), which were obtained with Ca 2ϩ -nonloaded vesicles. The rate and amplitude of the fluorescence drop were unaffected by the presence of 30% (v/v) Me 2 SO in the whole range of ATP concentrations tested (Fig. 1B). Similar results were obtained with 30% (v/v) DMF (Fig. 1C) or 30% (v/v) glycerol (Fig. 1D), although the fluorescence drop in the presence of DMF or glycerol was slightly slower than that in the absence of organic solvents.

Effects of Me
Effects of Me 2 SO, DMF, and Glycerol on the Kinetics of EP Formation-EP formation with 100 M ATP was followed in the presence of different concentrations of Me 2 SO (Fig. 2) agreement with the previously reported results (10,11). When 40% (v/v) Me 2 SO was present, an increase in the ATP concentration from 100 to 300 M gave no increase in the rate of EP formation and in the steady-state level of EP. It is, therefore, clear that EP formation at saturating concentrations of ATP was greatly retarded by Me 2 SO. For comparison, the EDANS fluorescence drop induced by 100 M ATP was followed by the stopped-flow method in the presence of 100 M Ca 2ϩ and 30% (v/v) (Fig. 2) or 40% (v/v) (not shown) Me 2 SO. The fluorescence drop was much faster than EP formation under the same conditions. All these findings demonstrate that Me 2 SO does not affect the rate of the ATP-induced fluorescence drop but greatly retards the subsequent phosphoryl transfer from ATP to the enzyme protein.
In order to see whether the observed effect of Me 2 SO on EP formation is due to reduction in the water activity or it is specific to this organic solvent, effects of two other organic solvents, DMF and glycerol, were investigated in the same way as described in Fig. 2 except that DMF or glycerol was used in place of Me 2 SO. A similar retardation of EP formation was found with either DMF (Fig. 3) or glycerol (Fig. 4), although glycerol was somewhat less effective than Me 2 SO and DMF. In fact, the rate of EP formation decreased 20-fold by 40% (v/v) DMF and 13-fold by 40% (v/v) glycerol. An increase in the ATP concentration from 100 M to 300 M again gave no increase in the rate of EP formation which was retarded by 40% (v/v) DMF or glycerol. The steady-state level of EP was unaffected by these organic solvents, being 3.65 nmol/mg at 40 s in the presence of 40% (v/v) DMF and 4.05 nmol/mg at 30 s in the presence of 40% (v/v) glycerol (not shown). The EDANS fluorescence drop in the presence of 30% (v/v) DMF or glycerol was much faster than EP formation under the same conditions. These results are essentially the same as those obtained with Me 2 SO, strongly suggesting that the observed retardation of EP formation is due to the reduction in water activity. It is evident that the observed effects of the organic solvents are not due to changes in the dielectric constant of the medium, because the dielectric constant decreases considerably upon addition of DMF or glycerol but only slightly upon addition of Me 2 SO. Indeed, the dielectric constants of water, 40% (v/v) Me 2 SO, 40% (v/v) DMF, and 40% (v/v) glycerol at 0°C are 88.1, 85.1, 77.1, and 75.7, respectively (29).
Effects of Me 2 SO, DMF, and Glycerol on the Affinity of Ca 2ϩ -ATPase for AMP-PCP and ADP-Concentration dependencies of the EDANS fluorescence drop on AMP-PCP (a nonhydrolyzable ATP analog which is incapable of phosphorylating the enzyme) and ADP were determined by the steady-state measurements in the absence and presence of 40% (v/v) Me 2 SO, 40% (v/v) DMF, or 40% (v/v) glycerol ( Fig. 5 and Table I).
When AMP-PCP was added in the presence of Ca 2ϩ and absence of organic solvents, the fluorescence decreased with increasing concentration of AMP-PCP (Fig. 5A). The K m value obtained is consistent with the previously reported affinity of the catalytic site of the enzyme for AMP-PCP (30,31). This is in accord with our previous conclusion (17) that the fluorescence drop reflects a conformational change occurring upon formation of the calcium-enzyme-substrate complex. When Me 2 SO was added to give 40% (v/v), the affinity of the enzyme for AMP-PCP increased greatly, and the K m became 50-fold lower than that in the absence of organic solvents. The K m value for ADP obtained in the presence of Ca 2ϩ and absence of organic solvents is also consistent with the previously reported affinity of the catalytic site for ADP (32). When Me 2 SO was added to give 40% (v/v), the K m for ADP again decreased greatly. Similar results were obtained with DMF, although DMF was less effective than Me 2 SO. On the other hand, 40% (v/v) glycerol caused no appreciable decrease in the K m for AMP-PCP and ADP.
The K m for AMP-PCP and ADP in the absence of Ca 2ϩ decreased only slightly when Me 2 SO was added to give 40% (v/v) and rather increased to some extent when DMF or glycerol was added to give 40% (v/v) ( Fig. 5B and Table I). These results are in contrast to those obtained in the presence of Ca 2ϩ . Accordingly, the conformational change responsible for the ATP (or its analog)-induced fluorescence drop in the presence of Ca 2ϩ is distinct from that in the absence of Ca 2ϩ . It is, therefore, very likely that the observed effects of Me 2 SO and DMF on the affinity for AMP-PCP or ADP in the presence of Ca 2ϩ are specific to the Ca 2ϩ -activated enzyme. DISCUSSION The mechanism of the phosphoryl transfer retardation induced by the reduction in water activity and a possible role of water in the early stage of the catalytic cycle may be conveniently discussed in terms of the following reaction scheme proposed previously (16 -18), where E, EЈ, and EЉ denote different conformational states of the Ca 2ϩ -activated enzyme, and S denotes the substrate (ATP or its analog). EЉP represents ADP-sensitive EP. Our previous findings (16 -18) revealed that most of the ATP-induced fluorescence drop (Fig. 1), as well as the whole of the fluorescence drop induced by nonhydrolyzable ATP analogs (Fig. 5) in the presence of Ca 2ϩ , occurs upon the conformational change in Step 1. The present results (Figs. [2][3][4] show that the reduction in water activity markedly increases the activation energy for the phosphoryl transfer in Step 2. This strongly suggests that in this early stage of the catalytic cycle water plays a critical role in ensuring the rapid turnover of the enzyme, although the stereochemical analysis of the phosphoryl transfer in the SR Ca 2ϩ -ATPase (33) previously presented convincing evidence for the in-line displacement mechanism of the phosphoryl transfer in which water molecules are not directly involved. The increase in the activation energy for the phosphoryl transfer by the reduction in water activity may be possibly due to unstabilization of the transition state in Step 2. This suggests that the transition state is stabilized by hydration when the water activity has not been reduced. The data (Table I) showing the lack of the glycerol-induced shift of the equilibrium between Ca 2 ⅐E ϩ S and Ca 2 ⅐EЈ⅐S imply that the energy levels of both Ca 2 ⅐E ϩ S and Ca 2 ⅐EЈ⅐S are equally raised, rather than equally lowered, by the addition of glycerol, because glycerol is less hydrophilic than water and thus unstabilizes hydrated substrates such as ATP and its analogs (cf. Ref. 11). This gives a support to the above possibility that the observed increase in the activation energy for the phosphoryl transfer is due to unstabilization of the transition state in Step 2 rather than stabilization of Ca 2 ⅐EЈ⅐S. However, it should be noted that the suggested hydration of the transition state is seemingly out of harmony with the well known tight binding of transition state analogs to amino acid residues within the catalytic site (34 -36).
The results (Fig. 1) show that the addition of any of the three organic solvents causes no change or only a slight decrease in the rate of the conformational change in Step 1. This finding indicates that the energy levels of Ca⅐E ϩ S and the transition state in Step 1 are almost equally raised by the reduction in water activity. In contrast, the Me 2 SO-or DMF-induced large reductions in the K m for formation of Ca 2 ⅐EЈ⅐S (Table I) indicate that the rise in the energy level of Ca 2 ⅐EЈ⅐S comparable with that in the energy level of Ca 2 ⅐E ϩ S is not induced by the addition of Me 2 SO and DMF. It is, therefore, likely that the energy level of Ca 2 ⅐EЈ⅐S is less sensitive to these two considerably hydrophobic solvents than that of Ca 2 ⅐E ϩ S. This is consistent with the possible hydrophobic tertiary structure of the ATP-binding site, which was proposed previously by Taylor and Green (37) on the basis of the predicted secondary structure of the enzyme. Although the reason why the effect of glycerol on the relative energy levels of Ca 2 ⅐E ϩ S and Ca 2 ⅐EЈ⅐S is different from those of Me 2 SO and DMF remains obscure, this difference might be possibly due to the fact that glycerol is less hydrophobic than Me 2 SO and DMF (11).