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• W2021663041 abstract "Cys-674 of the sarcoplasmic reticulum Ca2+-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 CaCl2 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. Cys-674 of the sarcoplasmic reticulum Ca2+-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 CaCl2 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. The SR ( 1The abbreviations used are: SRsarcoplasmic reticulumEPphosphoenzymeDMFN,N-dimethylformamideEDANSN-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamineI-EDANSN-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamineMOPS3-(N-morpholino)propanesulfonic acidAMP-PCPadenosine 5′-(β,γ-methylene)triphosphate.) Ca2+-ATPase catalyzes ATP hydrolysis coupled to Ca2 transport(1.Hasselbach W. Makinose M. Biochem. Z. 1961; 333: 518-528PubMed Google Scholar, 2.Ebashi S. Lipmann F. J. Cell Biol. 1962; 14: 389-400Crossref PubMed Scopus (360) Google Scholar). In the initial step of the catalytic cycle, the enzyme is activated through Ca2 binding to the high affinity transport site. Theγ-phosphoryl group of ATP is transferred to Asp-351 in the catalytic site of the activated enzyme (3.Degani C. Boyer P.D. J. Biol. Chem. 1973; 248: 8222-8226Abstract Full Text PDF PubMed Google Scholar, 4.Bastide F. Meissner G. Fleischer S. Post R.L. J. Biol. Chem. 1973; 248: 8385-8391Abstract Full Text PDF PubMed Google Scholar, 5.Allen G. Green N.M. FEBS Lett. 1976; 63: 188-192Crossref PubMed Scopus (69) Google Scholar, 6.Brandl C.J. Green N.M. Korczak B. MacLennan D.H. Cell. 1986; 44: 597-607Abstract Full Text PDF PubMed Scopus (593) Google Scholar) to form ADP-sensitive EP(7.Makinose M. Pflügers Arch. Gesamte Physiol. Menschen Tiere. 1967; 294: R82-R83Google Scholar, 8.Yamamoto T. Tonomura Y. J. Biochem. (Tokyo). 1967; 62: 558-575Crossref PubMed Scopus (202) Google Scholar, 9.Kanazawa T. Yamada S. Tonomura Y. J. Biochem. (Tokyo). 1970; 68: 593-595Crossref PubMed Scopus (22) Google Scholar). In the subsequent conformational transition, this EP is converted to ADP-insensitive form. Concurrently, the affinity of the transport site for Ca2 is greatly reduced, and the Ca2 is released into the lumen. Finally, ADP-insensitive EP is hydrolyzed to liberate Pi. This catalytic cycle is fully reversible. sarcoplasmic reticulum phosphoenzyme N,N-dimethylformamide N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine 3-(N-morpholino)propanesulfonic acid adenosine 5′-(β,γ-methylene)triphosphate. The and Hasselbach (10.The R. Hasselbach W. Eur. J. Biochem. 1977; 74: 611-621Crossref PubMed Scopus (40) Google Scholar) showed previously that the rate of EP hydrolysis is markedly reduced by Me2SO. Later, de Meis et al. (11.de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar, 12.de Meis L. Inesi G. J. Biol. Chem. 1982; 257: 1289-1294Abstract Full Text PDF PubMed Google Scholar, 13.de Meis L. de Souza Otero A. Martins O.B. Alves E.W. Inesi G. Nakamoto R. J. Biol. Chem. 1982; 257: 4993-4998Abstract Full Text PDF PubMed Google Scholar) found that EP formation from Pi in the reverse reaction is greatly favored by substituting organic solvents such as Me2SO, 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.de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar), 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.de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar, 14.Dupont Y. Pougeois R. FEBS Lett. 1983; 156: 93-98Crossref PubMed Scopus (72) Google Scholar, 15.de Meis L. Biochim. Biophys. Acta. 1989; 973: 333-349Crossref PubMed Scopus (140) Google Scholar). 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.Suzuki H. Obara M. Kuwayama H. Kanazawa T. J. Biol. Chem. 1987; 262: 15448-15456Abstract Full Text PDF PubMed Google Scholar, 17.Kubo K. Suzuki H. Kanazawa T. Biochim. Biophys. Acta. 1990; 1040: 251-259Crossref PubMed Scopus (20) Google Scholar, 18.Suzuki H. Nakamura S. Kanazawa T. Biochemistry. 1994; 33: 8240-8246Crossref PubMed Scopus (17) Google Scholar). This fluorescence drop reflects a conformational change in the vicinity of the ATP-binding site, because Cys-674 is surrounded by amino acid residues which contribute the conformation of the ATP-binding site(19.Mitchinson C. Wilderspin A.F. Trinnaman B.J. Green N.M. FEBS Lett. 1982; 146: 87-92Crossref PubMed Scopus (185) Google Scholar, 20.Yamamoto H. Imamura Y. Tagaya M. Fukui T. Kawakita M. J. Biochem. (Tokyo). 1989; 106: 1121-1125Crossref PubMed Scopus (51) Google Scholar, 21.Clarke D.M. Loo T.W. MacLennan D.H. J. Biol. Chem. 1990; 265: 22223-22227Abstract Full Text PDF PubMed Google Scholar, 22.McIntosh D.B. Woolley D.G. Berman M.C. J. Biol. Chem. 1992; 267: 5301-5309Abstract Full Text PDF PubMed Google Scholar, 23.Lacapère J.-J. Garin J. Trinnaman B. Green N.M. Biochemistry. 1993; 32: 3414-3421Crossref PubMed Scopus (28) Google Scholar, 24.Yamagata K. Daiho T. Kanazawa T. J. Biol. Chem. 1993; 268: 20930-20936Abstract Full Text PDF PubMed Google Scholar, 25.Yamasaki K. Daiho T. Kanazawa T. J. Biol. Chem. 1994; 269: 4129-4134Abstract Full Text PDF PubMed Google Scholar). This conformational change is immediately followed by the phosphoryl transfer from ATP to the enzyme protein (16.Suzuki H. Obara M. Kuwayama H. Kanazawa T. J. Biol. Chem. 1987; 262: 15448-15456Abstract Full Text PDF PubMed Google Scholar, 17.Kubo K. Suzuki H. Kanazawa T. Biochim. Biophys. Acta. 1990; 1040: 251-259Crossref PubMed Scopus (20) Google Scholar, 18.Suzuki H. Nakamura S. Kanazawa T. Biochemistry. 1994; 33: 8240-8246Crossref PubMed Scopus (17) Google Scholar). In this study, we have investigated the effects of organic solvents (Me2SO, 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. SR vesicles were prepared from rabbit skeletal muscle and stored at −80°C as described previously (26.Nakamura S. Suzuki H. Kanazawa T. J. Biol. Chem. 1994; 269: 16015-16019Abstract Full Text PDF PubMed Google Scholar). The Ca2+-dependent ATPase activity determined at 25°C in a mixture containing 0.01 mg of SR vesicles/ml, 1.33 μM A23187, 0.2 mM [♦-32P]ATP, 5 mM MgCl2, 0.5 mM CaCl2, 0.4 mM EGTA, 0.1 M KCl, and 20 mM MOPS/Tris (pH 7.0) was 2.51 ± 0.05 μmol of Pi/mg/min (n = 3). The content of phosphorylation site in this preparation was 4.46 ± 0.08 nmol/mg (n = 6) when determined with [♦-32P]ATP according to Barrabin et al.(27.Barrabin H. Scofano H.M. Inesi G. Biochemistry. 1984; 23: 1542-1548Crossref PubMed Scopus (83) Google Scholar). SR vesicles were labeled with I-EDANS as described previously(16.Suzuki H. Obara M. Kuwayama H. Kanazawa T. J. Biol. Chem. 1987; 262: 15448-15456Abstract Full Text PDF PubMed Google Scholar). The content of bound EDANS was about 1 mol per mole of phosphorylation site. The Ca2+-dependent ATPase activity was not impaired by this labeling, being 2.53 ± 0.04 μmol of Pi/mg/min (n = 3). The content of the phosphorylation site determined as described above was 4.31 ± 0.06 nmol/mg (n = 6). EDANS-labeled SR vesicles were passively loaded with Ca2+ by incubation for 12-16 h at 4°C in a mixture containing 10 mg of EDANS-labeled SR vesicles/ml, 20 mM CaCl2, 84 mM KCl, 0.25 M sucrose, and 20 mM MOPS/Tris (pH 7.0). The steady-state fluorescence intensity of bound EDANS was measured on a computer-interfaced spectrofluorometer as described previously(17.Kubo K. Suzuki H. Kanazawa T. Biochim. Biophys. Acta. 1990; 1040: 251-259Crossref PubMed Scopus (20) Google Scholar). The excitation and emission wavelengths were set to 380 and 475 nm, respectively. 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 previously(17.Kubo K. Suzuki H. Kanazawa T. Biochim. Biophys. Acta. 1990; 1040: 251-259Crossref PubMed Scopus (20) Google Scholar). 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 Ca2-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. Rapid kinetic measurements of EP formation were made by using a continuous-flow rapid quenching system as described previously(17.Kubo K. Suzuki H. Kanazawa T. Biochim. Biophys. Acta. 1990; 1040: 251-259Crossref PubMed Scopus (20) Google Scholar). The reaction was started by mixing equal volumes of solutions from two syringes, one containing Ca2-loaded, EDANS-labeled SR vesicles and the other containing [β-32P]ATP, and quenched with trichloroacetic acid containing carrier ATP and Pi. 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 Pi and PPi, dissolved in 0.5 N NaOH containing 1% SDS, and the radioactivity was measured. Protein concentrations were determined by the method of Lowry et al. (28.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as a standard. Data were analyzed by the nonlinear least squares method as described previously(26.Nakamura S. Suzuki H. Kanazawa T. J. Biol. Chem. 1994; 269: 16015-16019Abstract Full Text PDF PubMed Google Scholar). ATP and ADP were from Yamasa Biochemicals (Choshi, Japan). AMP-PCP was from Sigma. I-EDANS was from Aldrich. [γ-32P]ATP was purchased from Amersham Corp. The ATP-induced change in the fluorescence of bound EDANS in the presence of 100 μM Ca2+ was followed by the stopped-flow method with Ca2+-loaded, EDANS-labeled SR vesicles in the absence of organic solvents or in the presence of 30% (v/v) Me2SO, 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.Suzuki H. Obara M. Kuwayama H. Kanazawa T. J. Biol. Chem. 1987; 262: 15448-15456Abstract Full Text PDF PubMed Google Scholar, 18.Suzuki H. Nakamura S. Kanazawa T. Biochemistry. 1994; 33: 8240-8246Crossref PubMed Scopus (17) Google Scholar), which were obtained with Ca2+-nonloaded vesicles. The rate and amplitude of the fluorescence drop were unaffected by the presence of 30% (v/v) Me2SO 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. EP formation with 100 μM ATP was followed in the presence of different concentrations of Me2SO (Fig. 2). The rate of EP formation decreased sharply with increasing concentration of Me2SO and thus became 20-fold lower in the presence of 40% (v/v) Me2SO than that in the absence of Me2SO. On the other hand, the steady-state level of EP was unaffected by 20-40% (v/v) Me2SO. Actually, the level of EP in the absence and presence of 40% (v/v) Me2SO was 4.04 nmol/mg at 4 s (Fig. 2) and 3.85 nmol/mg at 50 s (not shown), respectively. The lack of the effect of Me2SO on the steady-state level of EP is in agreement with the previously reported results(10.The R. Hasselbach W. Eur. J. Biochem. 1977; 74: 611-621Crossref PubMed Scopus (40) Google Scholar, 11.de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar). When 40% (v/v) Me2SO was present, an increase in the ATP concentration from 100 to 300 μM 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 Me2SO. For comparison, the EDANS fluorescence drop induced by 100 μM ATP was followed by the stopped-flow method in the presence of 100 μM Ca2+ and 30% (v/v) (Fig. 2) or 40% (v/v) (not shown) Me2SO. The fluorescence drop was much faster than EP formation under the same conditions. All these findings demonstrate that Me2SO 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 MeSO 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 Me2SO. A similar retardation of EP formation was found with either DMF (Fig. 3) or glycerol (Fig. 4), although glycerol was somewhat less effective than Me2SO 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 μM to 300 μM 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 Me2SO, 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 Me2SO. Indeed, the dielectric constants of water, 40% (v/v) Me2SO, 40% (v/v) DMF, and 40% (v/v) glycerol at 0°C are 88.1, 85.1, 77.1, and 75.7, respectively(29.Travers F. Douzou P. Biochimie (Paris). 1974; 56: 509-514Crossref PubMed Scopus (37) Google Scholar).Figure 4:Effect of glycerol on the kinetics of EP formation. EP formation was performed as in Fig. 2 except that Me2SO was replaced by glycerol. Glycerol concentrations used were 0% (○), 20% (•), 30% (▵), and 40% (♦, ×) (v/v). [β-32P]ATP concentrations after the mixing were 100 (○, •, ▵, ♦) and 300 (×) μM. For comparison, the ATP-induced fluorescence change (•••) was determined in the presence of 30% (v/v) glycerol, otherwise same as described in the legend to Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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) Me2SO, 40% (v/v) DMF, or 40% (v/v) glycerol (Fig. 5 and Table 1).Tabled 1 Open table in a new tab When AMP-PCP was added in the presence of Ca2+ and absence of organic solvents, the fluorescence decreased with increasing concentration of AMP-PCP (Fig. 5A). The Km value obtained is consistent with the previously reported affinity of the catalytic site of the enzyme for AMP-PCP(30.Cable M.B. Feher J.J. Briggs F.N. Biochemistry. 1985; 24: 5612-5619Crossref PubMed Scopus (33) Google Scholar, 31.Ogawa Y. Kurebayashi N. Harafuji H. J. Biochem. (Tokyo). 1986; 100: 1305-1318Crossref PubMed Scopus (17) Google Scholar). This is in accord with our previous conclusion (17.Kubo K. Suzuki H. Kanazawa T. Biochim. Biophys. Acta. 1990; 1040: 251-259Crossref PubMed Scopus (20) Google Scholar) that the fluorescence drop reflects a conformational change occurring upon formation of the calcium-enzyme-substrate complex. When Me2SO was added to give 40% (v/v), the affinity of the enzyme for AMP-PCP increased greatly, and the Km became 50-fold lower than that in the absence of organic solvents. The Km value for ADP obtained in the presence of Ca2+ and absence of organic solvents is also consistent with the previously reported affinity of the catalytic site for ADP (32.Arav R. Aderem A.A. Berman M.C. J. Biol. Chem. 1983; 258: 10433-10438Abstract Full Text PDF PubMed Google Scholar). When Me2SO was added to give 40% (v/v), the Km for ADP again decreased greatly. Similar results were obtained with DMF, although DMF was less effective than Me2SO. On the other hand, 40% (v/v) glycerol caused no appreciable decrease in the Km for AMP-PCP and ADP. The Km for AMP-PCP and ADP in the absence of Ca2+ decreased only slightly when Me2SO 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 1). These results are in contrast to those obtained in the presence of Ca2+. Accordingly, the conformational change responsible for the ATP (or its analog)-induced fluorescence drop in the presence of Ca2+ is distinct from that in the absence of Ca2+. It is, therefore, very likely that the observed effects of Me2SO and DMF on the affinity for AMP-PCP or ADP in the presence of Ca2+ are specific to the Ca2+-activated enzyme. 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.Suzuki H. Obara M. Kuwayama H. Kanazawa T. J. Biol. Chem. 1987; 262: 15448-15456Abstract Full Text PDF PubMed Google Scholar, 17.Kubo K. Suzuki H. Kanazawa T. Biochim. Biophys. Acta. 1990; 1040: 251-259Crossref PubMed Scopus (20) Google Scholar, 18.Suzuki H. Nakamura S. Kanazawa T. Biochemistry. 1994; 33: 8240-8246Crossref PubMed Scopus (17) Google Scholar), where E, E′, and E″ denote different conformational states of the Ca2+-activated enzyme, and S denotes the substrate (ATP or its analog). E″P represents ADP-sensitive EP. Our previous findings(16.Suzuki H. Obara M. Kuwayama H. Kanazawa T. J. Biol. Chem. 1987; 262: 15448-15456Abstract Full Text PDF PubMed Google Scholar, 17.Kubo K. Suzuki H. Kanazawa T. Biochim. Biophys. Acta. 1990; 1040: 251-259Crossref PubMed Scopus (20) Google Scholar, 18.Suzuki H. Nakamura S. Kanazawa T. Biochemistry. 1994; 33: 8240-8246Crossref PubMed Scopus (17) Google Scholar) 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 Ca2+, occurs upon the conformational change in Step 1. The present results (Figure 2:, Figure 3:, Figure 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 Ca2+-ATPase (33.Webb M.R. Trentham D.R. J. Biol. Chem. 1981; 256: 4884-4887Abstract Full Text PDF PubMed Google Scholar) 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 1) showing the lack of the glycerol-induced shift of the equilibrium between Ca2+•E + S and Ca2+•E′•S imply that the energy levels of both Ca2+•E + S and Ca2+•E′•S 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.(11.de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar)). 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 Ca2+•E′•S. 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.Frieden C. Kurz L.C. Gilbert H.R. Biochemistry. 1980; 19: 5303-5309Crossref PubMed Scopus (123) Google Scholar, 35.Fersht A. Enzyme Structure and Mechanism. 2nd Ed. W. H. Freeman and Co., New York1985Google Scholar, 36.Morrison J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar). 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 Ca•E + S and the transition state in Step 1 are almost equally raised by the reduction in water activity. In contrast, the Me2SO- or DMF-induced large reductions in the Km for formation of Ca2+•E′•S (Table 1) indicate that the rise in the energy level of Ca2+•E′•S comparable with that in the energy level of Ca2+•E + S is not induced by the addition of Me2SO and DMF. It is, therefore, likely that the energy level of Ca2+•E′•S is less sensitive to these two considerably hydrophobic solvents than that of Ca2+•E + S. This is consistent with the possible hydrophobic tertiary structure of the ATP-binding site, which was proposed previously by Taylor and Green (37.Taylor W.R. Green N.M. Eur. J. Biochem. 1989; 179: 241-248Crossref PubMed Scopus (118) Google Scholar) 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 Ca2+•E + S and Ca2+•E′•S is different from those of Me2SO and DMF remains obscure, this difference might be possibly due to the fact that glycerol is less hydrophobic than Me2SO and DMF(11.de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar)." @default.
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• W2021663041 date "1996-03-01" @default.
• W2021663041 modified "2023-10-07" @default.
• W2021663041 title "Reduction in Water Activity Greatly Retards the Phosphoryl Transfer from ATP to Enzyme Protein in the Catalytic Cycle of Sarcoplasmic Reticulum Ca2+-ATPase" @default.
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