- Email: [email protected]
Conformational changes and inactivation of calf intestinal alkaline phosphatase in trifluoroethanol solutions
The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894 www.elsevier.com/locate/ijbcb
Conformational changes and inactivation of calf intestinal alkaline phosphatase in tri¯uoroethanol solutions Ying-Xia Zhang a, Ying Zhu a, Hai-Meng Zhou b,* a Department of Chemistry, Capital University of Medical Sciences, Beijing 100054, People's Republic of China Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China
b
Received 27 January 2000; accepted 20 April 2000
Abstract The changes in activity and unfolding of calf intestinal alkaline phosphatase (CIP) during denaturation in dierent concentrations of tri¯uoroethanol (TFE) have been investigated by far-ultraviolet circular dichroism and ¯uorescence emission spectra. Unfolding and activation rate constants were measured and compared, the activation and inactivation courses were much faster than that of unfolding, which suggests that the active site of CIP containing two zinc ions and one magnesium ion is situated in a limited and ¯exible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole. However, compared to other metalloenzymes, CIP is inactivated at higher concentrations of TFE as denaturant. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Alkaline phosphatase; Tri¯uoroethanol; Unfolding; Inactivation
1. Introduction Alkaline phosphatase (EC3.1.3.1), which is widely distributed in nature, is characterized by a high pH optima and a broad substrate speci®city. It is a zinc-containing metalloprotein. The enzyme from Escherichia coli has been extensively Abbreviations: TFE, tri¯uoroethanol; CIP, calf intestinal alkaline phosphatase; CD, circular dichroism; pNPP, p-nitrophenylphosphate. * Corresponding author. Tel.: +861-255-2451; fax: +861256-2768.
studied [1]. The X-ray crystal structure of bacterial alkaline phosphatase has been reported to 2.0 AÊ resolution in the presence of inorganic phosphate [2].The active site is a tight cluster of two zinc ions (3.9 AÊ separation) and one magnesium ion (5 and 7 AÊ from the two zinc ions). Calf intestinal alkaline phosphatase (CIP) is also a dimeric metalloenzyme containing zinc and magnesium ions, and the structure of its active site is probably similar to that of bacterial alkaline phosphatase. During the denaturation of a number of enzymes by guanidinium chloride, urea, or or-
1357-2725/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 0 ) 0 0 0 2 5 - X
888
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
ganic solvents, inactivation occurs before noticeable conformational change of the enzyme molecule as a whole [3±5]. Tsou [6,7] suggested that enzyme active sites are usually situated in a limited region of the enzyme molecule which is more ¯exible than the enzyme molecule as a whole. Some authors [8,9] have studied the relationship between the inactivation and unfolding of enzymes containing metal ions as prosthetic groups [8,9]. The metal ions as prosthetic groups are usually situated in the enzyme active site and help to keep the conformation of the active site in a strained state [10], which is known as the entactic state. CIP is a typical metalloenzyme containing zinc and magnesium ions as the prosthetic group with a total of six metal ions. In the present paper, conformational changes of CIP during TFE denaturation were studied and compared with the activity changes in TFE solutions of dierent concentrations. The results suggested that although CIP contains four zinc ions and two magnesium ions, its active sites are more fragile to denaturants than the protein as a whole.
using the intensity change of the maximum intrinsic protein ¯uorescence emission. The data was plotted using the semilogarithmic graphical method to obtain the ®rst order rate constants for unfolding and activation (or inactivation) for the enzyme. Circular dichroism (CD) spectra were recorded with a Jasco 500C CD spectropolarimeter. The cell path length for CD measurements is 0.2 cm. In both the ¯uorescence and CD experiments the samples were incubated at 258C for 40 min, before the spectra measurements were performed. All measurements were carried out in 0.1 M NaHCO3/Na2CO3 buer solution, pH 10.0 at 258C.
2. Material and methods CIP was obtained from Promega. 2,2,2-tri¯uoroethanol was a Sigma product. The speci®c activity of the puri®ed enzyme was 1600 u/mg. The p-nitrophenylphosphate ( pNPP) was also from Promega. All other reagents were local products of analytical grade. Enzyme concentration was determined using the absorption coecient A1% 278 of 7.60 [11]. Enzyme activity was determined at 258C by following the increasing absorbence at 405 nm accompanying the hydrolysis of the substrate ( pNPP) with a molar absorption coecient of 1:73 10 ÿ4 Mÿ1 cmÿ1. The kinetic course of the activation (or inactivation) reaction was also followed by the absorbence at 405 nm. Kinetic measurements were carried out on a Shimadzu UV-2201 spectrophotometer. Fluorescence spectrum measurements were made with a Shimadzu RF5000 spectro¯uorometer. The excitation wavelength was 295 nm. The unfolding rate was measured
Fig. 1. Fluorescence emission spectra of CIP denatured in TFE solutions of dierent concentrations. CIP was dissolved in 0.1 M NaHCO3/Na2CO3 buer solution of pH 10.0 containing TFE at the desired concentration at 258C. The solutions were allowed to stand 30 minutes before ¯uorescence measurements with an excitation wavelength of 295 nm. TFE concentrations for curves 1±8 were 0, 5, 10, 20, 26, 30, 40 and 70%, respectively. The ®nal CIP concentration was 0.70 mM.
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
3. Results 3.1. Changes in activity and conformation of CIP during denaturation in TFE solutions of dierent concentrations Changes in the ¯uorescence emission spectra of CIP after denaturation for 30 min in TFE solutions of increasing concentrations are shown in Fig. 1. The maximum intrinsic protein ¯uorescence emission is 331 nm. Starting, at 0% (v/v) TFE concentration, increasing the TFE concentration caused the ¯uorescence emission intensity to decrease some with no noticeable shift of the emission peak. When the TFE concentration exceeded 20%, the ¯uorescence emission intensity began to increase. Further increases in the TFE concentration caused the ¯uorescence emission intensity increase to become more noticeable
889
until the maximum emission intensity was reached at a TFE concentration of 70% (v/v). Secondary structural changes of CIP containing 1123 amino acid residues [11] during TFE denaturaton were studied using the far-ultraviolet CD spectrum. Fig. 2 shows the far-ultraviolet CD spectra of CIP denatured in TFE solutions of dierent concentrations under similar conditions. For TFE concentrations from 0 to 10% (v/v), the average molecular ellipticity had no noticeable change. However for TFE concentrations above 30%, the average molecular ellipticity began to increase noticeably with a maximum value at 70% (v/v) TFE. The method described by Chen et al. [12] was used to estimate the secondary structure of denatured CIP. The calculated results are listed in Table 1. The extent of CIP activation increased with increasing TFE concentration and reached a
Fig. 2. Far-ultraviolet CD spectra of CIP during denaturation in TFE solutions of dierent concentrations. Experimental conditions were as for Fig. 1. Enzyme concentrations was 0.70 mM. Final TFE concentrations 1±6 were 0, 5, 10, 30, 40 and 70%, respectively, with the ®rst two curves nearly identical.
890
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
Table 1 Secondary structure of CIP for various TFE concentrations TFE % (v/v)
0 5 10 30 40 70
Secondary structure parameters a-Helix (%)
b-Sheet (%)
Turn and coil (%)
24.3 24.2 26.4 27.8 27.9 31.2
1.0 0 0 0 5.9 26.0
74.7 75.8 73.6 72.2 66.2 42.8
maximum at a TFE concentration of 10% (v/v), then decreased with further additions of TFE. When the TFE concentration exceeded 30%, the presence of TFE led to inactivation of CIP, Fig. 3. 3.2. Measurements of activation and inactivation rate constants of CIP in TFE solutions The temporal variation of the absorbence at
Table 2 Comparison of CIP activation and conformational change rate constants (10ÿ3, sÿ1) TFE concentration Activation Conformational change A/Da (%) (A ) (D ) 2 5 10 15 20 25 30 40
1.74 3.31 1.66 9.90 1.58 1.77 1.72 0.84 b
ÿ0.12 ÿ0.31 0.14 1.37 0.90
82.0 5.1 12.6 0.61
a
The ratio of rate constants of activation to conformational changes. b Inactivation rate constant was too fast to be measured by conventional method.
405 nm for dierent TFE concentration, which re¯ects the product concentration during substrate hydrolysis, is shown in Fig. 1. Within 20 min the kinetic course of the substrate reaction
Fig. 3. Courses of activation and inactivation of CIP in TFE solutions of dierent concentrations. Final substrate ( pNPP) concentrations was 2.0 mM and the ®nal enzyme concentration was 0.005 unit in 0.1 M NaHCO3/Na2CO3 buer solution pH 10.0 at 258C. TFE concentrations (v/v) for curves 1±7 were 0, 2.5, 5, 10, 30, 50 and 70%, respectively.
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
891
Fig. 4. Kinetics of activation of CIP in TFE solutions of dierent concentrations. The measurement conditions were as for Fig. 3. (a) Courses of activation of CIP in TFE solutions. TFE concentrations (v/v) for curves *, ^, q, R and r were 10, 5, 20, 15 and 2%, respectively. Ln(at/a0) was calculated as a function of time from data shown in (a). TFE concentrations (v/v) for curves in (b)±(e) are 2, 10, 15 and 20%, respectively.
892
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
approached a straight line for each TFE concentration, the slope of which increased with increasing TFE concentration until reaching the maximum at a TFE concentration of 10% (v/v). The slope then decreased with further increase in the TFE concentration until reaching the minimum at a TFE concentration of 70%, indicating that the denatured CIP retained very little residual activity. Plots of CIP activity versus time at each TFE concentration give a series of curves, Fig. 4a. Semilogarithmic plots of the activity versus time give a series of straight lines, indicating that the activation or inactivation process is a ®rst order reaction. The slopes of these curves give the activation or inactivation rate constants, Fig. 4b±e, which are listed in Table 2. 3.3. Rates of conformational changes of CIP during denaturation in TFE solutions The unfolding course of CIP in the TFE solutions of dierent concentrations was followed by changes in intrinsic ¯uorescence of the protein
Fig. 5. Kinetics of conformational changes of CIP in TFE solutions of dierent concentrations. The measurement conditions were as for Fig. 1. TFE concentrations (v/v) for curves ^, r, * and R were 30, 40, 20 and 10%, respectively.
Fig. 6. CIP activity changes in TFE solutions of dierent concentrations. The data is same as for Fig. 3. The circles represent the activity changes of aminoacylase in TFE solutions, Ref. [3].
molecule. The ¯uorescence emission spectra were measured at constant time intervals after addition of the enzyme into the denaturation system. The results showed that for TFE concentrations, less than 10%, the gradual unfolding of the molecule caused the emission intensity to decrease, while for TFE concentrations greater than 20%, the unfolding of the enzyme molecule caused the emission intensity to increase. Fig. 5 shows the course of the change in the ¯uorescence emission intensity for various TFE concentrations. A semilogarithmic plot of the change of the emission maximum versus time gives a straight line indicating that the unfolding process is a ®rst-order reaction. The unfolding rate constants, obtained from the slopes of the straight lines, are also listed in Table 2. Fig. 6 compares the activity changes of CIP and aminoacylase for various TFE concentrations. 4. Discussion It is well known that enzyme activity is
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
dependent on conformational integrity. The eects of structural changes on the activity of enzymes have been extensively studied in the literature. Tsou [6,7] suggested that active sites are usually situated in a limited region of the enzyme and more fragile to denaturants than the proteins as a whole. This conclusion is mainly based on investigations of enzymes not containing metal ions. The enzymes containing metal ions as prosthetic groups have not been thoroughly explored. It has been suggested by Vallee and Williams [10] that the presence of Zn2+ helps to keep the conformation of the active site in a strained state required for the catalysis of the enzyme. Therefore, it appears doubtful whether the active site has more conformational ¯exibility than the molecule as a whole. Therefore, some recent studies have concentrated on metalloproteins [8,9,14±19]. Although the X-ray crystal structure of CIP is still unknown, the structure of the active site of CIP is probably similar to that of bacterial alkaline phosphatase. The metal ions are essential to the conformational integrity and catalysis of the substrate reactions [20]. In the present study, the secondary structure of the enzyme, as followed by its far-ultraviolet CD spectra, underwent a noticeable change during TFE denaturation, showing a marked unfolding of the enzyme, which coincides with the reports [12,13,21] that TFE induces helical structure in polypeptides. These changes were also indicated by the changes in the ¯uorescence emission spectra. The conformation changes of the enzyme in TFE solutions measured by both ¯uorescence emission spectra and CD spectra increased with increasing TFE concentration. The activation and inactivation of CIP have been compared with the unfolding of CIP during denaturation in TFE solutions. As shown in Table 2, for any TFE concentration except for 30% (v/v), the activation rate constants of the enzyme are greater than the rate constants of conformational changes. The activation and inactivation rate constants are at least ®ve times greater than the conformational rate constants, with the greatest ratio at 40% TFE concen-
893
tration. The 30% TFE concentration is the critical concentration where the eect of TFE on the enzyme changes from activation to inactivation. Similar results were obtained for the denaturation of another zinc-containing enzyme, aminoacylase, in TFE solutions [3]. CIP, which contains multiple metal ions per subunit has much greater ability to withstand denaturation in organic solvents like TFE, as compared to aminoacylase which contains only a single metal ion per subunit of the molecule. As shown in Table 1, at a TFE concentration of 10% the secondary structure variation is much smaller than that of aminoacylase. For the same conditions, the a-helix content of aminoacylase increased [3] by 50%. Moreover, the CIP residual activity is still 70% at a TFE concentration of 50%, Fig. 6, while 10% TFE resulted in nearly complete loss of the aminoacylase activity.
Acknowledgements The present investigation was supported by National Key Basic Research Special Funds, P.R. China, No. G 1999075607.
References [1] J.E. Coleman, A Rev. Biophys. Biomol. Struct. 24 (1992) 441±483. [2] E.E. Kim, H.W. Wycko, J. Mol. Biol. 281 (1991) 449± 454. [3] Y.X. Zhang, L.Y. Shu, H.M. Zhou, J. of Protein Chemistry 15 (1996) 631±637. [4] S.L. Chen, Q.X. Chen, P.Z. Yang, W.J. Qiu, L.T. Wang, H.M. Zhou, Biochemistry and Molecular Biology International 42 (1997) 517±526. [5] S.M. Kelly, N.C. Price, Biochem. J. 275 (1991) 745±749. [6] C.L. Tsou, Science 268 (1993) 380±382. [7] C.L. Tsou, Trends Biochem. Sci. 11 (1986) 427±429. [8] B. He, M. Yan, H.M. Zhou, Chin. Sci. Bull. 39 (1994) 1122±1127. [9] H.R. Wang, T. Zhang, H.M. Zhou, Biochem. Biophys. Acta 1248 (1995) 97±106. [10] B.L. Vallee, R.J.P. Williams, Proc. Natl. Acad. Sci. USA 59 (1968) 498±505. [11] M. Fosset, D. Chappelet-Tordo, M. Lazdunski, Biochemistry 13 (1974) 1783±1787.
894
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
[12] Y.H. Chen, J.T. Yang, H.M. Martiner, Biochemistry 11 (1972) 4120±4131. [13] D.V. Waterhous, W.C. Johnson Jr., Biochemistry 33 (1994) 2121±2128. [14] M.Q. Huang, H.M. Zhou, Protein and Enzyme 48 (4) (1995) 229±237. [15] H.R. Wang, X.C. Wang, T. Zhang, H.M. Zhou, Science in China (Series B) 38 (3) (1995) 328±335. [16] B. He, T. Zhang, H.M. Zhou, Int. J. Biol. Macromol. 20 (1997) 53±62.
[17] T. Zhang, H.M. Zhou, Int. J. Biol. Macromol. 19 (1996) 113±119. [18] Z.X. Wang, H.R. Wang, H.M. Zhou, Biochemistry 34 (1995) 6863±6868. [19] J.H. Bai, D. Xu, H.R. Wang, S.Y. Zheng, H.M. Zhou, Bichim. Biophys. Acta 1430 (1999) 39±45. [20] Q.X. Chen, W.Z. Zheng, H.R. Wang, H.M. Zhou, Int. J. Biol. Macromol. 19 (1996) 257±261. [21] Y. Yang, X.S. Zhang, H.M. Zhou, Biochemistry (Moscow) 63 (1998) 1537±1542.
Conformational changes and inactivation of calf intestinal alkaline phosphatase in tri¯uoroethanol solutions Ying-Xia Zhang a, Ying Zhu a, Hai-Meng Zhou b,* a Department of Chemistry, Capital University of Medical Sciences, Beijing 100054, People's Republic of China Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China
b
Received 27 January 2000; accepted 20 April 2000
Abstract The changes in activity and unfolding of calf intestinal alkaline phosphatase (CIP) during denaturation in dierent concentrations of tri¯uoroethanol (TFE) have been investigated by far-ultraviolet circular dichroism and ¯uorescence emission spectra. Unfolding and activation rate constants were measured and compared, the activation and inactivation courses were much faster than that of unfolding, which suggests that the active site of CIP containing two zinc ions and one magnesium ion is situated in a limited and ¯exible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole. However, compared to other metalloenzymes, CIP is inactivated at higher concentrations of TFE as denaturant. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Alkaline phosphatase; Tri¯uoroethanol; Unfolding; Inactivation
1. Introduction Alkaline phosphatase (EC3.1.3.1), which is widely distributed in nature, is characterized by a high pH optima and a broad substrate speci®city. It is a zinc-containing metalloprotein. The enzyme from Escherichia coli has been extensively Abbreviations: TFE, tri¯uoroethanol; CIP, calf intestinal alkaline phosphatase; CD, circular dichroism; pNPP, p-nitrophenylphosphate. * Corresponding author. Tel.: +861-255-2451; fax: +861256-2768.
studied [1]. The X-ray crystal structure of bacterial alkaline phosphatase has been reported to 2.0 AÊ resolution in the presence of inorganic phosphate [2].The active site is a tight cluster of two zinc ions (3.9 AÊ separation) and one magnesium ion (5 and 7 AÊ from the two zinc ions). Calf intestinal alkaline phosphatase (CIP) is also a dimeric metalloenzyme containing zinc and magnesium ions, and the structure of its active site is probably similar to that of bacterial alkaline phosphatase. During the denaturation of a number of enzymes by guanidinium chloride, urea, or or-
1357-2725/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 0 ) 0 0 0 2 5 - X
888
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
ganic solvents, inactivation occurs before noticeable conformational change of the enzyme molecule as a whole [3±5]. Tsou [6,7] suggested that enzyme active sites are usually situated in a limited region of the enzyme molecule which is more ¯exible than the enzyme molecule as a whole. Some authors [8,9] have studied the relationship between the inactivation and unfolding of enzymes containing metal ions as prosthetic groups [8,9]. The metal ions as prosthetic groups are usually situated in the enzyme active site and help to keep the conformation of the active site in a strained state [10], which is known as the entactic state. CIP is a typical metalloenzyme containing zinc and magnesium ions as the prosthetic group with a total of six metal ions. In the present paper, conformational changes of CIP during TFE denaturation were studied and compared with the activity changes in TFE solutions of dierent concentrations. The results suggested that although CIP contains four zinc ions and two magnesium ions, its active sites are more fragile to denaturants than the protein as a whole.
using the intensity change of the maximum intrinsic protein ¯uorescence emission. The data was plotted using the semilogarithmic graphical method to obtain the ®rst order rate constants for unfolding and activation (or inactivation) for the enzyme. Circular dichroism (CD) spectra were recorded with a Jasco 500C CD spectropolarimeter. The cell path length for CD measurements is 0.2 cm. In both the ¯uorescence and CD experiments the samples were incubated at 258C for 40 min, before the spectra measurements were performed. All measurements were carried out in 0.1 M NaHCO3/Na2CO3 buer solution, pH 10.0 at 258C.
2. Material and methods CIP was obtained from Promega. 2,2,2-tri¯uoroethanol was a Sigma product. The speci®c activity of the puri®ed enzyme was 1600 u/mg. The p-nitrophenylphosphate ( pNPP) was also from Promega. All other reagents were local products of analytical grade. Enzyme concentration was determined using the absorption coecient A1% 278 of 7.60 [11]. Enzyme activity was determined at 258C by following the increasing absorbence at 405 nm accompanying the hydrolysis of the substrate ( pNPP) with a molar absorption coecient of 1:73 10 ÿ4 Mÿ1 cmÿ1. The kinetic course of the activation (or inactivation) reaction was also followed by the absorbence at 405 nm. Kinetic measurements were carried out on a Shimadzu UV-2201 spectrophotometer. Fluorescence spectrum measurements were made with a Shimadzu RF5000 spectro¯uorometer. The excitation wavelength was 295 nm. The unfolding rate was measured
Fig. 1. Fluorescence emission spectra of CIP denatured in TFE solutions of dierent concentrations. CIP was dissolved in 0.1 M NaHCO3/Na2CO3 buer solution of pH 10.0 containing TFE at the desired concentration at 258C. The solutions were allowed to stand 30 minutes before ¯uorescence measurements with an excitation wavelength of 295 nm. TFE concentrations for curves 1±8 were 0, 5, 10, 20, 26, 30, 40 and 70%, respectively. The ®nal CIP concentration was 0.70 mM.
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
3. Results 3.1. Changes in activity and conformation of CIP during denaturation in TFE solutions of dierent concentrations Changes in the ¯uorescence emission spectra of CIP after denaturation for 30 min in TFE solutions of increasing concentrations are shown in Fig. 1. The maximum intrinsic protein ¯uorescence emission is 331 nm. Starting, at 0% (v/v) TFE concentration, increasing the TFE concentration caused the ¯uorescence emission intensity to decrease some with no noticeable shift of the emission peak. When the TFE concentration exceeded 20%, the ¯uorescence emission intensity began to increase. Further increases in the TFE concentration caused the ¯uorescence emission intensity increase to become more noticeable
889
until the maximum emission intensity was reached at a TFE concentration of 70% (v/v). Secondary structural changes of CIP containing 1123 amino acid residues [11] during TFE denaturaton were studied using the far-ultraviolet CD spectrum. Fig. 2 shows the far-ultraviolet CD spectra of CIP denatured in TFE solutions of dierent concentrations under similar conditions. For TFE concentrations from 0 to 10% (v/v), the average molecular ellipticity had no noticeable change. However for TFE concentrations above 30%, the average molecular ellipticity began to increase noticeably with a maximum value at 70% (v/v) TFE. The method described by Chen et al. [12] was used to estimate the secondary structure of denatured CIP. The calculated results are listed in Table 1. The extent of CIP activation increased with increasing TFE concentration and reached a
Fig. 2. Far-ultraviolet CD spectra of CIP during denaturation in TFE solutions of dierent concentrations. Experimental conditions were as for Fig. 1. Enzyme concentrations was 0.70 mM. Final TFE concentrations 1±6 were 0, 5, 10, 30, 40 and 70%, respectively, with the ®rst two curves nearly identical.
890
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
Table 1 Secondary structure of CIP for various TFE concentrations TFE % (v/v)
0 5 10 30 40 70
Secondary structure parameters a-Helix (%)
b-Sheet (%)
Turn and coil (%)
24.3 24.2 26.4 27.8 27.9 31.2
1.0 0 0 0 5.9 26.0
74.7 75.8 73.6 72.2 66.2 42.8
maximum at a TFE concentration of 10% (v/v), then decreased with further additions of TFE. When the TFE concentration exceeded 30%, the presence of TFE led to inactivation of CIP, Fig. 3. 3.2. Measurements of activation and inactivation rate constants of CIP in TFE solutions The temporal variation of the absorbence at
Table 2 Comparison of CIP activation and conformational change rate constants (10ÿ3, sÿ1) TFE concentration Activation Conformational change A/Da (%) (A ) (D ) 2 5 10 15 20 25 30 40
1.74 3.31 1.66 9.90 1.58 1.77 1.72 0.84 b
ÿ0.12 ÿ0.31 0.14 1.37 0.90
82.0 5.1 12.6 0.61
a
The ratio of rate constants of activation to conformational changes. b Inactivation rate constant was too fast to be measured by conventional method.
405 nm for dierent TFE concentration, which re¯ects the product concentration during substrate hydrolysis, is shown in Fig. 1. Within 20 min the kinetic course of the substrate reaction
Fig. 3. Courses of activation and inactivation of CIP in TFE solutions of dierent concentrations. Final substrate ( pNPP) concentrations was 2.0 mM and the ®nal enzyme concentration was 0.005 unit in 0.1 M NaHCO3/Na2CO3 buer solution pH 10.0 at 258C. TFE concentrations (v/v) for curves 1±7 were 0, 2.5, 5, 10, 30, 50 and 70%, respectively.
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
891
Fig. 4. Kinetics of activation of CIP in TFE solutions of dierent concentrations. The measurement conditions were as for Fig. 3. (a) Courses of activation of CIP in TFE solutions. TFE concentrations (v/v) for curves *, ^, q, R and r were 10, 5, 20, 15 and 2%, respectively. Ln(at/a0) was calculated as a function of time from data shown in (a). TFE concentrations (v/v) for curves in (b)±(e) are 2, 10, 15 and 20%, respectively.
892
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
approached a straight line for each TFE concentration, the slope of which increased with increasing TFE concentration until reaching the maximum at a TFE concentration of 10% (v/v). The slope then decreased with further increase in the TFE concentration until reaching the minimum at a TFE concentration of 70%, indicating that the denatured CIP retained very little residual activity. Plots of CIP activity versus time at each TFE concentration give a series of curves, Fig. 4a. Semilogarithmic plots of the activity versus time give a series of straight lines, indicating that the activation or inactivation process is a ®rst order reaction. The slopes of these curves give the activation or inactivation rate constants, Fig. 4b±e, which are listed in Table 2. 3.3. Rates of conformational changes of CIP during denaturation in TFE solutions The unfolding course of CIP in the TFE solutions of dierent concentrations was followed by changes in intrinsic ¯uorescence of the protein
Fig. 5. Kinetics of conformational changes of CIP in TFE solutions of dierent concentrations. The measurement conditions were as for Fig. 1. TFE concentrations (v/v) for curves ^, r, * and R were 30, 40, 20 and 10%, respectively.
Fig. 6. CIP activity changes in TFE solutions of dierent concentrations. The data is same as for Fig. 3. The circles represent the activity changes of aminoacylase in TFE solutions, Ref. [3].
molecule. The ¯uorescence emission spectra were measured at constant time intervals after addition of the enzyme into the denaturation system. The results showed that for TFE concentrations, less than 10%, the gradual unfolding of the molecule caused the emission intensity to decrease, while for TFE concentrations greater than 20%, the unfolding of the enzyme molecule caused the emission intensity to increase. Fig. 5 shows the course of the change in the ¯uorescence emission intensity for various TFE concentrations. A semilogarithmic plot of the change of the emission maximum versus time gives a straight line indicating that the unfolding process is a ®rst-order reaction. The unfolding rate constants, obtained from the slopes of the straight lines, are also listed in Table 2. Fig. 6 compares the activity changes of CIP and aminoacylase for various TFE concentrations. 4. Discussion It is well known that enzyme activity is
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
dependent on conformational integrity. The eects of structural changes on the activity of enzymes have been extensively studied in the literature. Tsou [6,7] suggested that active sites are usually situated in a limited region of the enzyme and more fragile to denaturants than the proteins as a whole. This conclusion is mainly based on investigations of enzymes not containing metal ions. The enzymes containing metal ions as prosthetic groups have not been thoroughly explored. It has been suggested by Vallee and Williams [10] that the presence of Zn2+ helps to keep the conformation of the active site in a strained state required for the catalysis of the enzyme. Therefore, it appears doubtful whether the active site has more conformational ¯exibility than the molecule as a whole. Therefore, some recent studies have concentrated on metalloproteins [8,9,14±19]. Although the X-ray crystal structure of CIP is still unknown, the structure of the active site of CIP is probably similar to that of bacterial alkaline phosphatase. The metal ions are essential to the conformational integrity and catalysis of the substrate reactions [20]. In the present study, the secondary structure of the enzyme, as followed by its far-ultraviolet CD spectra, underwent a noticeable change during TFE denaturation, showing a marked unfolding of the enzyme, which coincides with the reports [12,13,21] that TFE induces helical structure in polypeptides. These changes were also indicated by the changes in the ¯uorescence emission spectra. The conformation changes of the enzyme in TFE solutions measured by both ¯uorescence emission spectra and CD spectra increased with increasing TFE concentration. The activation and inactivation of CIP have been compared with the unfolding of CIP during denaturation in TFE solutions. As shown in Table 2, for any TFE concentration except for 30% (v/v), the activation rate constants of the enzyme are greater than the rate constants of conformational changes. The activation and inactivation rate constants are at least ®ve times greater than the conformational rate constants, with the greatest ratio at 40% TFE concen-
893
tration. The 30% TFE concentration is the critical concentration where the eect of TFE on the enzyme changes from activation to inactivation. Similar results were obtained for the denaturation of another zinc-containing enzyme, aminoacylase, in TFE solutions [3]. CIP, which contains multiple metal ions per subunit has much greater ability to withstand denaturation in organic solvents like TFE, as compared to aminoacylase which contains only a single metal ion per subunit of the molecule. As shown in Table 1, at a TFE concentration of 10% the secondary structure variation is much smaller than that of aminoacylase. For the same conditions, the a-helix content of aminoacylase increased [3] by 50%. Moreover, the CIP residual activity is still 70% at a TFE concentration of 50%, Fig. 6, while 10% TFE resulted in nearly complete loss of the aminoacylase activity.
Acknowledgements The present investigation was supported by National Key Basic Research Special Funds, P.R. China, No. G 1999075607.
References [1] J.E. Coleman, A Rev. Biophys. Biomol. Struct. 24 (1992) 441±483. [2] E.E. Kim, H.W. Wycko, J. Mol. Biol. 281 (1991) 449± 454. [3] Y.X. Zhang, L.Y. Shu, H.M. Zhou, J. of Protein Chemistry 15 (1996) 631±637. [4] S.L. Chen, Q.X. Chen, P.Z. Yang, W.J. Qiu, L.T. Wang, H.M. Zhou, Biochemistry and Molecular Biology International 42 (1997) 517±526. [5] S.M. Kelly, N.C. Price, Biochem. J. 275 (1991) 745±749. [6] C.L. Tsou, Science 268 (1993) 380±382. [7] C.L. Tsou, Trends Biochem. Sci. 11 (1986) 427±429. [8] B. He, M. Yan, H.M. Zhou, Chin. Sci. Bull. 39 (1994) 1122±1127. [9] H.R. Wang, T. Zhang, H.M. Zhou, Biochem. Biophys. Acta 1248 (1995) 97±106. [10] B.L. Vallee, R.J.P. Williams, Proc. Natl. Acad. Sci. USA 59 (1968) 498±505. [11] M. Fosset, D. Chappelet-Tordo, M. Lazdunski, Biochemistry 13 (1974) 1783±1787.
894
Y.-X. Zhang et al. / The International Journal of Biochemistry & Cell Biology 32 (2000) 887±894
[12] Y.H. Chen, J.T. Yang, H.M. Martiner, Biochemistry 11 (1972) 4120±4131. [13] D.V. Waterhous, W.C. Johnson Jr., Biochemistry 33 (1994) 2121±2128. [14] M.Q. Huang, H.M. Zhou, Protein and Enzyme 48 (4) (1995) 229±237. [15] H.R. Wang, X.C. Wang, T. Zhang, H.M. Zhou, Science in China (Series B) 38 (3) (1995) 328±335. [16] B. He, T. Zhang, H.M. Zhou, Int. J. Biol. Macromol. 20 (1997) 53±62.
[17] T. Zhang, H.M. Zhou, Int. J. Biol. Macromol. 19 (1996) 113±119. [18] Z.X. Wang, H.R. Wang, H.M. Zhou, Biochemistry 34 (1995) 6863±6868. [19] J.H. Bai, D. Xu, H.R. Wang, S.Y. Zheng, H.M. Zhou, Bichim. Biophys. Acta 1430 (1999) 39±45. [20] Q.X. Chen, W.Z. Zheng, H.R. Wang, H.M. Zhou, Int. J. Biol. Macromol. 19 (1996) 257±261. [21] Y. Yang, X.S. Zhang, H.M. Zhou, Biochemistry (Moscow) 63 (1998) 1537±1542.