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Zinc-mediated thermal stabilization of carboxypeptidase A
Biomolecular Engineering 18 (2001) 179– 183 www.elsevier.com/locate/geneanabioeng
Zinc-mediated thermal stabilization of carboxypeptidase A Xinhui Li, Beka Solomon * Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-A6i6 Uni6ersity, Ramat A6i6 69978, Tel-A6i6, Israel Received 8 November 2000; received in revised form 10 July 2001; accepted 18 July 2001
Abstract In this study we investigated the contribution of Zn ions to the catalytic and structural thermostability of carboxypeptidase A (CPA). Structural studies on CPA molecule, performed in the presence of a number of ligands, demonstrated the multiple binding models around Zn ions which may affect the enzyme functions. Zinc was reported to bind at various sites in the CPA molecule at room temperature leading to inhibition of its enzymic activity. In this study we found that binding of Zn to CPA molecule followed by exposure to 50 °C did not inhibit the enzymic activity but activates and protects it against heat denaturation. The stabilization effect was found to be dependent on the increasing Zn/CPA ratios. The moderate changes of CPA activity as well as the UV and fluorescence spectra analyses indicate that the main function of the newly introduced zinc atoms is structural rather than catalytical. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Carboxypeptidase A; Stabilization; Activation; Zinc; Binding site; Catalytical; Structural
1. Introduction Zinc, one of the metal ions widely distributed in living systems, is an integral component of a large number of enzymes and proteins [1]. The role of zinc in proteins has been defined as catalytic and/or structural; structural zinc atoms being involved by association into large aggregates, in compaction and/or stabilization of proteins while catalytic zinc participates in the biological activity of the respective proteins [2–4]. Carboxypeptidase A (CPA) occupies a prominent position in the literature of metalloenzymes being a well-characterized zinc exopeptidase that exhibits both peptidase and esterase activity [5 – 7]. The enzyme is composed of 307 amino acid residues and one zinc ion in the active site, ligated to two histidines (His 69 and 196), glutamic acid, Glu 72, and one molecule of water [8]. Structural studies of CPA, in the presence of a number of inhibitors and transition state analogs, have demonstrated multiple binding models around the Zn * Corresponding author. Tel.: + 972-3-640-9836; fax: +972-3-6409407. E-mail address: [email protected] (B. Solomon).
ions, suggesting that flexibility in ligation may be an important factor for enzyme function [9]. X-ray adsorption fine structure (XAFC) studies indicate that the metal binding sites can have different conformations in solution from those in crystal [10,11]. The suggestion that zinc binds at multiple sites in CPA, besides in the active site, is supported by reported findings that an excess of zinc, when bound at room temperature, inhibits enzymic activity of CPA [12]. In the present study, we investigated thermostabilization of CPA occurring in the presence of an excess of Zn ions. The results indicated that addition of excess zinc ions to CPA before exposure to 50 °C protected the enzyme from heat denaturation which in turn had a stabilizing effect on the whole molecule.
2. Materials and methods CPA (Sigma Chemical Co., St. Louis, MO, USA) was obtained as an aqueous crystalline suspension. The crystals were washed with double-distilled water, centrifuged and dissolved in PBS to a 4 mg/ml stock CPA solution. The enzyme concentration derived from the absorbancy at 278 nm, using an extinction coefficient of E1% at 278 nm= 19.4, was measured with a UV/Visi-
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X. Li, B. Solomon / Biomolecular Engineering 18 (2001) 179–183
ble Spectrophotometer (Pharmacia, Pharmacia LKBUltrospec III). Fresh stock solution of ZnCl2 (Merck, Darmstadt, Germany) in water was prepared daily with different concentrations, ranged between 0.1 and 100 mM.
2.1. Preparation of Zn– CPA complexes Zn –CPA complexes were prepared in phosphate buffer at different pH values and zinc content. Samples were prepared using buffers composed of 0.65 M NaCl, 100 mM phosphate at three different pH values: 6.0, 6.8 and 7.5. The molar ratio of Zn to CPA was 0, 1, 5, 10, 50, 100, 500 and 1000, respectively. In each experiment, the samples were prepared in duplicate.
2.2. Denaturation of Zn– CPA complex The samples of Zn– CPA were exposed to a temperature of 50 °C for 1 h. After heating, the samples were kept at a temperature of 4 °C. Before further analysis, all Zn –CPA samples were centrifuged at 10 000 rpm for 10 min. In order to study the characteristics of heated Zn –CPA, fresh native CPA was prepared in each case under the same experimental conditions and the same volume of double-distilled water takes the place of the ZnCl2 solution used for the preparation of Zn–CPA.
2.3. Characterization of Zn– CPA complexes exposed to 50 °C 2.3.1. Protein assay The residual amounts of heat denatured CPA or Zn – CPA were measured using the absorbance at 278 nm and related to the unheated sample prepared in the phosphate buffer without zinc, as well as using the Biorad Assay (Cat. No. 500-006, Biorad Laboratories GmbH). The assay is proceeded on ELISA microplate and in each well were added 150 ml of five times diluted Biorad reagent and 100 ml CPA sample. The absorbance at 690 nm of the samples was read on EAR 400 FW (from SLT, Labinstruments, Austria). 2.3.2. Esterase acti6ity assay Zn –CPA residual activity was measured using esterase substrate (Hippuryl-DL-Phenyllactic Acid (Sigma Chemical Co., USA)) and reported to the native CPA prepared under the same conditions [13]. Specific activity of Zn –CPA and/or denatured CPA was calculated from residual activity versus residual amount of protein and related to the activity of native CPA prepared under the same conditions. 2.3.3. Fluorescence emission analysis The fluorescence emission spectra of native CPA, denatured Zn–CPA and CPA were followed by an
LS-50 luminescence spectrometer at an excitation wavelength of 280 nm at a scan range from 300 to 400 nm. Excitation and emission slits are 3 and 5 nm, respectively.
2.3.4. UV analysis All the UV spectra were scanned at room temperature on Hewlett Packard HP 8452 Diode Array Spectrophotometer (with HP89531A MS-DOS-UV/Vis operation software). The samples are exposed to 50 °C for 1 h, cooled and spectra recorded at room temperature. 2.3.5. Atomic adsorption measurements The remaining free zinc from the reaction mixtures was removed by dialysis using a membrane with 12–14 kDa cut-off against PBS 0.5 M NaCl overnight at 4 °C, followed by desalting by passing it over a column of PD-10 (Biorad, Richmond, CN), according to manufacturer’s instructions. The amount of Zn bound to CPA after exposure at 50 °C for 1 h was measured by flame atomic adsorption spectroscopy with Perkin–Elmer 2280 spectrophotometer.
3. Results and discussion CPA requires zinc for its catalytic activity but is inhibited by the addition of an excess of zinc at room temperature in the concentration range of 10 − 4 –10 − 6 M [12]. We found that the addition of increasing amounts of zinc to CPA followed by heating the complex at 50 °C for 1 h stabilizes and activates the enzyme. Under the experimental conditions used, if zinc is added to the enzyme without heating, no changes occur in its catalytic activity. Barium and calcium added to the CPA under the same experimental conditions had no effect on the enzyme stability [14].
3.1. Effect of zinc ions on the thermal stability of CPA Heat stability of CPA and/or Zn –CPA preparations was followed by measuring the residual amount of protein and specific enzymic activity remaining after heating at 50 °C for 1 h in 100 mM phosphate buffer with different zinc content. Addition of zinc prior to heating affects the stability of heat exposed enzyme and increases the residual amount of soluble CPA (Fig. 1). At the same Zn/CPA ratio in the range of 0–100 Zn/CPA, the complexes prepared at pH 6.8 have higher specific activity than the corresponding samples of pH 6.0 and pH 7.5 (Fig. 1). The sharp fall in specific activities can be attributed to precipitation of enzyme or ‘‘salt out’’ from solution at ratio of 1000 of Zn/CPA, or may be due to subtle differences in the surface amino acid residues involved in new revealed sites.
X. Li, B. Solomon / Biomolecular Engineering 18 (2001) 179–183
181
The specific enzymic activity of Zn– CPA increases with the ratio of Zn/CPA and the complexes exhibit a higher specific activity than CPA samples prepared under the same experimental conditions without zinc. A superactivation effect on the specific enzyme activity occurred on addition of zinc anions.
Fig. 3. UV difference spectrum of CPA and Zn – CPA. (A) Native CPA containing 100 mM phosphate and 0.65 M NaCl at pH 6.8, at room temperature; (B) heated CPA and Zn – CPA, prepared as described in (A) after 1 h heating at 50 °C. All UV measurements are proceeded at room temperature. Fig. 1. Specific esterase activity of CPA as function of zinc content and various pH values. The specific enzymatic activity of all the samples are related to that of denatured CPA without zinc in the sample preparation. The samples were prepared in 100 mM phosphate buffer which contained 0.65 M NaCl, pH 6.8. All data presented represent the means of triplicate determinations. The S.D. values for the intra-assay and inter-assays were less than 5% in all cases.
3.2. Fluorescence analysis of Zn–CPA complexes CPA molecule contains 42 fluorophores, as follows: 16 Phe, 19 Tyr and 7 Trp residues [15,16]. When CPA is excitated at 280 nm, the seven Trp residues will mainly contribute to fluorescence emission at 330 nm (Fig. 2). Most of these seven Trp residues are located on the surface of the CPA molecule. Heating of CPA to 50 °C in the absence of zinc in 100 mM phosphate buffer at pH 7.5 led to maximum shift of tryptophan from 330 to 335 nm (Fig. 2). This phenomenon suggested shielding of tryptophan fluorophores and implies a structural reorganization in heated CPA molecule. In fact, the reorganized structure may be an intermediate stable structure, as the ones known for IgGs when exposed to acidic pHs [17]. When CPA was heated in the presence of different amounts of zinc, this red shift decreased and became close to 330 nm, similar to native CPA as the ratio of Zn/CPA increased.
3.3. UV analysis of the Zn/CPA complexes
Fig. 2. Fluorescence emission wavelength variations of denatured CPA and Zn/CPA after 1 h at 50 °C. The samples were prepared with increasing amounts of Zn as compared to native, unheated enzyme.
UV spectra of native and various Zn–CPA complexes are shown in Fig. 3. The UV maximum wavelength (umax) variation pattern of soluble denatured CPA and Zn/CPA are presented in Table 1. The difference of umax between heated and native CPA is smaller after Zn complexation with CPA.
X. Li, B. Solomon / Biomolecular Engineering 18 (2001) 179–183
182
Table 1 UV wavelength variation pattern of denatured CPA and Zn–CPA compared to native enzyme A. Peak I Zn/CPA pH 7.5 pH 6.8 pH 6.0
0 212 206 212
1 212 206 210
5 210 206 210
10 210 206 208
50 210 206 210
100 210 208 212
1000 210 208 214
Native 208 206 206
B. Peak II Zn/CPA pH 7.5 pH 6.8 pH 6.0
0 280 278 280
1 282 278 280
5 278 278 280
10 276 278 280
50 276 278 280
100 276 278 280
1000 276 276 280
Native 280 279 280
Table 2 The putative binding site of zinc ion in the structure of Zn–CPA Site atom
Coordination residues
Zn-13a Zn-29 Zn-166 Zn-303
His His His His
13,Asp 16, His 120 29, Glu 88 166, Asp 142, Glu 173 303, Asp 215, Glu 218
a The numbers are related to the position of the close histidine residue.
under these experimental conditions rather than a reversible inhibition by phosphate anions. The present findings suggest that heating of CPA at 50 °C in the presence of excess of Zn exposes new zinc binding sites, which increase enzyme stability and activity. We investigated the effect of Zn after exposure to 50 °C and propose that the Zn stabilization effect can be due to a new revealed Zn-binding site, in spite of inhibitory effect reported at room temperature. Another metal, like barium and calcium, has no effect on enzyme stability of CPA. The effect seems to be similar to the stabilization of proteins through engineered metal sites [19]. His, Asp and Glu are putative ligands that bind zinc but His remained the preferred ligand which is combined with zinc most easily [20,21]. An examination of the primary structure of CPA [8] suggests the location of putative metal binding configurations which may contain histidine, aspartate and glutamate residues (Table 2), (Fig. 4). The changes of CPA activity as well as the UV and fluorescence spectra analyses indicate that the main function of the newly introduced zinc atoms is structural rather than catalytical.
Fig. 4. Putative sites on CPA molecule for zinc binding. Zinc ions are assumed to bind to one of the four His residues, as follows: His 13, His 29, His 166 and/or His 303 in CPA molecule and connect to other possible ligands like His, Asp or Glu which are located at distances of approximately 10 A, . The structure of Zn–CPA is then minimized in CHARMn using Quanta 4.0 until 0.02 rms.
Acknowledgements
4. Atomic adsorption measurements
References
After dialysis and desalination procedure, atomic adsorption analysis showed that the amount of zinc atoms in the CPA molecule ranged between 1 and 5 according to the initial amount of Zn added to CPA before heat exposure. In a previous paper we showed the protective effect of phosphate under similar experimental conditions after heating to 50 °C [18]. It seems to be a cooperative or synergistic stabilizing effect of zinc and phosphate
The authors wish to express thanks to Dr. Dorit Arad for assistance in modeling studies.
[1] [2] [3] [4] [5] [6] [7] [8]
Vallee BL, Auld DS. Faraday Discuss 1992;93:47 – 65. Regan L. Annu Rev Biophys Biomol Struct 1993;22:57 –81. Arnold FH, Zhang J-H. Trends Biotechnol 1994;12:189 –92. Ochiai T, Hoshina S, Usuki I. Biochem Biophys Acta 1993;1203:310 – 4. Vallee BL, Wacker WEC. In: Neurath H, editor. The Proteins, vol. 5. New York: Academic Press, 1970:1 – 192. Quiocho FA, Lipscomb WN. Adv Prot Chem 1971;25:1 –49. Christianson DW, Lipscomb WN. Acc Chem Res 1989;22:62 – 9. Rees DC, Lewis M, Lipscomb WN. J Mol Biol 1983;168:367 – 87.
X. Li, B. Solomon / Biomolecular Engineering 18 (2001) 179–183 [9] Stote RH, Karplus M. Proteins Structure Function Genet 1995;28:12 – 31. [10] Zhang K, Chance B, Auld DS, Larsen KS, Vallee BL. Biochemistry 1992;31:1159 – 68. [11] Zhang K, Auld DS. Biochemistry 1993;32:13844 – 51. [12] Larsen KS, Auld DS. Biochemistry 1991;30:2613 –8. [13] Whitaker JR, Menger F, Bender ML. Biochemistry 1966;5:386 – 92. [14] Katzav-Gojanski T. M.Sc. Thesis. Tel-Aviv University; 1994. [15] Hartsuck J, Lipscomb WN. In: Boyer PD, editor. The Enzymes,
183
vol. 5. New York: Academic Press, 1971:1 – 56. [16] Valley BL, Galdes A, Auld DS, Riordan JR. In: Spiro TG, editor. zinc enzymes. Wiley, 1983:26 – 74. [17] Buchner J, Renner M, Lilie H, Hinz HJ, Jaenicke R, Kiefhabel T, Rudolph R. Biochemistry 1991;30(28):6922 – 9. [18] Li X, Solomon B. Biochem Molec Biol Int 1997;43:601 –11. [19] Arnold FH, Zhang ZH. Trends Biotechnol 1994;12:189 –92. [20] Rees DC, Lipscomb WN. Proc Natl Acad Sci USA 1983;83:7151 – 4. [21] Holmes MA, Mathews BW. J Mol Biol 1982;160:623 –39.
Zinc-mediated thermal stabilization of carboxypeptidase A Xinhui Li, Beka Solomon * Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-A6i6 Uni6ersity, Ramat A6i6 69978, Tel-A6i6, Israel Received 8 November 2000; received in revised form 10 July 2001; accepted 18 July 2001
Abstract In this study we investigated the contribution of Zn ions to the catalytic and structural thermostability of carboxypeptidase A (CPA). Structural studies on CPA molecule, performed in the presence of a number of ligands, demonstrated the multiple binding models around Zn ions which may affect the enzyme functions. Zinc was reported to bind at various sites in the CPA molecule at room temperature leading to inhibition of its enzymic activity. In this study we found that binding of Zn to CPA molecule followed by exposure to 50 °C did not inhibit the enzymic activity but activates and protects it against heat denaturation. The stabilization effect was found to be dependent on the increasing Zn/CPA ratios. The moderate changes of CPA activity as well as the UV and fluorescence spectra analyses indicate that the main function of the newly introduced zinc atoms is structural rather than catalytical. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Carboxypeptidase A; Stabilization; Activation; Zinc; Binding site; Catalytical; Structural
1. Introduction Zinc, one of the metal ions widely distributed in living systems, is an integral component of a large number of enzymes and proteins [1]. The role of zinc in proteins has been defined as catalytic and/or structural; structural zinc atoms being involved by association into large aggregates, in compaction and/or stabilization of proteins while catalytic zinc participates in the biological activity of the respective proteins [2–4]. Carboxypeptidase A (CPA) occupies a prominent position in the literature of metalloenzymes being a well-characterized zinc exopeptidase that exhibits both peptidase and esterase activity [5 – 7]. The enzyme is composed of 307 amino acid residues and one zinc ion in the active site, ligated to two histidines (His 69 and 196), glutamic acid, Glu 72, and one molecule of water [8]. Structural studies of CPA, in the presence of a number of inhibitors and transition state analogs, have demonstrated multiple binding models around the Zn * Corresponding author. Tel.: + 972-3-640-9836; fax: +972-3-6409407. E-mail address: [email protected] (B. Solomon).
ions, suggesting that flexibility in ligation may be an important factor for enzyme function [9]. X-ray adsorption fine structure (XAFC) studies indicate that the metal binding sites can have different conformations in solution from those in crystal [10,11]. The suggestion that zinc binds at multiple sites in CPA, besides in the active site, is supported by reported findings that an excess of zinc, when bound at room temperature, inhibits enzymic activity of CPA [12]. In the present study, we investigated thermostabilization of CPA occurring in the presence of an excess of Zn ions. The results indicated that addition of excess zinc ions to CPA before exposure to 50 °C protected the enzyme from heat denaturation which in turn had a stabilizing effect on the whole molecule.
2. Materials and methods CPA (Sigma Chemical Co., St. Louis, MO, USA) was obtained as an aqueous crystalline suspension. The crystals were washed with double-distilled water, centrifuged and dissolved in PBS to a 4 mg/ml stock CPA solution. The enzyme concentration derived from the absorbancy at 278 nm, using an extinction coefficient of E1% at 278 nm= 19.4, was measured with a UV/Visi-
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X. Li, B. Solomon / Biomolecular Engineering 18 (2001) 179–183
ble Spectrophotometer (Pharmacia, Pharmacia LKBUltrospec III). Fresh stock solution of ZnCl2 (Merck, Darmstadt, Germany) in water was prepared daily with different concentrations, ranged between 0.1 and 100 mM.
2.1. Preparation of Zn– CPA complexes Zn –CPA complexes were prepared in phosphate buffer at different pH values and zinc content. Samples were prepared using buffers composed of 0.65 M NaCl, 100 mM phosphate at three different pH values: 6.0, 6.8 and 7.5. The molar ratio of Zn to CPA was 0, 1, 5, 10, 50, 100, 500 and 1000, respectively. In each experiment, the samples were prepared in duplicate.
2.2. Denaturation of Zn– CPA complex The samples of Zn– CPA were exposed to a temperature of 50 °C for 1 h. After heating, the samples were kept at a temperature of 4 °C. Before further analysis, all Zn –CPA samples were centrifuged at 10 000 rpm for 10 min. In order to study the characteristics of heated Zn –CPA, fresh native CPA was prepared in each case under the same experimental conditions and the same volume of double-distilled water takes the place of the ZnCl2 solution used for the preparation of Zn–CPA.
2.3. Characterization of Zn– CPA complexes exposed to 50 °C 2.3.1. Protein assay The residual amounts of heat denatured CPA or Zn – CPA were measured using the absorbance at 278 nm and related to the unheated sample prepared in the phosphate buffer without zinc, as well as using the Biorad Assay (Cat. No. 500-006, Biorad Laboratories GmbH). The assay is proceeded on ELISA microplate and in each well were added 150 ml of five times diluted Biorad reagent and 100 ml CPA sample. The absorbance at 690 nm of the samples was read on EAR 400 FW (from SLT, Labinstruments, Austria). 2.3.2. Esterase acti6ity assay Zn –CPA residual activity was measured using esterase substrate (Hippuryl-DL-Phenyllactic Acid (Sigma Chemical Co., USA)) and reported to the native CPA prepared under the same conditions [13]. Specific activity of Zn –CPA and/or denatured CPA was calculated from residual activity versus residual amount of protein and related to the activity of native CPA prepared under the same conditions. 2.3.3. Fluorescence emission analysis The fluorescence emission spectra of native CPA, denatured Zn–CPA and CPA were followed by an
LS-50 luminescence spectrometer at an excitation wavelength of 280 nm at a scan range from 300 to 400 nm. Excitation and emission slits are 3 and 5 nm, respectively.
2.3.4. UV analysis All the UV spectra were scanned at room temperature on Hewlett Packard HP 8452 Diode Array Spectrophotometer (with HP89531A MS-DOS-UV/Vis operation software). The samples are exposed to 50 °C for 1 h, cooled and spectra recorded at room temperature. 2.3.5. Atomic adsorption measurements The remaining free zinc from the reaction mixtures was removed by dialysis using a membrane with 12–14 kDa cut-off against PBS 0.5 M NaCl overnight at 4 °C, followed by desalting by passing it over a column of PD-10 (Biorad, Richmond, CN), according to manufacturer’s instructions. The amount of Zn bound to CPA after exposure at 50 °C for 1 h was measured by flame atomic adsorption spectroscopy with Perkin–Elmer 2280 spectrophotometer.
3. Results and discussion CPA requires zinc for its catalytic activity but is inhibited by the addition of an excess of zinc at room temperature in the concentration range of 10 − 4 –10 − 6 M [12]. We found that the addition of increasing amounts of zinc to CPA followed by heating the complex at 50 °C for 1 h stabilizes and activates the enzyme. Under the experimental conditions used, if zinc is added to the enzyme without heating, no changes occur in its catalytic activity. Barium and calcium added to the CPA under the same experimental conditions had no effect on the enzyme stability [14].
3.1. Effect of zinc ions on the thermal stability of CPA Heat stability of CPA and/or Zn –CPA preparations was followed by measuring the residual amount of protein and specific enzymic activity remaining after heating at 50 °C for 1 h in 100 mM phosphate buffer with different zinc content. Addition of zinc prior to heating affects the stability of heat exposed enzyme and increases the residual amount of soluble CPA (Fig. 1). At the same Zn/CPA ratio in the range of 0–100 Zn/CPA, the complexes prepared at pH 6.8 have higher specific activity than the corresponding samples of pH 6.0 and pH 7.5 (Fig. 1). The sharp fall in specific activities can be attributed to precipitation of enzyme or ‘‘salt out’’ from solution at ratio of 1000 of Zn/CPA, or may be due to subtle differences in the surface amino acid residues involved in new revealed sites.
X. Li, B. Solomon / Biomolecular Engineering 18 (2001) 179–183
181
The specific enzymic activity of Zn– CPA increases with the ratio of Zn/CPA and the complexes exhibit a higher specific activity than CPA samples prepared under the same experimental conditions without zinc. A superactivation effect on the specific enzyme activity occurred on addition of zinc anions.
Fig. 3. UV difference spectrum of CPA and Zn – CPA. (A) Native CPA containing 100 mM phosphate and 0.65 M NaCl at pH 6.8, at room temperature; (B) heated CPA and Zn – CPA, prepared as described in (A) after 1 h heating at 50 °C. All UV measurements are proceeded at room temperature. Fig. 1. Specific esterase activity of CPA as function of zinc content and various pH values. The specific enzymatic activity of all the samples are related to that of denatured CPA without zinc in the sample preparation. The samples were prepared in 100 mM phosphate buffer which contained 0.65 M NaCl, pH 6.8. All data presented represent the means of triplicate determinations. The S.D. values for the intra-assay and inter-assays were less than 5% in all cases.
3.2. Fluorescence analysis of Zn–CPA complexes CPA molecule contains 42 fluorophores, as follows: 16 Phe, 19 Tyr and 7 Trp residues [15,16]. When CPA is excitated at 280 nm, the seven Trp residues will mainly contribute to fluorescence emission at 330 nm (Fig. 2). Most of these seven Trp residues are located on the surface of the CPA molecule. Heating of CPA to 50 °C in the absence of zinc in 100 mM phosphate buffer at pH 7.5 led to maximum shift of tryptophan from 330 to 335 nm (Fig. 2). This phenomenon suggested shielding of tryptophan fluorophores and implies a structural reorganization in heated CPA molecule. In fact, the reorganized structure may be an intermediate stable structure, as the ones known for IgGs when exposed to acidic pHs [17]. When CPA was heated in the presence of different amounts of zinc, this red shift decreased and became close to 330 nm, similar to native CPA as the ratio of Zn/CPA increased.
3.3. UV analysis of the Zn/CPA complexes
Fig. 2. Fluorescence emission wavelength variations of denatured CPA and Zn/CPA after 1 h at 50 °C. The samples were prepared with increasing amounts of Zn as compared to native, unheated enzyme.
UV spectra of native and various Zn–CPA complexes are shown in Fig. 3. The UV maximum wavelength (umax) variation pattern of soluble denatured CPA and Zn/CPA are presented in Table 1. The difference of umax between heated and native CPA is smaller after Zn complexation with CPA.
X. Li, B. Solomon / Biomolecular Engineering 18 (2001) 179–183
182
Table 1 UV wavelength variation pattern of denatured CPA and Zn–CPA compared to native enzyme A. Peak I Zn/CPA pH 7.5 pH 6.8 pH 6.0
0 212 206 212
1 212 206 210
5 210 206 210
10 210 206 208
50 210 206 210
100 210 208 212
1000 210 208 214
Native 208 206 206
B. Peak II Zn/CPA pH 7.5 pH 6.8 pH 6.0
0 280 278 280
1 282 278 280
5 278 278 280
10 276 278 280
50 276 278 280
100 276 278 280
1000 276 276 280
Native 280 279 280
Table 2 The putative binding site of zinc ion in the structure of Zn–CPA Site atom
Coordination residues
Zn-13a Zn-29 Zn-166 Zn-303
His His His His
13,Asp 16, His 120 29, Glu 88 166, Asp 142, Glu 173 303, Asp 215, Glu 218
a The numbers are related to the position of the close histidine residue.
under these experimental conditions rather than a reversible inhibition by phosphate anions. The present findings suggest that heating of CPA at 50 °C in the presence of excess of Zn exposes new zinc binding sites, which increase enzyme stability and activity. We investigated the effect of Zn after exposure to 50 °C and propose that the Zn stabilization effect can be due to a new revealed Zn-binding site, in spite of inhibitory effect reported at room temperature. Another metal, like barium and calcium, has no effect on enzyme stability of CPA. The effect seems to be similar to the stabilization of proteins through engineered metal sites [19]. His, Asp and Glu are putative ligands that bind zinc but His remained the preferred ligand which is combined with zinc most easily [20,21]. An examination of the primary structure of CPA [8] suggests the location of putative metal binding configurations which may contain histidine, aspartate and glutamate residues (Table 2), (Fig. 4). The changes of CPA activity as well as the UV and fluorescence spectra analyses indicate that the main function of the newly introduced zinc atoms is structural rather than catalytical.
Fig. 4. Putative sites on CPA molecule for zinc binding. Zinc ions are assumed to bind to one of the four His residues, as follows: His 13, His 29, His 166 and/or His 303 in CPA molecule and connect to other possible ligands like His, Asp or Glu which are located at distances of approximately 10 A, . The structure of Zn–CPA is then minimized in CHARMn using Quanta 4.0 until 0.02 rms.
Acknowledgements
4. Atomic adsorption measurements
References
After dialysis and desalination procedure, atomic adsorption analysis showed that the amount of zinc atoms in the CPA molecule ranged between 1 and 5 according to the initial amount of Zn added to CPA before heat exposure. In a previous paper we showed the protective effect of phosphate under similar experimental conditions after heating to 50 °C [18]. It seems to be a cooperative or synergistic stabilizing effect of zinc and phosphate
The authors wish to express thanks to Dr. Dorit Arad for assistance in modeling studies.
[1] [2] [3] [4] [5] [6] [7] [8]
Vallee BL, Auld DS. Faraday Discuss 1992;93:47 – 65. Regan L. Annu Rev Biophys Biomol Struct 1993;22:57 –81. Arnold FH, Zhang J-H. Trends Biotechnol 1994;12:189 –92. Ochiai T, Hoshina S, Usuki I. Biochem Biophys Acta 1993;1203:310 – 4. Vallee BL, Wacker WEC. In: Neurath H, editor. The Proteins, vol. 5. New York: Academic Press, 1970:1 – 192. Quiocho FA, Lipscomb WN. Adv Prot Chem 1971;25:1 –49. Christianson DW, Lipscomb WN. Acc Chem Res 1989;22:62 – 9. Rees DC, Lewis M, Lipscomb WN. J Mol Biol 1983;168:367 – 87.
X. Li, B. Solomon / Biomolecular Engineering 18 (2001) 179–183 [9] Stote RH, Karplus M. Proteins Structure Function Genet 1995;28:12 – 31. [10] Zhang K, Chance B, Auld DS, Larsen KS, Vallee BL. Biochemistry 1992;31:1159 – 68. [11] Zhang K, Auld DS. Biochemistry 1993;32:13844 – 51. [12] Larsen KS, Auld DS. Biochemistry 1991;30:2613 –8. [13] Whitaker JR, Menger F, Bender ML. Biochemistry 1966;5:386 – 92. [14] Katzav-Gojanski T. M.Sc. Thesis. Tel-Aviv University; 1994. [15] Hartsuck J, Lipscomb WN. In: Boyer PD, editor. The Enzymes,
183
vol. 5. New York: Academic Press, 1971:1 – 56. [16] Valley BL, Galdes A, Auld DS, Riordan JR. In: Spiro TG, editor. zinc enzymes. Wiley, 1983:26 – 74. [17] Buchner J, Renner M, Lilie H, Hinz HJ, Jaenicke R, Kiefhabel T, Rudolph R. Biochemistry 1991;30(28):6922 – 9. [18] Li X, Solomon B. Biochem Molec Biol Int 1997;43:601 –11. [19] Arnold FH, Zhang ZH. Trends Biotechnol 1994;12:189 –92. [20] Rees DC, Lipscomb WN. Proc Natl Acad Sci USA 1983;83:7151 – 4. [21] Holmes MA, Mathews BW. J Mol Biol 1982;160:623 –39.