/. Biochem. 85, 1509-1517 (1979)
from Thermus thermophilus Strain HB8 Hiroshi NOJIMA,* Tairo OSHIMA,** and Haruhiko NODA* •Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Hongo, Tokyo 113, and "MitsubishiKasei Institute of Life Sciences, Machida, Tokyo 194 Received for publication, December 19, 1978
(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10-20 min at 79°C. (2) Catalytic properties such as pH optimum (pH 6-8.5), kinetic parameters (A"m=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0. The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% a-helix and 1 1 % pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.
It is known that proteins of thermophilic organisms are generally more resistant to heat than the corresponding ones from mesophilic sources (1, 2). Studies on the thermal stabilities of proteins extracted from thermophiles offer an opportunity to understand the forces that determine protein structure in general. In this context, many enzymes have been extracted from a moderately thermophilic bacterium, Bacillus stearothermophilus, and extremely thermophilic bacteria belonging to the genus Thermus, and their properties have been studied extensively (1-3). None of Abbreviations: G-3-P, 3-phosphoglycerate; SDS, sodium dodecyl sulfate; DPG, 1,3-bisphosphoglycerate. Vol. 85, No. 6, 1979
1509
them, however, have given any unequivocal indication of the molecular basis of their unusual thermostability. Phosphoglycerate kinase [EC 2.7.2.3] should represent good experimental material for the study of the structure-stability relationship of thermophilic proteins, because (i) only phosphoglycerate kinase is present in a monomeric form among glycolytic enzymes (4), (ii) the enzyme has neither tightly bound metal nor a prosthetic group (4), and (iii) the three-dimensional structures of horse and yeast phosphoglycerate kinases were determined by crystallographic studies (5-8). It should be easier to interpret the thermodynamic properties of thermal denaturation of an enzyme composed
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Purification and Properties of Phosphoglycerate Kinase
1510
H. NOJIMA, T. OSHIMA, and H. NODA
MATERIALS AND METHODS Bacteria—Thermus thermophilus strain HB8 (=ATCC 27634) was cultured according to Oshima and Imahori (9, 10). The cells were collected at the late log phase of growth and stored frozen at —20°C until use. Enzyme Assay—The enzyme activity was assayed by means of the back reaction, leading from 3-phosphoglycerate to 1,3-diphosphoglycerate, according to Krietsch and Biicher (11). The standard reaction mixture consisted of 0.1 M TrisHC1, pH 7.5, IITIM EDTA, 10 mM magnesium acetate, 10 mM 2-mercaptoethanol, 10 mM glycerate 3-phosphate, 1 mM ATP, 0.2 mM NADH, 0.34 [IM rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [EC 1.2.1.12]. Absorbancy changes at 340 nm were recorded on a Gilford 2400S spectrophotometer. In experiments on Michaelis constants, the magnesium acetate level in the mixture was lowered to 2 mM, and the concentrations of ATP and glycerate 3-phosphate were varied. Estimation of Protein Concentration—The protein concentration was estimated from the absorption coefficient at 280 nm, £^,^,,,=0.45, which was determined by direct weighing of the dried enzyme protein. The enzyme solution was freeze-dried to determine the absorption coefficient after thorough dialysis against distilled water. The lyophilized protein was further dried at 60°C over PSO6 under reduced pressure before weighing. Electrophoresis—Polyacrylamide disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis were carried out according to Davis (12) and Weber and Osborn (13), respectively. Isoelectric Focusing—Isoelectric focusing was carried out at 4°C as described by Vesterberg and Svenson (14) using LKB Ampholine carrier ampholytes and an LKB 8100 analytical electro-
focusing column of 110 ml capacity. To prepare a pH gradient of pH 3 to 7, 10 ml of carrier ampholytes was used in a sucrose solution. The anode solution was 0.2 ml of 36 N H,SO4 in 14 ml of water containing 12 g of sucrose. The cathode solution consisted of 0.2 ml of ethylene diamine in 10 ml of water. The electrophoresis was carried out at 400 V. After 26 h, the current decreased from 5 mA to 0.75 mA, and the solution in the column was removed with a peristaltic pump at a flow rate of 50 ml per h. Amino Acid Analysis—The enzyme protein was denatured with 8 M urea and carboxymethylated according to Crestfield et al. (15). An acid hydrolysate of the carboxymethylated sample was analyzed using a JEOL JL-6AH automatic amino acid analyzer. Tryptophan content was estimated according to Edelhoch (16). Fluorescence Spectrum—Fluorescence spectra were recorded on a Hitachi MPF-3 spectrofluorometer at 20°C. The quantum yield () was calculated from the following formula (17). , Y
1-10-Dr 1-10-DP
An Ar
. Yq
where A represents the area under the emission spectrum and D is the absorbancy at the exciting wavelength. The subscripts p and r refer to the protein and the reference substance (quinine sulfate in 1 M H2SO4 in our experiments). A value of 0.55 for the fluorescence quantum yield of the reference substance (j6q) was used (18). Circular Dichroism—Circular dichroic spectra were recorded on a JASCO J-20 spectropolarimeter at room temperature. The ordered secondary structure contents were estimated according to Chen et al. (19). Analytical Ultracentrifugation—Sedimentation equilibrium experiments were carried out using a Beckman model E ultracentrifuge equipped with a scanner at 15,220 rpm (20). The partial specific volume of T. thermophilus phosphoglycerate kinase (0.748 ml/g) was estimated from its amino acid composition according to Cohn and Edsall (21). pH—The pH values of the solutions were measured with a Hitachi-Horiba F-7ss pH meter equipped with a No. 6028 glass electrode at 25°C. Materials—DEAE cellulose was a product of Whatman (DE-32). Calcium phosphate gel was prepared according to the literature (22) using J. Biochem.
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of a single polypeptide chain without tightly bound cofactors, especially if the structures of the corresponding enzyme from other sources have been extensively studied. For this reason, we are interested in phosphoglycerate Jdnase of an extreme thermophile, Thermus thermophilus. The isolated enzyme was unusually stable to heat, but other enzymatic properties were similar to those of the enzyme from mesophilic sources.
T. thermophilus PHOSPHOGLYCERATE KINASE
period of an hour with gentle stirring. After 30 min, the precipitate was removed by centrifugation at 10,000 x g for 30 min. T o each 100 ml of the supernatant, 9.0 g of ammonium sulfate was added with stirring. After standing overnight, the precipitate was collected by centrifugation at 10,000 X g for 30 min. The collected precipitate was dissolved in a minimum volume of Tris-HCI buffer. (Step 4 ) Sephadex G-100 gel jiltration: The RESULTS solution thus obtained was again cleared by Purification Procedure-All steps in the follow- centrifugation at 10,500 x g for I0 min. The ing procedure were carried out in a cold room clear supernatant was applied on a Sephadex (4"C), unless otherwise indicated. During the G-100 column ($6 x 120 cm) which had been following purification procedure, 50 mM Tris-HC1 equilibrated with Tris-HC1 buffer. The protein buffer, pH 7.5, containing 10 mM EDTA and 10 mM was then eluted with the same buffer at a flow 2-mercaptoethanol was used. This buffer solution rate of 60 ml per h. In some cases the collected will be designated as Tris-HC1 buffer hereafter. fraction showed a major band with some minor (Step I ) Extraction and ammonium sulfate bands on SDS-polyacrylamide gel suggesting that fractionation: The cells (2 kg) were ground with 5 kg the preparation was fairly homogeneous, and the of aluminium oxide in order to disrupt the cell crystallization of the enzyme was carried out envelopes. Four liters of 50 mM Tris-HC1 buffer without the calcium phosphate gel column chrowas added and the insoluble material was removed matography described below. However, in most by centrifugation at 10,000 x g for 30 min. The cases the homogeneity of the enzyme fraction was supernatant was ultracentrifuged at 2 3 , 0 0 0 ~g for insufficient. The fractions were dialyzed against 15 h. After the centrifugation, a clear brown 5 mM potassium phosphate buffer (pH 6.4), and applied to a calcium phosphate gel column ($3 x supernatant was obtained. To each lOOml portion of the supernatant, 10cm) which had been equilibrated with the 29.1 g of ammonium sulfate was added slowly with potassium phosphate buffer. The enzyme was stirring (50% saturation of ammonium sulfate). eluted with a linear gradient of 5 to 40 mM potasAfter standing overnight, the precipitate was sium phosphate buffer. (Step 5 ) Crystallization: The fraction conremoved by centrifugation at 10,000xg for 30 min. To each 100 ml portion of the supernatant, taining phosphoglycerate kinase was precipitated 9.2 g of ammonium sulfate was added carefully by adding excess ammonium sulfate and the over a period of an hour with stirring (65 % satura- precipitate was dissolved in the minimum volume tion of ammonium sulfate). After standing for an of Tris-HCI buffer. A small amount of finely hour, the precipitate was collected by centrifugation ground solid ammonium sulfate was added at at 10,000 x g for 30 min. The collected precipitate 4°C until slight turbidity was noticed. The was dissolved in Tris-HCI buffer and dialyzed temperature of this sample was then gradually raised to room temperature during overnight against the same buffer. (Step 2) DEAE-cellulose chromatography: standing. After this treatment, the solution The dialyzed solution (257 ml) was then applied displayed a silk-like shimmer which is characteristic on a DEAE-cellulose column ($4.5 x 30 cm) which of protein crystals. Crystals obtained in the had been equilibrated with Tris-HCI buffer. The presence of ATP were hexagonal plates as shown enzyme was eluted with 4 liters of the Tris-HC1 in Fig. 1. The results of the purification are buffer containing a linear gradient of 0 to 0.2 M summarized in Table I . The enzyme thus purified was completely NaCI. homogeneous as judged by polyacrylamide disc (Step 3) Ammonium sulfate fractionation: T o the fractions collected from step 2, 31 g of gel electrophoresis and SDS-polyacrylamide gel ammonium sulfate per 100 ml was added over a electrophoresis. Vol. 85, No. 6, 1979
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Whatman CF-I I cellulose powder. Horse heart cytochrome c, chymotrypsinogen, ovalbumin, and beef liver catalase used as markers in molecular weight determinations and glycerate 3-phosphate were purchased from Boehringer Mannheim Co. Aluminium oxide W800 was purchased from Wako Co. Other reagents used in the experiments were all of reagent grade.
1512
H. NOJIMA, T. OSHIMA, and H. NODA
TABLE I. Purification of T. thermophilus phosphoglycerate kinase.* Sample (la) (lb) (2 ) (3 ) (4 ) (5)
Ultracentrifugal supernatant Ammonium sulfate fractionation After DEAE-cellulose column chromatography After second ammonium sulfate fractionation After Sephadex G-100 chromatography Crystallization
Total volume (ml)
Total activity (unit)
Total protein (mg)
Specific activity (unit/mg)
Yield
3,700 257 578 25 117 1
48,700 33,100 33,000 15,500 12,700 7,500
87,000 12,400 1,170 330 56 29
0.56 2.67 28.2
100 67 65 30 ' 25 15
47.1 227 259
» In this purification experiment, calcium phosphate gel column chromatography was omitted since the preparation after Sephadex G-100 chromatography was highly homogeneous as judged by disc gel electrophoresis.
Characterization of Phosphoglycerate Kinase— (J) Substrate specificity and inhibition studies: The substrate specificity of the enzyme was studied, and the results are summarized in Table II. Little or no activity was observed when ATP was replaced by other nudeotides. Compared with the substrate specificities of yeast, rabbit (77) and B. stearothermophilus (23) phosphoglycerate kinases, the T. thermophilus enzyme showed the highest specificity.
TABLE II. Nucleotide specificity of T. thermophilus phosphoglycerate kinase. Nucleotide (1 mM) ATP GTP ITP CTP UTP ADP
Enzyme activity (%) 100 8 4 7 0 0
J. Biochem.
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Fig. 1. Crystals of T. thermophilus phosphoglycerate kinase formed in the presence of 2 mM ATP.
T. thermophilus PHOSPHOGLYCERATE KINASE
TABLE HI. Inhibitory effects of various nucleotides on T. thermophilus phosphoglycerate kinase. The enzyme activity was measured after the inhibitor indicated had been added to the reaction mixture containing 10 mM ATP.
B. stearothermophilus (pH 5.3-8.5) (23), and pea seed (pH 6.7-9.7) (24) phosphoglycerate kinases. (4) Salt and ion effects: The effect of salts on the enzyme activity was examined, as shown in Fig. 3. It was found that ammonium sulfate had a marked inhibitory effect and sodium chloride was slightly inhibitory. The effect of magnesium ion concentration was also examined (Fig. 4). It was found that the optimum concentration of magnesium ion for the reaction was between 8 and 16 mM. NaCI MOLARITY (•—•) 0I 1 °i2
0
0,3
100-
50-
Enzyme activity (%)
Inhibitor (10 mM) None GTP ITP CTP UTP ADP DPG
100 69
°
0-
88
10 20 (NH,)2S04 SATURATION (c
69 83 21 78
5
6
8
10
PH
Fig. 2. Optimum pH of T. thermophilus phosphoglycerate kinase. The buffers used were: A, citric acid-sodium citrate buffer (pH 4.3-7.0); B, Tris-HCl buffer (pH 7.0-8.5); C, glycine-NaOH buffer (pH 8.5-10.0). Vol. 85, No. 6, 1979
—a
o 30 %
o)
Fig. 3. Effects of ammonium sulfate (A) and NaCI (B) on T. thermophilus phosphoglycerate kinase. NaCI or (NH,)iSO4 was added to the standard assay mixture described in "MATERIALS AND METHODS."
0
5 10 15 mM MAGNESIUM ACETATE MOLARITY
Fig. 4. The effect of magnesium ion on T. thermophilus phosphoglycerate kinase. Except for the concentration of magnesium acetate, the assay conditions were as described in "MATERIALS AND METHODS."
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The inhibitory effects of various nucleotides on the enzyme reaction were tested and the results are shown in Table m . ADP was the most potent inhibitor among the nucleotides tested. (2) Kinetic studies: Michaelis constants for ATP and G-3-P were estimated to be 0.28 mM and 1.79 mM, respectively. These values are similar to those reported for yeast, rabbit (11), and B. stearothermophilus (23) phosphoglycerate kinases. (3) pH optimum: The pH dependence of the enzyme activity was studied and the results are shown in Fig. 2. Similar broad pH optima were reported for yeast, rabbit (pH 6.0-9.2) (11),
1513
1514
H. NOJIMA, T. OSfflMA, and H. NODA
0 5 10 15 HEAT TREATMENT TIME
20 Min.
mum of native phosphoglycerate kinase was at 320 nm and that of the denatured enzyme was at 350 nm when excited at 280 nm. The emission maxima of native and denatured T. thermophilus phosphoglycerate kinase were at 328 nm and 355 nm, respectively, with excitation at 295 nm. The quantum yield was estimated to be 0.19 (excited at 280 nm) and 0.23 (excited at 295 nm) for the native enzyme. A value of 0.09 (excited at 280 nm or 295 nm) was obtained for the denatured enzyme in the presence of 5 M guanidine hydrochloride. The emission maximum and the quantum yield suggest that the fluorescence of T. thermophilus phosphoglycerate kinase was due to tryptophyl residues. (10) CD spectrum: The circular dichroic spectrum of the thermophile enzyme was measured and the secondary structure contents of the enzyme were estimated from the observed spectrum. The CD spectrum of the native enzyme resembled that of yeast phosphoglycerate kinase, as shown in Fig. 7. According to Chen et al. (19), the aTABLE IV. Amino acid composition of T. thermophilus phosphoglycerate kinase. Amino acid
Mol percentage
Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cys Val Met
2.4 1.5 8.6 6.1 3.3 3.8 10.7 6.9 11.5 12.8
from Thermus thermophilus Strain HB8 Hiroshi NOJIMA,* Tairo OSHIMA,** and Haruhiko NODA* •Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Hongo, Tokyo 113, and "MitsubishiKasei Institute of Life Sciences, Machida, Tokyo 194 Received for publication, December 19, 1978
(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10-20 min at 79°C. (2) Catalytic properties such as pH optimum (pH 6-8.5), kinetic parameters (A"m=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0. The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% a-helix and 1 1 % pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.
It is known that proteins of thermophilic organisms are generally more resistant to heat than the corresponding ones from mesophilic sources (1, 2). Studies on the thermal stabilities of proteins extracted from thermophiles offer an opportunity to understand the forces that determine protein structure in general. In this context, many enzymes have been extracted from a moderately thermophilic bacterium, Bacillus stearothermophilus, and extremely thermophilic bacteria belonging to the genus Thermus, and their properties have been studied extensively (1-3). None of Abbreviations: G-3-P, 3-phosphoglycerate; SDS, sodium dodecyl sulfate; DPG, 1,3-bisphosphoglycerate. Vol. 85, No. 6, 1979
1509
them, however, have given any unequivocal indication of the molecular basis of their unusual thermostability. Phosphoglycerate kinase [EC 2.7.2.3] should represent good experimental material for the study of the structure-stability relationship of thermophilic proteins, because (i) only phosphoglycerate kinase is present in a monomeric form among glycolytic enzymes (4), (ii) the enzyme has neither tightly bound metal nor a prosthetic group (4), and (iii) the three-dimensional structures of horse and yeast phosphoglycerate kinases were determined by crystallographic studies (5-8). It should be easier to interpret the thermodynamic properties of thermal denaturation of an enzyme composed
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1509/2186081 by university of winnipeg user on 17 January 2019
Purification and Properties of Phosphoglycerate Kinase
1510
H. NOJIMA, T. OSHIMA, and H. NODA
MATERIALS AND METHODS Bacteria—Thermus thermophilus strain HB8 (=ATCC 27634) was cultured according to Oshima and Imahori (9, 10). The cells were collected at the late log phase of growth and stored frozen at —20°C until use. Enzyme Assay—The enzyme activity was assayed by means of the back reaction, leading from 3-phosphoglycerate to 1,3-diphosphoglycerate, according to Krietsch and Biicher (11). The standard reaction mixture consisted of 0.1 M TrisHC1, pH 7.5, IITIM EDTA, 10 mM magnesium acetate, 10 mM 2-mercaptoethanol, 10 mM glycerate 3-phosphate, 1 mM ATP, 0.2 mM NADH, 0.34 [IM rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [EC 1.2.1.12]. Absorbancy changes at 340 nm were recorded on a Gilford 2400S spectrophotometer. In experiments on Michaelis constants, the magnesium acetate level in the mixture was lowered to 2 mM, and the concentrations of ATP and glycerate 3-phosphate were varied. Estimation of Protein Concentration—The protein concentration was estimated from the absorption coefficient at 280 nm, £^,^,,,=0.45, which was determined by direct weighing of the dried enzyme protein. The enzyme solution was freeze-dried to determine the absorption coefficient after thorough dialysis against distilled water. The lyophilized protein was further dried at 60°C over PSO6 under reduced pressure before weighing. Electrophoresis—Polyacrylamide disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis were carried out according to Davis (12) and Weber and Osborn (13), respectively. Isoelectric Focusing—Isoelectric focusing was carried out at 4°C as described by Vesterberg and Svenson (14) using LKB Ampholine carrier ampholytes and an LKB 8100 analytical electro-
focusing column of 110 ml capacity. To prepare a pH gradient of pH 3 to 7, 10 ml of carrier ampholytes was used in a sucrose solution. The anode solution was 0.2 ml of 36 N H,SO4 in 14 ml of water containing 12 g of sucrose. The cathode solution consisted of 0.2 ml of ethylene diamine in 10 ml of water. The electrophoresis was carried out at 400 V. After 26 h, the current decreased from 5 mA to 0.75 mA, and the solution in the column was removed with a peristaltic pump at a flow rate of 50 ml per h. Amino Acid Analysis—The enzyme protein was denatured with 8 M urea and carboxymethylated according to Crestfield et al. (15). An acid hydrolysate of the carboxymethylated sample was analyzed using a JEOL JL-6AH automatic amino acid analyzer. Tryptophan content was estimated according to Edelhoch (16). Fluorescence Spectrum—Fluorescence spectra were recorded on a Hitachi MPF-3 spectrofluorometer at 20°C. The quantum yield () was calculated from the following formula (17). , Y
1-10-Dr 1-10-DP
An Ar
. Yq
where A represents the area under the emission spectrum and D is the absorbancy at the exciting wavelength. The subscripts p and r refer to the protein and the reference substance (quinine sulfate in 1 M H2SO4 in our experiments). A value of 0.55 for the fluorescence quantum yield of the reference substance (j6q) was used (18). Circular Dichroism—Circular dichroic spectra were recorded on a JASCO J-20 spectropolarimeter at room temperature. The ordered secondary structure contents were estimated according to Chen et al. (19). Analytical Ultracentrifugation—Sedimentation equilibrium experiments were carried out using a Beckman model E ultracentrifuge equipped with a scanner at 15,220 rpm (20). The partial specific volume of T. thermophilus phosphoglycerate kinase (0.748 ml/g) was estimated from its amino acid composition according to Cohn and Edsall (21). pH—The pH values of the solutions were measured with a Hitachi-Horiba F-7ss pH meter equipped with a No. 6028 glass electrode at 25°C. Materials—DEAE cellulose was a product of Whatman (DE-32). Calcium phosphate gel was prepared according to the literature (22) using J. Biochem.
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of a single polypeptide chain without tightly bound cofactors, especially if the structures of the corresponding enzyme from other sources have been extensively studied. For this reason, we are interested in phosphoglycerate Jdnase of an extreme thermophile, Thermus thermophilus. The isolated enzyme was unusually stable to heat, but other enzymatic properties were similar to those of the enzyme from mesophilic sources.
T. thermophilus PHOSPHOGLYCERATE KINASE
period of an hour with gentle stirring. After 30 min, the precipitate was removed by centrifugation at 10,000 x g for 30 min. T o each 100 ml of the supernatant, 9.0 g of ammonium sulfate was added with stirring. After standing overnight, the precipitate was collected by centrifugation at 10,000 X g for 30 min. The collected precipitate was dissolved in a minimum volume of Tris-HCI buffer. (Step 4 ) Sephadex G-100 gel jiltration: The RESULTS solution thus obtained was again cleared by Purification Procedure-All steps in the follow- centrifugation at 10,500 x g for I0 min. The ing procedure were carried out in a cold room clear supernatant was applied on a Sephadex (4"C), unless otherwise indicated. During the G-100 column ($6 x 120 cm) which had been following purification procedure, 50 mM Tris-HC1 equilibrated with Tris-HC1 buffer. The protein buffer, pH 7.5, containing 10 mM EDTA and 10 mM was then eluted with the same buffer at a flow 2-mercaptoethanol was used. This buffer solution rate of 60 ml per h. In some cases the collected will be designated as Tris-HC1 buffer hereafter. fraction showed a major band with some minor (Step I ) Extraction and ammonium sulfate bands on SDS-polyacrylamide gel suggesting that fractionation: The cells (2 kg) were ground with 5 kg the preparation was fairly homogeneous, and the of aluminium oxide in order to disrupt the cell crystallization of the enzyme was carried out envelopes. Four liters of 50 mM Tris-HC1 buffer without the calcium phosphate gel column chrowas added and the insoluble material was removed matography described below. However, in most by centrifugation at 10,000 x g for 30 min. The cases the homogeneity of the enzyme fraction was supernatant was ultracentrifuged at 2 3 , 0 0 0 ~g for insufficient. The fractions were dialyzed against 15 h. After the centrifugation, a clear brown 5 mM potassium phosphate buffer (pH 6.4), and applied to a calcium phosphate gel column ($3 x supernatant was obtained. To each lOOml portion of the supernatant, 10cm) which had been equilibrated with the 29.1 g of ammonium sulfate was added slowly with potassium phosphate buffer. The enzyme was stirring (50% saturation of ammonium sulfate). eluted with a linear gradient of 5 to 40 mM potasAfter standing overnight, the precipitate was sium phosphate buffer. (Step 5 ) Crystallization: The fraction conremoved by centrifugation at 10,000xg for 30 min. To each 100 ml portion of the supernatant, taining phosphoglycerate kinase was precipitated 9.2 g of ammonium sulfate was added carefully by adding excess ammonium sulfate and the over a period of an hour with stirring (65 % satura- precipitate was dissolved in the minimum volume tion of ammonium sulfate). After standing for an of Tris-HCI buffer. A small amount of finely hour, the precipitate was collected by centrifugation ground solid ammonium sulfate was added at at 10,000 x g for 30 min. The collected precipitate 4°C until slight turbidity was noticed. The was dissolved in Tris-HCI buffer and dialyzed temperature of this sample was then gradually raised to room temperature during overnight against the same buffer. (Step 2) DEAE-cellulose chromatography: standing. After this treatment, the solution The dialyzed solution (257 ml) was then applied displayed a silk-like shimmer which is characteristic on a DEAE-cellulose column ($4.5 x 30 cm) which of protein crystals. Crystals obtained in the had been equilibrated with Tris-HCI buffer. The presence of ATP were hexagonal plates as shown enzyme was eluted with 4 liters of the Tris-HC1 in Fig. 1. The results of the purification are buffer containing a linear gradient of 0 to 0.2 M summarized in Table I . The enzyme thus purified was completely NaCI. homogeneous as judged by polyacrylamide disc (Step 3) Ammonium sulfate fractionation: T o the fractions collected from step 2, 31 g of gel electrophoresis and SDS-polyacrylamide gel ammonium sulfate per 100 ml was added over a electrophoresis. Vol. 85, No. 6, 1979
Downloaded from https://academic.oup.com/jb/article-abstract/85/6/1509/2186081 by university of winnipeg user on 17 January 2019
Whatman CF-I I cellulose powder. Horse heart cytochrome c, chymotrypsinogen, ovalbumin, and beef liver catalase used as markers in molecular weight determinations and glycerate 3-phosphate were purchased from Boehringer Mannheim Co. Aluminium oxide W800 was purchased from Wako Co. Other reagents used in the experiments were all of reagent grade.
1512
H. NOJIMA, T. OSHIMA, and H. NODA
TABLE I. Purification of T. thermophilus phosphoglycerate kinase.* Sample (la) (lb) (2 ) (3 ) (4 ) (5)
Ultracentrifugal supernatant Ammonium sulfate fractionation After DEAE-cellulose column chromatography After second ammonium sulfate fractionation After Sephadex G-100 chromatography Crystallization
Total volume (ml)
Total activity (unit)
Total protein (mg)
Specific activity (unit/mg)
Yield
3,700 257 578 25 117 1
48,700 33,100 33,000 15,500 12,700 7,500
87,000 12,400 1,170 330 56 29
0.56 2.67 28.2
100 67 65 30 ' 25 15
47.1 227 259
» In this purification experiment, calcium phosphate gel column chromatography was omitted since the preparation after Sephadex G-100 chromatography was highly homogeneous as judged by disc gel electrophoresis.
Characterization of Phosphoglycerate Kinase— (J) Substrate specificity and inhibition studies: The substrate specificity of the enzyme was studied, and the results are summarized in Table II. Little or no activity was observed when ATP was replaced by other nudeotides. Compared with the substrate specificities of yeast, rabbit (77) and B. stearothermophilus (23) phosphoglycerate kinases, the T. thermophilus enzyme showed the highest specificity.
TABLE II. Nucleotide specificity of T. thermophilus phosphoglycerate kinase. Nucleotide (1 mM) ATP GTP ITP CTP UTP ADP
Enzyme activity (%) 100 8 4 7 0 0
J. Biochem.
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Fig. 1. Crystals of T. thermophilus phosphoglycerate kinase formed in the presence of 2 mM ATP.
T. thermophilus PHOSPHOGLYCERATE KINASE
TABLE HI. Inhibitory effects of various nucleotides on T. thermophilus phosphoglycerate kinase. The enzyme activity was measured after the inhibitor indicated had been added to the reaction mixture containing 10 mM ATP.
B. stearothermophilus (pH 5.3-8.5) (23), and pea seed (pH 6.7-9.7) (24) phosphoglycerate kinases. (4) Salt and ion effects: The effect of salts on the enzyme activity was examined, as shown in Fig. 3. It was found that ammonium sulfate had a marked inhibitory effect and sodium chloride was slightly inhibitory. The effect of magnesium ion concentration was also examined (Fig. 4). It was found that the optimum concentration of magnesium ion for the reaction was between 8 and 16 mM. NaCI MOLARITY (•—•) 0I 1 °i2
0
0,3
100-
50-
Enzyme activity (%)
Inhibitor (10 mM) None GTP ITP CTP UTP ADP DPG
100 69
°
0-
88
10 20 (NH,)2S04 SATURATION (c
69 83 21 78
5
6
8
10
PH
Fig. 2. Optimum pH of T. thermophilus phosphoglycerate kinase. The buffers used were: A, citric acid-sodium citrate buffer (pH 4.3-7.0); B, Tris-HCl buffer (pH 7.0-8.5); C, glycine-NaOH buffer (pH 8.5-10.0). Vol. 85, No. 6, 1979
—a
o 30 %
o)
Fig. 3. Effects of ammonium sulfate (A) and NaCI (B) on T. thermophilus phosphoglycerate kinase. NaCI or (NH,)iSO4 was added to the standard assay mixture described in "MATERIALS AND METHODS."
0
5 10 15 mM MAGNESIUM ACETATE MOLARITY
Fig. 4. The effect of magnesium ion on T. thermophilus phosphoglycerate kinase. Except for the concentration of magnesium acetate, the assay conditions were as described in "MATERIALS AND METHODS."
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The inhibitory effects of various nucleotides on the enzyme reaction were tested and the results are shown in Table m . ADP was the most potent inhibitor among the nucleotides tested. (2) Kinetic studies: Michaelis constants for ATP and G-3-P were estimated to be 0.28 mM and 1.79 mM, respectively. These values are similar to those reported for yeast, rabbit (11), and B. stearothermophilus (23) phosphoglycerate kinases. (3) pH optimum: The pH dependence of the enzyme activity was studied and the results are shown in Fig. 2. Similar broad pH optima were reported for yeast, rabbit (pH 6.0-9.2) (11),
1513
1514
H. NOJIMA, T. OSfflMA, and H. NODA
0 5 10 15 HEAT TREATMENT TIME
20 Min.
mum of native phosphoglycerate kinase was at 320 nm and that of the denatured enzyme was at 350 nm when excited at 280 nm. The emission maxima of native and denatured T. thermophilus phosphoglycerate kinase were at 328 nm and 355 nm, respectively, with excitation at 295 nm. The quantum yield was estimated to be 0.19 (excited at 280 nm) and 0.23 (excited at 295 nm) for the native enzyme. A value of 0.09 (excited at 280 nm or 295 nm) was obtained for the denatured enzyme in the presence of 5 M guanidine hydrochloride. The emission maximum and the quantum yield suggest that the fluorescence of T. thermophilus phosphoglycerate kinase was due to tryptophyl residues. (10) CD spectrum: The circular dichroic spectrum of the thermophile enzyme was measured and the secondary structure contents of the enzyme were estimated from the observed spectrum. The CD spectrum of the native enzyme resembled that of yeast phosphoglycerate kinase, as shown in Fig. 7. According to Chen et al. (19), the aTABLE IV. Amino acid composition of T. thermophilus phosphoglycerate kinase. Amino acid
Mol percentage
Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cys Val Met
2.4 1.5 8.6 6.1 3.3 3.8 10.7 6.9 11.5 12.8
