134
KINASES
[23]
lian enzymes are rather less acidic than those of fish. The yeast enzyme is electrophoretically only slightly acid, but the chloroplast enzyme from silver beet is much more acid. TM A second phosphoglycerate kinase allele, expressed only in animal testes, has been described, and this appears to be less conservative electrophoretically, a9 The purified enzyme has a specific activity in the conditions described close to 1000 units/mg--this applies to all species tested. The yeast, silver beet, and erythrocyte enzymes have similar specific activities. In the other direction, i.e., in the direction of glycolysis, the activity is some 2-2.5 times greater, ~ but relatively little work has been carried out on the kinetics in this direction because of the lability of the substrate, 1,3-diphosphoglycerate. Extensive kinetic studies in the gluconeogenic direction have been carried out only with the yeast enzyme. In view of the marked similarity of the reported Michaelis constants and other properties for the muscle and yeast enzymes, 4 it is most likely that the mechanisms involved are the same in each case. '~J. L. Vandeberg, D. W. Cooper, and P. J. Close, Nature (London) New Biol. 243, 48 (1973).
[23] 3 - P h o s p h o g l y c e r a t e
Kinase of Baker's Yeast
B y R. K. ScoPEs
MgATP + 3-phosphoglycerate ~ MgADP + 1,3-diphosphoglycerate
Assay The assay principle and procedure is as given in the preceding article.
Purification Preparation of a yeast extract can be carried out by any of the standard methods, e.g., toluene autolyzate, or autolyzate of air-dried cells, but probably the easiest and least messy is to make the extract by cytolysis with ammonia. ~,2 Although fresh yeast can be used, it is most convenient to use vacuum- or nitrogen-packed cans of "active dried" yeast available commercially from bakeries. 1E. de la Morena, I. Santos, and S. Grisolia, Biochim. Biophys. Acta 151, 526 (1968). 2R. K. Scopes, Biochem. J. 122, 89 (1971).
[23]
3-PHOSPHOGLYCERATE KINASE OF BAKER'S YEAST
135
All stages of this isolation procedure are carried out at room temperature. Step 1. Extraction. One liter of 0.5 M NH~OH containing 1 g of EDTA (disodium salt) is stirred vigorously while 450 g of granulated dried yeast is added slowly over a period of 30-60 min. Any lumps forming are dispersed, and stirring is continued for several hours; then the preparation is allowed to stand overnight. The optimum temperature for this procedure is around 20°; in the cold room cytolysis is not always complete. The next day, stirring is recommenced, and 800 ml of 0.5 M lactic acid are mixed in. The pH is then measured and adjusted to 7.0_+ 0.5 using 5 M lactic acid that has been titrated to pH 3.5 with NH~0H. The mixture is then centrifuged at at least 6000 g for 30 min (preferably about 20,000 g for 15 min), and the orange-brown supernarant is decanted from the residue. The extract should be about 1250 ml, and contain on an activity basis over 2 g of phosphoglycerate kinase. Step 2. Ammonium Sul]ate Fractionation. The original procedure 2 made use of two successive ammonium sulfate fractionations, which will be described here. However, equally good recovery of enzyme has been made by a simpler procedure, which in addition also allows another enzyme, glucose-6-phosphate dehydrogenase, to be separated into a different fraction. 3 Ammonium sulfate, 100 g/liter, is dissolved into the extract, then the pH is further lowered, to 4.3 (checking with a 10X diluted sample) using cold 5 M lactate, pH 3.5. After 10-15 rain, the mixture is centrifuged (as above) and the precipitate discarded. A further 250 g ammonium sulfate is then dissolved per liter of supernatant; after stirring for 30 min, the precipitate is collected by centrifugation. This precipitate is dissolved in 800 ml of 40 mM Tris base + 1 mM EDTA, to raise the pH to 7.5 -+- 0.2 (further 1 M Tris is added if necessary) ; 340 g of ammonium sulfate per liter of solution is dissolved into the preparation, and after 30 rain of stirring the precipitate is removed by centrifugation. To the supernatant, a further 150 g of ammonium sulfate per liter is added, and the precipitate is collected as before. These two fractionations, at pH 4.3 and 7.5, represent ammonium sulfate cuts of 18-56% and 60-82% saturation, respectively, indicating completely different solubility char3 This simpler procedure has not been tested frequently. The pH of the extract is adjusted to 5.5 with 5 M lactate pH 3.5, and an ammonium sulfate fraction precipitating between 50% and 63% saturation is removed for preparation of glucose-6-phosphate dehydrogenase2 The 63% supernatant is then adjusted to pH 4.3 using the 5 M lactate, and checking the pH on a diluted sample. The precipitate forming is collected and dissolved in a little 1 m M E D T A ; the pH is brought to 7.0 with 1 M Tris. This preparation is then ready for dialysis and step 3. 4 R. I(. Scopes, Biochem. J. 134, 197 (1973).
136
KINASES
[23]
acteristics of the enzyme at the two pH's. The final precipitate is dissolved in a little 1 m M E D T A , pH 7.0, and dialyzed extensively against 2 or 3 changes of 5 liters of 1 m M E D T A pH 7.0. Step 3. Removal o] Nucleic Acids. If the dialyzed fraction from step 2 is adjusted to p H 6.0 prior to ion-exchange chromatography, turbidity develops due to interactions of nucleic acids. This can be avoided by treating with protamine sulfate. After dialysis, a solution of 1 g of profamine sulfate in 50 ml of water, adjusted to pH 7.0 with Tris, is added; the resulting precipitate is removed by centrifugation. 5 Then 0.01 volume of 1 M Tris is added to the solution, and the p H lowered to 6.0 with cacodylic acid2 The solution is then diluted to about 800 ml with the chromatography buffer, which consists of l0 m M Tris plus 1 m M E D T A , adjusted to pH 6.0 with cacodylic acid2 Step 4. CM-Cellulose Chromatography. A column packed with CMcellulose equilibrated in the T r i s - E D T A - c a c o d y l a t e buffer is prepared. The dimensions should be about 5 cm in diameter and 20-30 cm long to accommodate the protein from up to 600 g of dried yeast; preparations on a smaller or larger scale must be scaled appropriately. The fraction is run on to the column at a flow rate of approximately 12 ml per hour per square centimeter of cross section, and washed in with a little buffer. A linear gradient in KC1 is commenced, with incremental KCI concentration of 0.1 m M / m l ; the flow rate is maintained at between 8 and 10 ml per hour per square centimeter of cross section. The enzyme is eluted between 50 and 70 m M KC1, preceded by a substantial quantity of protein which merges with the enzyme peak. Tubes containing more than 300 units of enzyme per milliliter are combined, and the protein is salted out by adding 60 g of ammonium sulfate per 100 ml. (If available, ultrafiltration equipment can achieve a more satisfactory concentration of the protein after the CM-cellulose chromatography.) The precipitated protein is collected by centrifugation and dissolved in a small volume of 1 mM E D T A , p H 7.0. The volume at this stage should be between 40 and 50 ml. Step 5. Gel Filtration. To accommodate the whole of the fraction from the preceding step, a column with a volume of at least 1.5 liters is necessary. If not available, the fraction must be divided for successive application to a smaller column. Sephadex G-100 or G-150 is most suitable for the gel filtration, and a buffer consisting of 0.15 M KC1 containing 1 m M E D T A and 10 m M imidazole pH 7.0 can be used ; the composition of this buffer is not critical, although the ionic strength should be at least 5Treatment with protamine is not an essential step, but it avoids clogging of the column in step 4 with precipitated material. Suceinic acid can be used as the buffering anion.
[23]
3-PHOSPHOGLYCERATE KINASE OF BAKER'S YEAST
137
0.1. Two peaks of protein are obtained, the enzyme being in the most retarded fraction. At the peak and subsequent tubes the enzyme is pure, with specific activity close to 1000, but some larger molecular weight contaminants are introduced by including the whole enzyme peak. For optimum yield and purity, the earlier tubes containing activity are retained separately, and rerun on the column. Step 6. Crystallization. The tubes containing the enzyme after gel filtration are pooled, and salted out with 60 g of ammonium sulfate per 100 ml. The precipitate is collected by centrifugation and dissolved in about 15 ml of 20 mM phosphate buffer, pH 6.5. Saturated ammonium sulfate is added until the solution becomes milky; a few drops of phosphate buffer are then added until this milkiness begins to clear. The solution is then centrifuged briefly, and the supernatant is placed in a 100-ml conical flask for crystallization at room temperature. If after a few hours the solution is still completely clear, a few drops of saturated ammonium sulfate may be added. Crystallization may take several days, and a thick suspension of fine needles is obtained. After collection of these crystals, any impurities are left in the supernatant; however, some loss of activity usually occurs, as with the muscle enzyme,7 after crystallization. Larger polyhedric crystals which have been used for X-ray crystallography2,s can be grown at a pH just over 7.0, in the presence of about 1% 1,4-dioxane, and an ammonium sulfate concentration of close to 68% saturation. A summary of the isolation procedure is shown in the table. ISOLATION OF 3-PHOSPHOGLYCERATE KINASE FROM 450 G OF GRANULATED BAKER'S YEAST
Fraction 1. Ammoniacal extract 2. (a) Ammonium sulfate pH, 4.3 (b) Ammonium sulfate pH, 7.5 3.' Protamine treatment 4. CM-cellulose 5. Gel filtration 6. Crystals
Total protein (mg)
Total Specific activity activity (units × l0 b) (units/mg)
Yield (%)
49,000 26,500
2300 1800
47 68
100 78
17,500
1620
92
70
17,200 2,400 1,420 1,180
1620 1220 1160 1000
94 510 820 850
70 53 50 43
7 R. K. Scopes, Biochem. J. 113, 551 (1969). P. L. Wendell, T. N. Bryant, and H. C. Watson, Nature (London) N e w Biol. 240, 134 (1972).
138
KINASES
[23]
Properties The properties of yeast phosphoglycerate kinase are very similar to those of the muscle enzyme in most respects. 9,~° X - R a y diffraction studies on the yeast enzyme have confirmed the bilobal structure found for the horse muscle protein, and demonstrate that the long divergence of evolution has not led to any major alteration of the tertiary structure. The molecular weight is not significantly different from that of mammalian enzymes (45,000-50,000). The amino acid compositions, although remarkably similar, differ in one major respect: the sulfur-containing amino acids are poorly represented in the yeast enzyme. Thus, there are only 3 methionines compared with 13 in the mammalian enzymes (rabbit muscle and human erythrocyte), and, more significantly, only 1 cysteine compared with 8-10.1° This cysteine residue, although close to the active site, is not required for enzyme activity, and treatment with PCMB, D T N B , or Hg 2+ does not result in loss of any activity. This contrasts with the rapid loss of activity by the muscle enzyme when treated with any thiolreacting compound. T r y p t o p h a n and tyrosine residue contents are 0.5 and 2 times the mammalian enzymes, respectively, otherwise differences in amino acid composition are hardly significant. Extensive kinetic studies have been carried out on the back reaction catalyzed by the yeast enzyme. The results can be summarized as follows: The true substrates for the reaction are M g A T P 2- and 3-phosphoglycerate ~-, with weak inhibitions by A T P 4-, Mg-phosphoglycerate, and Mg2+. 11,~2 Other cations activating the enzyme (by forming the substrate M e A T P ~-) include Mn ~+, Ca 2÷, Zn ~÷, Co 2÷, Cd 2÷, and Ni 2÷, but free Zn 2÷ ions were particularly potent at inhibiting the reaction. ~- The evidence from these kinetic studies favors a rapid random equilibrium type of reaction mechanism; further support for this mechanism comes from inhibitor studies on the erythrocyte enzyme. ~ 9W. K. G. Krietsch and T. Biicher, Eur. J. Biochem. 17, 568 (1970). lo R. tC Scopes, in "The Enzymes" (P. J. Boyer, ed.), 3rd ed., Vol. 8, p. 335. Academic Press, New York, 1973. ~1M. Larsson-Raznikiewicz, Biochim. Biophys. Acta 85, 60 (1964). ~ M. Larsson-Raznikiewicz, Eur. J. Biochem. 17, 183 (1970). ~3C. S. Lee and W. J. O'Sullivan, Proc. Aust. Biochem. Soc. 6, 18 (1973).
KINASES
[23]
lian enzymes are rather less acidic than those of fish. The yeast enzyme is electrophoretically only slightly acid, but the chloroplast enzyme from silver beet is much more acid. TM A second phosphoglycerate kinase allele, expressed only in animal testes, has been described, and this appears to be less conservative electrophoretically, a9 The purified enzyme has a specific activity in the conditions described close to 1000 units/mg--this applies to all species tested. The yeast, silver beet, and erythrocyte enzymes have similar specific activities. In the other direction, i.e., in the direction of glycolysis, the activity is some 2-2.5 times greater, ~ but relatively little work has been carried out on the kinetics in this direction because of the lability of the substrate, 1,3-diphosphoglycerate. Extensive kinetic studies in the gluconeogenic direction have been carried out only with the yeast enzyme. In view of the marked similarity of the reported Michaelis constants and other properties for the muscle and yeast enzymes, 4 it is most likely that the mechanisms involved are the same in each case. '~J. L. Vandeberg, D. W. Cooper, and P. J. Close, Nature (London) New Biol. 243, 48 (1973).
[23] 3 - P h o s p h o g l y c e r a t e
Kinase of Baker's Yeast
B y R. K. ScoPEs
MgATP + 3-phosphoglycerate ~ MgADP + 1,3-diphosphoglycerate
Assay The assay principle and procedure is as given in the preceding article.
Purification Preparation of a yeast extract can be carried out by any of the standard methods, e.g., toluene autolyzate, or autolyzate of air-dried cells, but probably the easiest and least messy is to make the extract by cytolysis with ammonia. ~,2 Although fresh yeast can be used, it is most convenient to use vacuum- or nitrogen-packed cans of "active dried" yeast available commercially from bakeries. 1E. de la Morena, I. Santos, and S. Grisolia, Biochim. Biophys. Acta 151, 526 (1968). 2R. K. Scopes, Biochem. J. 122, 89 (1971).
[23]
3-PHOSPHOGLYCERATE KINASE OF BAKER'S YEAST
135
All stages of this isolation procedure are carried out at room temperature. Step 1. Extraction. One liter of 0.5 M NH~OH containing 1 g of EDTA (disodium salt) is stirred vigorously while 450 g of granulated dried yeast is added slowly over a period of 30-60 min. Any lumps forming are dispersed, and stirring is continued for several hours; then the preparation is allowed to stand overnight. The optimum temperature for this procedure is around 20°; in the cold room cytolysis is not always complete. The next day, stirring is recommenced, and 800 ml of 0.5 M lactic acid are mixed in. The pH is then measured and adjusted to 7.0_+ 0.5 using 5 M lactic acid that has been titrated to pH 3.5 with NH~0H. The mixture is then centrifuged at at least 6000 g for 30 min (preferably about 20,000 g for 15 min), and the orange-brown supernarant is decanted from the residue. The extract should be about 1250 ml, and contain on an activity basis over 2 g of phosphoglycerate kinase. Step 2. Ammonium Sul]ate Fractionation. The original procedure 2 made use of two successive ammonium sulfate fractionations, which will be described here. However, equally good recovery of enzyme has been made by a simpler procedure, which in addition also allows another enzyme, glucose-6-phosphate dehydrogenase, to be separated into a different fraction. 3 Ammonium sulfate, 100 g/liter, is dissolved into the extract, then the pH is further lowered, to 4.3 (checking with a 10X diluted sample) using cold 5 M lactate, pH 3.5. After 10-15 rain, the mixture is centrifuged (as above) and the precipitate discarded. A further 250 g ammonium sulfate is then dissolved per liter of supernatant; after stirring for 30 min, the precipitate is collected by centrifugation. This precipitate is dissolved in 800 ml of 40 mM Tris base + 1 mM EDTA, to raise the pH to 7.5 -+- 0.2 (further 1 M Tris is added if necessary) ; 340 g of ammonium sulfate per liter of solution is dissolved into the preparation, and after 30 rain of stirring the precipitate is removed by centrifugation. To the supernatant, a further 150 g of ammonium sulfate per liter is added, and the precipitate is collected as before. These two fractionations, at pH 4.3 and 7.5, represent ammonium sulfate cuts of 18-56% and 60-82% saturation, respectively, indicating completely different solubility char3 This simpler procedure has not been tested frequently. The pH of the extract is adjusted to 5.5 with 5 M lactate pH 3.5, and an ammonium sulfate fraction precipitating between 50% and 63% saturation is removed for preparation of glucose-6-phosphate dehydrogenase2 The 63% supernatant is then adjusted to pH 4.3 using the 5 M lactate, and checking the pH on a diluted sample. The precipitate forming is collected and dissolved in a little 1 m M E D T A ; the pH is brought to 7.0 with 1 M Tris. This preparation is then ready for dialysis and step 3. 4 R. I(. Scopes, Biochem. J. 134, 197 (1973).
136
KINASES
[23]
acteristics of the enzyme at the two pH's. The final precipitate is dissolved in a little 1 m M E D T A , pH 7.0, and dialyzed extensively against 2 or 3 changes of 5 liters of 1 m M E D T A pH 7.0. Step 3. Removal o] Nucleic Acids. If the dialyzed fraction from step 2 is adjusted to p H 6.0 prior to ion-exchange chromatography, turbidity develops due to interactions of nucleic acids. This can be avoided by treating with protamine sulfate. After dialysis, a solution of 1 g of profamine sulfate in 50 ml of water, adjusted to pH 7.0 with Tris, is added; the resulting precipitate is removed by centrifugation. 5 Then 0.01 volume of 1 M Tris is added to the solution, and the p H lowered to 6.0 with cacodylic acid2 The solution is then diluted to about 800 ml with the chromatography buffer, which consists of l0 m M Tris plus 1 m M E D T A , adjusted to pH 6.0 with cacodylic acid2 Step 4. CM-Cellulose Chromatography. A column packed with CMcellulose equilibrated in the T r i s - E D T A - c a c o d y l a t e buffer is prepared. The dimensions should be about 5 cm in diameter and 20-30 cm long to accommodate the protein from up to 600 g of dried yeast; preparations on a smaller or larger scale must be scaled appropriately. The fraction is run on to the column at a flow rate of approximately 12 ml per hour per square centimeter of cross section, and washed in with a little buffer. A linear gradient in KC1 is commenced, with incremental KCI concentration of 0.1 m M / m l ; the flow rate is maintained at between 8 and 10 ml per hour per square centimeter of cross section. The enzyme is eluted between 50 and 70 m M KC1, preceded by a substantial quantity of protein which merges with the enzyme peak. Tubes containing more than 300 units of enzyme per milliliter are combined, and the protein is salted out by adding 60 g of ammonium sulfate per 100 ml. (If available, ultrafiltration equipment can achieve a more satisfactory concentration of the protein after the CM-cellulose chromatography.) The precipitated protein is collected by centrifugation and dissolved in a small volume of 1 mM E D T A , p H 7.0. The volume at this stage should be between 40 and 50 ml. Step 5. Gel Filtration. To accommodate the whole of the fraction from the preceding step, a column with a volume of at least 1.5 liters is necessary. If not available, the fraction must be divided for successive application to a smaller column. Sephadex G-100 or G-150 is most suitable for the gel filtration, and a buffer consisting of 0.15 M KC1 containing 1 m M E D T A and 10 m M imidazole pH 7.0 can be used ; the composition of this buffer is not critical, although the ionic strength should be at least 5Treatment with protamine is not an essential step, but it avoids clogging of the column in step 4 with precipitated material. Suceinic acid can be used as the buffering anion.
[23]
3-PHOSPHOGLYCERATE KINASE OF BAKER'S YEAST
137
0.1. Two peaks of protein are obtained, the enzyme being in the most retarded fraction. At the peak and subsequent tubes the enzyme is pure, with specific activity close to 1000, but some larger molecular weight contaminants are introduced by including the whole enzyme peak. For optimum yield and purity, the earlier tubes containing activity are retained separately, and rerun on the column. Step 6. Crystallization. The tubes containing the enzyme after gel filtration are pooled, and salted out with 60 g of ammonium sulfate per 100 ml. The precipitate is collected by centrifugation and dissolved in about 15 ml of 20 mM phosphate buffer, pH 6.5. Saturated ammonium sulfate is added until the solution becomes milky; a few drops of phosphate buffer are then added until this milkiness begins to clear. The solution is then centrifuged briefly, and the supernatant is placed in a 100-ml conical flask for crystallization at room temperature. If after a few hours the solution is still completely clear, a few drops of saturated ammonium sulfate may be added. Crystallization may take several days, and a thick suspension of fine needles is obtained. After collection of these crystals, any impurities are left in the supernatant; however, some loss of activity usually occurs, as with the muscle enzyme,7 after crystallization. Larger polyhedric crystals which have been used for X-ray crystallography2,s can be grown at a pH just over 7.0, in the presence of about 1% 1,4-dioxane, and an ammonium sulfate concentration of close to 68% saturation. A summary of the isolation procedure is shown in the table. ISOLATION OF 3-PHOSPHOGLYCERATE KINASE FROM 450 G OF GRANULATED BAKER'S YEAST
Fraction 1. Ammoniacal extract 2. (a) Ammonium sulfate pH, 4.3 (b) Ammonium sulfate pH, 7.5 3.' Protamine treatment 4. CM-cellulose 5. Gel filtration 6. Crystals
Total protein (mg)
Total Specific activity activity (units × l0 b) (units/mg)
Yield (%)
49,000 26,500
2300 1800
47 68
100 78
17,500
1620
92
70
17,200 2,400 1,420 1,180
1620 1220 1160 1000
94 510 820 850
70 53 50 43
7 R. K. Scopes, Biochem. J. 113, 551 (1969). P. L. Wendell, T. N. Bryant, and H. C. Watson, Nature (London) N e w Biol. 240, 134 (1972).
138
KINASES
[23]
Properties The properties of yeast phosphoglycerate kinase are very similar to those of the muscle enzyme in most respects. 9,~° X - R a y diffraction studies on the yeast enzyme have confirmed the bilobal structure found for the horse muscle protein, and demonstrate that the long divergence of evolution has not led to any major alteration of the tertiary structure. The molecular weight is not significantly different from that of mammalian enzymes (45,000-50,000). The amino acid compositions, although remarkably similar, differ in one major respect: the sulfur-containing amino acids are poorly represented in the yeast enzyme. Thus, there are only 3 methionines compared with 13 in the mammalian enzymes (rabbit muscle and human erythrocyte), and, more significantly, only 1 cysteine compared with 8-10.1° This cysteine residue, although close to the active site, is not required for enzyme activity, and treatment with PCMB, D T N B , or Hg 2+ does not result in loss of any activity. This contrasts with the rapid loss of activity by the muscle enzyme when treated with any thiolreacting compound. T r y p t o p h a n and tyrosine residue contents are 0.5 and 2 times the mammalian enzymes, respectively, otherwise differences in amino acid composition are hardly significant. Extensive kinetic studies have been carried out on the back reaction catalyzed by the yeast enzyme. The results can be summarized as follows: The true substrates for the reaction are M g A T P 2- and 3-phosphoglycerate ~-, with weak inhibitions by A T P 4-, Mg-phosphoglycerate, and Mg2+. 11,~2 Other cations activating the enzyme (by forming the substrate M e A T P ~-) include Mn ~+, Ca 2÷, Zn ~÷, Co 2÷, Cd 2÷, and Ni 2÷, but free Zn 2÷ ions were particularly potent at inhibiting the reaction. ~- The evidence from these kinetic studies favors a rapid random equilibrium type of reaction mechanism; further support for this mechanism comes from inhibitor studies on the erythrocyte enzyme. ~ 9W. K. G. Krietsch and T. Biicher, Eur. J. Biochem. 17, 568 (1970). lo R. tC Scopes, in "The Enzymes" (P. J. Boyer, ed.), 3rd ed., Vol. 8, p. 335. Academic Press, New York, 1973. ~1M. Larsson-Raznikiewicz, Biochim. Biophys. Acta 85, 60 (1964). ~ M. Larsson-Raznikiewicz, Eur. J. Biochem. 17, 183 (1970). ~3C. S. Lee and W. J. O'Sullivan, Proc. Aust. Biochem. Soc. 6, 18 (1973).
