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[72] A phosphoenolpyruvate-hexose phosphotransferase system from Escherichia coli
396
KINASES AND TRANSPHOSPHORYLASES
[72]
Stability. The most highly purified fractions of phosphotransferase have been stored at --20 ° with repeated thawing and freezing with no detectable loss of activity.
[ 72 ] A P h o s p h o e n o l p y r u v a t e - H e x o s e P h o s p h o t r a n s f e r a s e S y s t e m f r o m E s c h e r i c l d a coli By WER~ER KUNDm and SAUL ROSEMAN (A)
enzyme
Phosphoenolpyruvate ~ heat-stable protein (HPr) . enzyme
(B)
PhosphooHPr nu hexose
(A -t- B)
Mg++
I
Mg++ phospho-HPr ~ pyruvate
II , hexose-6-P ~ H P r
Phosphoenolpyruvate -I- hexose
enzymes I + II , hexose-6-P ~ pyruvate HPr. Mg++
Assay Method Principle. The phosphotransferase system was isolated from Escherichia coli, 1 and has been detected in Aerobacter, Lactobacillus, and Bacillus subtilis. 2 The system catalyzes the transfer of phosphate from phosphoenol pyruvate (PEP) to a variety of sugars and sugar derivatives, and requires three protein fractions, enzymes I and II, and a heatstable protein, called HPr. The latter acts as a "phosphate carrier" in the overall reaction; in phospho-HPr, the phosphate group is covalently linked to protein-bound histidine. 1 The complete system involves two discrete reactions: enzyme I catalyzes the transfer of phosphate from P E P to ttPr; enzyme II catalyzes the transfer of phosphate from phospho-HPr to suitable sugar acceptors. The specificity toward the sugars is confined to enzyme II. At least three different enzymes II have been demonstrated; they are located in the membrane fraction obtained from lysed E. coli spheroplasts, and B. subtilis protoplastsY Enzyme I and H P r are found primarily in the supernatant fractions of the lysed preparations. Although the rates of the individual reactions can be determined with purified preparations, a more convenient procedure involves determination of the rate of the complete system where one of the three protein fractions is used in rate-limiting concentration. With purified H P r as the ' W . Kundig, S. Ghosh, and S. Roseman, Proc. Natl. Acad. Scl. U.8. 52, 1073 (1964). 2 W. Kundig, F. Dodyk Kundig, B. Anderson, and S. Roseman, Federation Proc. 24, 6.58 (1965).
[72]
P~OSPHOENOLPYRUVATE-HEXOSE PHOSPHOTRANSFERASE
397
rate-limiting protein, using the conditions described below, product formation is proportional to time of incubation in the range 5-15 ~g. The assay mixtures contain either 14C-labeled hexose and unlabeled PEP, or 32P-labeled P E P and unlabeled hexosc. Labeled products are separated from excess substrate by high voltage paper electrophoresis and determined by counting in the Packard Tri-Carb liquid scintillation spectrometer. A useful acceptor with crude extracts is ~4C-acetyl labeled N-acetylmannosamine, ~ since the product N-acetylmannosamine-6-P is not further metabolized with preparations obtained from glucose- or glycerolgrown cells. With more purified preparations, a number of sugars can be used as substrates. Reagents Phosphate buffers (K ÷) , 1 M, pH 7.6 and 6.5 Tris-HC1 buffers, 1 M, pH 7.6 and 7.4 Sodium glycinate buffer, 1 M, pH 8.0 EDTA, 0.1 M, pH 7.6 MgC12, 0.1 M Hexose, 0.1 M; hexosamine, 0.1 M; or N-acylhexosamine, 0.1M Phosphoenolpyruvate, sodium salt, 0.1 M 14C-labeled hexose or hexosamine, 0.1 M. The preferred substrate for crude extracts is N-acetyl-(14C)-D-mannosamine (specific activity, 5 X 10~ cpm per mieromole) (Vol. VIII [26]) a2P-labeled PEP, 0.1 M, prepared by incubation of chicken liver mitochondria with 82p~, in the presence of K malate (specific activity, 5 X 105 cpm per micromole) ~ Tergitol-4, 1%, in water (purchased from Union Carbide, New York) Procedure The rate of phosphorylation is determined with either 14C-labeled sugar (such as N-aeetylmannosamine) and unlabeled PEP, or with unlabeled sugar and s2P-labeled PEP. The incubation mixtures contain the following components in final volumes of 0.18 mh 0.020 ml hexose, hexosamine or N-acetylmannosamine; 0.020 ml PEP; 0.010 ml MgC12; 0.010 ml of either Tris-HC1 or the glycine buffer (pH 7.4 and 8.0, respectively) ; purified HPr, 20/zg; 0.010 ml Tergitol-4; one unit of enzymes I and II. To estimate the concentration of enzyme I, an excess of enzyme II (4 units) is added, while the reverse is true for estimating the concentration of enzyme II. The concentration of H P r given above represents sj. Mendicino and M. F. Utter, J. Biol. Chem. 237, 1716 (1962).
398
KINASES AND TRANSPHOSPHORYLASES
[72]
an excess for the complete system. To assay for HPr, 4 units of enzymes I and II are employed, with the equivalent of 5-15 #g of purified HPr. After 30 minutes of incubation at 37 °, the reaction is stopped by heating for 2 minutes at 100 °, and an aliquot is subjected to electrophoresis on Whatman 3 MM paper saturated with 0.05 M pyridinium-acetate buffer, pH 6.5 (50 volts/cm). When the labeled substrate is the hexose, electrophoresis is conducted for 15 minutes, and 30 minutes is employed when the substrate is 82P-labeled P E P ; these conditions will separate the product (a sugar-P), P,, and PEP. The control incubation mixtures contain heat-inactivated in place of active enzyme, or are incubated 0 minute, or lack hexose in the case of the 8~P-labeled PEP. In addition, standard samples are electrophoresed at the same time as the incubation mixtures. The determination of 8~p, in the s2p assay system permits quantitation of the phosphatases that are present in the crude extracts. Definition o] Unit and Specific Activity. One unit of enzyme I is the amount that will catalyze the synthesis of 1 micromole of N-acetylmannosamine-6-P when incubated for 30 minutes at 37 ° under the conditions described above, in the presence of excess enzyme II and HPr. A similar definition applies to enzyme II. Specific activity is defined as the units of enzyme I, or II, per milligram of protein. The method of Lowry et al. ~ is used to determine protein concentration. Purification Procedure
Growth o] Organism. A number of E. coli strains, such as B, K12, contain the phosphotransferase system, Most of the kinetic studies were conducted with fractions obtained from E. coli K235. 5 The organism is grown in Todd-Hewitt broth supplemented with 1.5% glucose,6 or in a mineral salts medium, 6 with either 0.4% glucose or 0.4% glycerol as the carbon source. With 600 ml quantities of medium, the cells are grown in 2-liter Erlenmeyer flasks on a New Brunswick Rotary Shaker at 37 ° (400 rpm). For larger quantities of cells, 14-liter New Brunswick fermenfor jars are used, containing up to 10 liters of medium; here, the maximum yield of the phosphotransferase system is obtained when the culture is stirred at approximately 200 rpm, but without passage of air through the sparger. In both cases, it is essential to maintain the pH O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 1@3, 265 (1951). G. W. Barry and W. F. Goebel, Nature 17@, 206 (1957). s The mineral salts medium contains per liter: 0.05 mole potassium phosphate, pH 7.3; 2 g (NH,)~SO4; 0.2 g MgSO4-7 H10; 0.5 mg FeSO,.7 H~O; 1 g Casamlno acids, (Difco). After sterilization 1 mg of thiamine is added. Todd-Hewitt broth is avail able from Difco Laboratories, Detroit, Michigan.
[72]
PHOSPHOENOLPYRUYATE-HEXOStl PHOSPHOTRANSFERASE
399
between 7.0 and 7.5 by adding 6 M NaOH as required. The culture is permitted to grow until it reaches two-thirds of maximum growth and is then rapidly chilled to stop growth; the yield of H P r is greatly decreased if the cells reach the stationary phase of growth. After harvesting, the cells are washed twice with cold 1% KC1, and the wet paste is stored at --18 ° .
Purification oJ Enzymes I and H Unless otherwise specified, all operations are conducted between 0 ° and 4 °, and all buffers contain 5 micromoles EDTA, and 1 mg 2-mercaptoethanol per milliliter. Step 1. Crude Extracts. The cells (1 g wet paste) are suspended in 20 ml of 0.025 M Tris-HC1 buffer, pH 7.6, and ruptured either by sonic vibration, or by passage through a French Pressure Cell. The mixture is centrifuged at 10,000 g for 20 minutes, and the residue is discarded. Step 2. Charcoal Treatment. The crude extract is treated with charcoal to remove HPr. Twenty milliliters of a 10% suspension of Darco G-60 charcoal is centrifuged for 5 minutes at 10,000 g, and the resulting precipitate is mixed with 20 ml of crude extract. The mixture is stirred for 5 minutes, then centrifuged at 10,000 g for 20 minutes; the residue is discarded. Step 3. Ammonium SuIJate. A saturated solution of ammonium sulfate (Enzyme Grade, Mann Research Lab., Inc.) is adjusted with N a 0 H until a 1:4 dilution with water is at pH 7.6 when measured with a glass electrode. The ammonium sulfate solution (20 ml) is slowly added, with stirring, to 20 ml of the supernatant fluid obtained in step 2, the mixture is occasionally stirred for 20 minutes, and the precipitate is collected by centrifugation at 35,000 g for 20 minutes. Five ml of 0.01 M K phosphate buffer, pH 7.6, is immediately added to the residue, and the turbid suspension is passed through a 100 ml column of Sephadex G-25 previously equilibrated with the same buffer. The column is eluted with the phosphate buffer, and the protein fractions are combined. Step 4. C~, Alumina Gel. A suspension containing C], alumina gel (2% solids; Alumina C), aged (Nutritional Biochemicals Corp.) is prepared, and 30 ml is centrifuged for 5 minutes at 35,000 g. The residue is mixed with 15 ml of the slightly turbid fraction obtained in the previous step, and after 5 minutes with occasional stirring, the suspension is centrifuged for 10 minutes at 35,000 g. The supernatant fraction contains enzyme II, while enzyme I is adsorbed to the gel. The pellet is first washed with 30 ml of 0.05 M K phosphate buffer, pH 7.6, and enzyme I is then eluted by twice extracting the gel with 10 ml portions of 0.10 M K phosphate buffer, pH 7.6. The washing and extractions are performed
400
KINASES AND TRANSPHOSPHORYLASES
[72]
by mixing the gel for 5 minutes with the buffers, followed by 10-minute centrifugations at 35,000 g. The extracts are combined and dialyzed against 0.01 M Tris-HC1 buffer, p H 7.6, for 12 hours. Step 5. DEAE-CelIulose Chromatography (Enzyme I). DEAE-cellulose is equilibrated with 0.01 M Tris-HC1, p H 7.6, and a column is prepared (2 X 15 cm) containing 60 ml of the cellulose packed by gravity filtration. The dialyzed gel eluate obtained in the previous step is transferred to the column, which is then eluted with 300-ml portions of 0.01 M Tris-HC1, p H 7.6, containing the increasing concentrations of KC1 in the following sequence: 0.00, 0.05, 0.10, and 0.20M. The 0.20 M eluate contains enzyme I, and is concentrated to 5 ml by pressure dialysis against 8 liters of 0.01 M Tris-HC1, p H 7.6. Step 6. Purification o] Enzyme II. The alumina gel supernatant fraction (step 4) contains enzyme II, which can be sedimented at 100,000 g (90 minutes). The pellet is washed with 20 ml 0.01 M Tris-HCl, p H 7.6, centrifuged, and resuspended in the same buffer. Enzyme II can also be purified by chromatography on DEAE-cellulose, phosphate form, previously equilibrated with 0.01 M K phosphate buffer, p H 7.6. The alumina gel supernatant fraction (15 ml) is placed on a 60-ml column of the cellulose (2 X 15 cm), and the column is eluted with 300 ml portions of phosphate buffer at the following concentrations: 0.01, 0.10, 0.20, and 0.30 M. The 0.30 M eluate contains enzyme I I which is concentrated to 5 ml by pressure dialysis against 8 liters of 0.01 M Tris-HCl, p H 7.6. At this stage, enzyme I I is sedimentable when subjected to centrifugation for 90 minutes at 100,000 g. PURIFICATION OF ENZYMES I AND I I
Specific activitya Fraction Crude extract Charcoal supernatant 50% Ammonium sulfate Alumina gel
Supernatant Eluate DEAE-cellulose Alumina gel supernatant Alumina gel eluate
I + II
I
II
Yield (%)
0.30 0.82 2.2
----
----
100 62 51
0 19
16 0
39 41
0 119
98 0
24 27
a Specific activity is defined as ~moles N-acetyl-mannosamine-6-P formed per milligram protein per 30 minutes. After the alumina gel step, either enzyme I or enzyme II is added in rate-limiting amounts and the other enzyme is added in excess. Zero values indicate no detectable activity (i.e., less than 0.05~).
[72]
PHOSPHOENOLPYRUVATE-HEXOSE PHOSPHOTRANSFERASE
401
The purification procedure for enzymes I and II is summarized in the table.
Purification o] Heat-stable Protein (HPr) These steps are conducted at room temperature, unless otherwise indieated. Step 1. Crude Extract. The cells (2.3 kg wet paste) are suspended in 10 liters of 0.025 M K phosphate buffer, pH 7.6, without 2-mereaptoethanol or EDTA. The cells are ruptured by passage through a continuous flow sonic oscillator, centrifuged at 35,000 g for 30 minutes, and 500-ml aliquots of the supernatant fluid are heated for 15 minutes in a boiling water bath. The coagulated protein is removed by centrifugation and discarded. Step 2. Acid Precipitation. The supernatant fluid of the preceding step is cooled to about 4 °, and adjusted to pH 1.0 with conc. HC1 solution. After 30 minutes, the resulting white precipitate is collected by centrifugation and washed once with cold 0.01 M HC1. The precipitate is then suspended in 2 liters of 0.5 M K phosphate buffer, pH 7.6, at room temperature, using a Waring blendor, and the suspension is stirred for 24 hours with a magnetic stirrer. After centrifugation at 35,000 g for 20 minutes, the precipitate is discarded, and the supernatant fluid is dialyzed against at least 4 changes of distilled water (10 liters each) for a total of 48 hours. The dialysis residue is concentrated to about 200 ml in a rotary evaporator, and dialyzed against 2-liter portions (4 changes) of 0.025 M K phospate buffer, pH 6.5. Step 3. ECTEOLA-Cellulose. The cellulose derivative is converted to the phosphate form, adjusted to pH 6.5, and equilibrated with 0.025 M K phosphate, pH 6.5; a column of the ECTEOLA-cellulose is prepared by gravity filtration (6 X 40 cm) and contains 1 liter of packed cellulose. The dialyzate obtained in the preceding step (200 ml) is placed on the column and eluted with the 0.025 M phosphate buffer. The protein-containing fractions, detected by a modified biuret method, 4 are combined, concentrated in a rotary evaporator to approximately 200 ml, and dialyzed against 5 changes of 0.01 M Tris-HC1 buffer, pH 7.6 (5 liters each). Step 4. DEAE-Cellulose, First Column. The eluate obtained in the previous step is placed on a 1 liter column of DEAE-cellulose (5 X 30 cm; previously converted to the C1- form and equilibrated with 0.01 M Tris-HC1, pH 7.6). The column is then eluted with 3-liter portions of 0.01 M Tris-HC1, pH 7.6, containing the following concentrations of KCI 0.0, 0.050, and 0.10 M. The last fraction contains HPr, and is con-
402
KINASES AND TRANSPHOSPHORYLASES
[72]
centrated to 100 ml in the rotary evaporator, and dialyzed against 4 changes of 0.01 M Tris-HC1, pH 7.6 (5 liters each). Step 5. DEAE-CeUulose, Second Column. A column containing 500 ml of DEAE-cellulose is pretreated as described in step 4; the H P r solution of the preceding step is transferred to the column. After the column has been washed with 0.01 M Tris-HCl buffer, pH 7.6, H P r is eluted using a linear gradient (total volume 6 liters) of KC1 with the initial concentration of 0.0M and the final concentration a~ O.12M, the gradient containing 0.01 M Tris-HC1 at all points. The fractions are assayed for protein (absorbance at 280 m~) and for H P r activity; inactive protein is eluted early after application of the gradient, while H P r is eluted in a single symmetrical peak at about 0.075 M KC1. The fractions containing H P r are combined, concentrated to 20 ml in a rotary evaporator, and dialyzed against 5 changes of distilled water (2 liters each). Based on analysis by a modified biuret method, 4 H P r is purified between 8000- and 10,000-fold by the procedure described above. The final yield is approximately 10-15 mg. Properties
Stability o] Enzymes I, II, and HPr. Purified enzyme I is stable for approximately 1 week when stored at 0 ° and is not stable to freezing and thawing; enzyme II is also inactivated by freezing and thawing and is stable for only 24-48 hours at 0% H P r is stable at 100° at neutral pH for at least 20 minutes, at pH 1 at room temperature for several hours, but is unstable in 0.1 M N a 0 H for 60 minutes at room temperature. It is completely resistant to prolonged digestion and dialysis with the following nucleases: purified venom phosphodiesterase, venom 5'-nucleotidase, polynucleotide phosphorylase, pancreatic RNAse and DNAse. On the other hand, it is very labile to the action of the following proteinases: chymotrypsin, trypsin, papain, pepsin, and pronase. H P r is either irreversibly adsorbed or inactivated by charcoal, but not by mixed-bed ion exchange resins. Properties o] the Complete System. The purified enzyme system is active only with P E P as the phosphate donor (Kin, 6 X 10-* M).~ The optimum divalent metal ion is Mg +÷, which can partially be replaced by Mn ÷÷, Zn +~, and Co ÷+. The following ions are strongly inhibitory: Cu% Fe ++, and Ca ÷÷. Other proteins cannot replace HPr. Preliminary experiments indicate that the components of the system ioslated from E . coli will substitute for the components of the system isolated from B. subtilis and other organisms.'-'
[73]
PHOSPHORAMIDATE-HEXOSE TRANSPHOSPHORYLASE
403
The sugar specificity of the system is confined to enzyme II, and there are at least three such enzymes present in the particulate fraction. The specificity of enzyme II is determined by the growth medium. For example, when a Todd-Hewitt glucose broth is used, active phosphate aeceptors are of the D-gluco and D-manno configuration (hexoses, hexitols, hexosamines, N-acylhexosamines, methyl a-D-glucopyranoside), while a number of pentoses, pentitols, other hexitols, fructose, galactose, and disaccharides are inactive. When the cells are grown in mineral medium containing the inactive compounds as the carbon source, then enzyme II activity is demonstrable with these substrates. Enzyme II from cells grown on a glycerol mineral salts medium shows a broad substrate specificity, and the activity toward galactose can be increased at least 4-fold when the nonmetabolized compound D-fucose is added to the medium at 0.001 M concentration. 2 When the following sugars are used as substrates, the corresponding 6-phosphate esters are formed in the reaction (all D-sugars): glucose, mannose, glucosamine, mannosamine, N-acetyl- and N-glycolylglucosamine, and the corresponding N-acyl mannosamines, galactose, and methyl fl-l-thiogalactoside. The pH optimum for the complete system is approximately 7.4 for hexoses of the gluco and manno configuration, and 8.0 for those of the galacto configuration.
[ 73 ] P h o s p h o r a m i d a t e - H e x o s e
Transphosphorylase
By ROBERTSA. SMITH and MYNA C. THEISEN KO3PN+H3 + glucose~ glucose 1-phosphate + NH~ Assay Method Principle. The most convenient and sensitive assay method is based on the formation of glucose 1-phosphate by phosphoryl transfer from 32P-NH2. The phosphorylated glucose is estimated after hydrolysis of the remaining ~2P-NH2 to 32p~ and its subsequent removal from solution by precipitation of the phosphomolybdate complex according to the procedure of Sugino and Miyoshi. 1 Phosphoramidate is so extremely acid labile ~ that it can be converted to P~ virtually quantitatively while glucose 1-phosphate remains unaltered. This conversion is accomplished by the addition of trichloroacetie acid to terminate the reaction.
'Y. Sugino and Y. Miyoshi, J. Biol. Chem. 239, 2360 (1964). ~"T. R'tlhlev anti T. Rosenberg, Arch. Biochim. Biophy.~. 65, 319 (1956).
KINASES AND TRANSPHOSPHORYLASES
[72]
Stability. The most highly purified fractions of phosphotransferase have been stored at --20 ° with repeated thawing and freezing with no detectable loss of activity.
[ 72 ] A P h o s p h o e n o l p y r u v a t e - H e x o s e P h o s p h o t r a n s f e r a s e S y s t e m f r o m E s c h e r i c l d a coli By WER~ER KUNDm and SAUL ROSEMAN (A)
enzyme
Phosphoenolpyruvate ~ heat-stable protein (HPr) . enzyme
(B)
PhosphooHPr nu hexose
(A -t- B)
Mg++
I
Mg++ phospho-HPr ~ pyruvate
II , hexose-6-P ~ H P r
Phosphoenolpyruvate -I- hexose
enzymes I + II , hexose-6-P ~ pyruvate HPr. Mg++
Assay Method Principle. The phosphotransferase system was isolated from Escherichia coli, 1 and has been detected in Aerobacter, Lactobacillus, and Bacillus subtilis. 2 The system catalyzes the transfer of phosphate from phosphoenol pyruvate (PEP) to a variety of sugars and sugar derivatives, and requires three protein fractions, enzymes I and II, and a heatstable protein, called HPr. The latter acts as a "phosphate carrier" in the overall reaction; in phospho-HPr, the phosphate group is covalently linked to protein-bound histidine. 1 The complete system involves two discrete reactions: enzyme I catalyzes the transfer of phosphate from P E P to ttPr; enzyme II catalyzes the transfer of phosphate from phospho-HPr to suitable sugar acceptors. The specificity toward the sugars is confined to enzyme II. At least three different enzymes II have been demonstrated; they are located in the membrane fraction obtained from lysed E. coli spheroplasts, and B. subtilis protoplastsY Enzyme I and H P r are found primarily in the supernatant fractions of the lysed preparations. Although the rates of the individual reactions can be determined with purified preparations, a more convenient procedure involves determination of the rate of the complete system where one of the three protein fractions is used in rate-limiting concentration. With purified H P r as the ' W . Kundig, S. Ghosh, and S. Roseman, Proc. Natl. Acad. Scl. U.8. 52, 1073 (1964). 2 W. Kundig, F. Dodyk Kundig, B. Anderson, and S. Roseman, Federation Proc. 24, 6.58 (1965).
[72]
P~OSPHOENOLPYRUVATE-HEXOSE PHOSPHOTRANSFERASE
397
rate-limiting protein, using the conditions described below, product formation is proportional to time of incubation in the range 5-15 ~g. The assay mixtures contain either 14C-labeled hexose and unlabeled PEP, or 32P-labeled P E P and unlabeled hexosc. Labeled products are separated from excess substrate by high voltage paper electrophoresis and determined by counting in the Packard Tri-Carb liquid scintillation spectrometer. A useful acceptor with crude extracts is ~4C-acetyl labeled N-acetylmannosamine, ~ since the product N-acetylmannosamine-6-P is not further metabolized with preparations obtained from glucose- or glycerolgrown cells. With more purified preparations, a number of sugars can be used as substrates. Reagents Phosphate buffers (K ÷) , 1 M, pH 7.6 and 6.5 Tris-HC1 buffers, 1 M, pH 7.6 and 7.4 Sodium glycinate buffer, 1 M, pH 8.0 EDTA, 0.1 M, pH 7.6 MgC12, 0.1 M Hexose, 0.1 M; hexosamine, 0.1 M; or N-acylhexosamine, 0.1M Phosphoenolpyruvate, sodium salt, 0.1 M 14C-labeled hexose or hexosamine, 0.1 M. The preferred substrate for crude extracts is N-acetyl-(14C)-D-mannosamine (specific activity, 5 X 10~ cpm per mieromole) (Vol. VIII [26]) a2P-labeled PEP, 0.1 M, prepared by incubation of chicken liver mitochondria with 82p~, in the presence of K malate (specific activity, 5 X 105 cpm per micromole) ~ Tergitol-4, 1%, in water (purchased from Union Carbide, New York) Procedure The rate of phosphorylation is determined with either 14C-labeled sugar (such as N-aeetylmannosamine) and unlabeled PEP, or with unlabeled sugar and s2P-labeled PEP. The incubation mixtures contain the following components in final volumes of 0.18 mh 0.020 ml hexose, hexosamine or N-acetylmannosamine; 0.020 ml PEP; 0.010 ml MgC12; 0.010 ml of either Tris-HC1 or the glycine buffer (pH 7.4 and 8.0, respectively) ; purified HPr, 20/zg; 0.010 ml Tergitol-4; one unit of enzymes I and II. To estimate the concentration of enzyme I, an excess of enzyme II (4 units) is added, while the reverse is true for estimating the concentration of enzyme II. The concentration of H P r given above represents sj. Mendicino and M. F. Utter, J. Biol. Chem. 237, 1716 (1962).
398
KINASES AND TRANSPHOSPHORYLASES
[72]
an excess for the complete system. To assay for HPr, 4 units of enzymes I and II are employed, with the equivalent of 5-15 #g of purified HPr. After 30 minutes of incubation at 37 °, the reaction is stopped by heating for 2 minutes at 100 °, and an aliquot is subjected to electrophoresis on Whatman 3 MM paper saturated with 0.05 M pyridinium-acetate buffer, pH 6.5 (50 volts/cm). When the labeled substrate is the hexose, electrophoresis is conducted for 15 minutes, and 30 minutes is employed when the substrate is 82P-labeled P E P ; these conditions will separate the product (a sugar-P), P,, and PEP. The control incubation mixtures contain heat-inactivated in place of active enzyme, or are incubated 0 minute, or lack hexose in the case of the 8~P-labeled PEP. In addition, standard samples are electrophoresed at the same time as the incubation mixtures. The determination of 8~p, in the s2p assay system permits quantitation of the phosphatases that are present in the crude extracts. Definition o] Unit and Specific Activity. One unit of enzyme I is the amount that will catalyze the synthesis of 1 micromole of N-acetylmannosamine-6-P when incubated for 30 minutes at 37 ° under the conditions described above, in the presence of excess enzyme II and HPr. A similar definition applies to enzyme II. Specific activity is defined as the units of enzyme I, or II, per milligram of protein. The method of Lowry et al. ~ is used to determine protein concentration. Purification Procedure
Growth o] Organism. A number of E. coli strains, such as B, K12, contain the phosphotransferase system, Most of the kinetic studies were conducted with fractions obtained from E. coli K235. 5 The organism is grown in Todd-Hewitt broth supplemented with 1.5% glucose,6 or in a mineral salts medium, 6 with either 0.4% glucose or 0.4% glycerol as the carbon source. With 600 ml quantities of medium, the cells are grown in 2-liter Erlenmeyer flasks on a New Brunswick Rotary Shaker at 37 ° (400 rpm). For larger quantities of cells, 14-liter New Brunswick fermenfor jars are used, containing up to 10 liters of medium; here, the maximum yield of the phosphotransferase system is obtained when the culture is stirred at approximately 200 rpm, but without passage of air through the sparger. In both cases, it is essential to maintain the pH O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 1@3, 265 (1951). G. W. Barry and W. F. Goebel, Nature 17@, 206 (1957). s The mineral salts medium contains per liter: 0.05 mole potassium phosphate, pH 7.3; 2 g (NH,)~SO4; 0.2 g MgSO4-7 H10; 0.5 mg FeSO,.7 H~O; 1 g Casamlno acids, (Difco). After sterilization 1 mg of thiamine is added. Todd-Hewitt broth is avail able from Difco Laboratories, Detroit, Michigan.
[72]
PHOSPHOENOLPYRUYATE-HEXOStl PHOSPHOTRANSFERASE
399
between 7.0 and 7.5 by adding 6 M NaOH as required. The culture is permitted to grow until it reaches two-thirds of maximum growth and is then rapidly chilled to stop growth; the yield of H P r is greatly decreased if the cells reach the stationary phase of growth. After harvesting, the cells are washed twice with cold 1% KC1, and the wet paste is stored at --18 ° .
Purification oJ Enzymes I and H Unless otherwise specified, all operations are conducted between 0 ° and 4 °, and all buffers contain 5 micromoles EDTA, and 1 mg 2-mercaptoethanol per milliliter. Step 1. Crude Extracts. The cells (1 g wet paste) are suspended in 20 ml of 0.025 M Tris-HC1 buffer, pH 7.6, and ruptured either by sonic vibration, or by passage through a French Pressure Cell. The mixture is centrifuged at 10,000 g for 20 minutes, and the residue is discarded. Step 2. Charcoal Treatment. The crude extract is treated with charcoal to remove HPr. Twenty milliliters of a 10% suspension of Darco G-60 charcoal is centrifuged for 5 minutes at 10,000 g, and the resulting precipitate is mixed with 20 ml of crude extract. The mixture is stirred for 5 minutes, then centrifuged at 10,000 g for 20 minutes; the residue is discarded. Step 3. Ammonium SuIJate. A saturated solution of ammonium sulfate (Enzyme Grade, Mann Research Lab., Inc.) is adjusted with N a 0 H until a 1:4 dilution with water is at pH 7.6 when measured with a glass electrode. The ammonium sulfate solution (20 ml) is slowly added, with stirring, to 20 ml of the supernatant fluid obtained in step 2, the mixture is occasionally stirred for 20 minutes, and the precipitate is collected by centrifugation at 35,000 g for 20 minutes. Five ml of 0.01 M K phosphate buffer, pH 7.6, is immediately added to the residue, and the turbid suspension is passed through a 100 ml column of Sephadex G-25 previously equilibrated with the same buffer. The column is eluted with the phosphate buffer, and the protein fractions are combined. Step 4. C~, Alumina Gel. A suspension containing C], alumina gel (2% solids; Alumina C), aged (Nutritional Biochemicals Corp.) is prepared, and 30 ml is centrifuged for 5 minutes at 35,000 g. The residue is mixed with 15 ml of the slightly turbid fraction obtained in the previous step, and after 5 minutes with occasional stirring, the suspension is centrifuged for 10 minutes at 35,000 g. The supernatant fraction contains enzyme II, while enzyme I is adsorbed to the gel. The pellet is first washed with 30 ml of 0.05 M K phosphate buffer, pH 7.6, and enzyme I is then eluted by twice extracting the gel with 10 ml portions of 0.10 M K phosphate buffer, pH 7.6. The washing and extractions are performed
400
KINASES AND TRANSPHOSPHORYLASES
[72]
by mixing the gel for 5 minutes with the buffers, followed by 10-minute centrifugations at 35,000 g. The extracts are combined and dialyzed against 0.01 M Tris-HC1 buffer, p H 7.6, for 12 hours. Step 5. DEAE-CelIulose Chromatography (Enzyme I). DEAE-cellulose is equilibrated with 0.01 M Tris-HC1, p H 7.6, and a column is prepared (2 X 15 cm) containing 60 ml of the cellulose packed by gravity filtration. The dialyzed gel eluate obtained in the previous step is transferred to the column, which is then eluted with 300-ml portions of 0.01 M Tris-HC1, p H 7.6, containing the increasing concentrations of KC1 in the following sequence: 0.00, 0.05, 0.10, and 0.20M. The 0.20 M eluate contains enzyme I, and is concentrated to 5 ml by pressure dialysis against 8 liters of 0.01 M Tris-HC1, p H 7.6. Step 6. Purification o] Enzyme II. The alumina gel supernatant fraction (step 4) contains enzyme II, which can be sedimented at 100,000 g (90 minutes). The pellet is washed with 20 ml 0.01 M Tris-HCl, p H 7.6, centrifuged, and resuspended in the same buffer. Enzyme II can also be purified by chromatography on DEAE-cellulose, phosphate form, previously equilibrated with 0.01 M K phosphate buffer, p H 7.6. The alumina gel supernatant fraction (15 ml) is placed on a 60-ml column of the cellulose (2 X 15 cm), and the column is eluted with 300 ml portions of phosphate buffer at the following concentrations: 0.01, 0.10, 0.20, and 0.30 M. The 0.30 M eluate contains enzyme I I which is concentrated to 5 ml by pressure dialysis against 8 liters of 0.01 M Tris-HCl, p H 7.6. At this stage, enzyme I I is sedimentable when subjected to centrifugation for 90 minutes at 100,000 g. PURIFICATION OF ENZYMES I AND I I
Specific activitya Fraction Crude extract Charcoal supernatant 50% Ammonium sulfate Alumina gel
Supernatant Eluate DEAE-cellulose Alumina gel supernatant Alumina gel eluate
I + II
I
II
Yield (%)
0.30 0.82 2.2
----
----
100 62 51
0 19
16 0
39 41
0 119
98 0
24 27
a Specific activity is defined as ~moles N-acetyl-mannosamine-6-P formed per milligram protein per 30 minutes. After the alumina gel step, either enzyme I or enzyme II is added in rate-limiting amounts and the other enzyme is added in excess. Zero values indicate no detectable activity (i.e., less than 0.05~).
[72]
PHOSPHOENOLPYRUVATE-HEXOSE PHOSPHOTRANSFERASE
401
The purification procedure for enzymes I and II is summarized in the table.
Purification o] Heat-stable Protein (HPr) These steps are conducted at room temperature, unless otherwise indieated. Step 1. Crude Extract. The cells (2.3 kg wet paste) are suspended in 10 liters of 0.025 M K phosphate buffer, pH 7.6, without 2-mereaptoethanol or EDTA. The cells are ruptured by passage through a continuous flow sonic oscillator, centrifuged at 35,000 g for 30 minutes, and 500-ml aliquots of the supernatant fluid are heated for 15 minutes in a boiling water bath. The coagulated protein is removed by centrifugation and discarded. Step 2. Acid Precipitation. The supernatant fluid of the preceding step is cooled to about 4 °, and adjusted to pH 1.0 with conc. HC1 solution. After 30 minutes, the resulting white precipitate is collected by centrifugation and washed once with cold 0.01 M HC1. The precipitate is then suspended in 2 liters of 0.5 M K phosphate buffer, pH 7.6, at room temperature, using a Waring blendor, and the suspension is stirred for 24 hours with a magnetic stirrer. After centrifugation at 35,000 g for 20 minutes, the precipitate is discarded, and the supernatant fluid is dialyzed against at least 4 changes of distilled water (10 liters each) for a total of 48 hours. The dialysis residue is concentrated to about 200 ml in a rotary evaporator, and dialyzed against 2-liter portions (4 changes) of 0.025 M K phospate buffer, pH 6.5. Step 3. ECTEOLA-Cellulose. The cellulose derivative is converted to the phosphate form, adjusted to pH 6.5, and equilibrated with 0.025 M K phosphate, pH 6.5; a column of the ECTEOLA-cellulose is prepared by gravity filtration (6 X 40 cm) and contains 1 liter of packed cellulose. The dialyzate obtained in the preceding step (200 ml) is placed on the column and eluted with the 0.025 M phosphate buffer. The protein-containing fractions, detected by a modified biuret method, 4 are combined, concentrated in a rotary evaporator to approximately 200 ml, and dialyzed against 5 changes of 0.01 M Tris-HC1 buffer, pH 7.6 (5 liters each). Step 4. DEAE-Cellulose, First Column. The eluate obtained in the previous step is placed on a 1 liter column of DEAE-cellulose (5 X 30 cm; previously converted to the C1- form and equilibrated with 0.01 M Tris-HC1, pH 7.6). The column is then eluted with 3-liter portions of 0.01 M Tris-HC1, pH 7.6, containing the following concentrations of KCI 0.0, 0.050, and 0.10 M. The last fraction contains HPr, and is con-
402
KINASES AND TRANSPHOSPHORYLASES
[72]
centrated to 100 ml in the rotary evaporator, and dialyzed against 4 changes of 0.01 M Tris-HC1, pH 7.6 (5 liters each). Step 5. DEAE-CeUulose, Second Column. A column containing 500 ml of DEAE-cellulose is pretreated as described in step 4; the H P r solution of the preceding step is transferred to the column. After the column has been washed with 0.01 M Tris-HCl buffer, pH 7.6, H P r is eluted using a linear gradient (total volume 6 liters) of KC1 with the initial concentration of 0.0M and the final concentration a~ O.12M, the gradient containing 0.01 M Tris-HC1 at all points. The fractions are assayed for protein (absorbance at 280 m~) and for H P r activity; inactive protein is eluted early after application of the gradient, while H P r is eluted in a single symmetrical peak at about 0.075 M KC1. The fractions containing H P r are combined, concentrated to 20 ml in a rotary evaporator, and dialyzed against 5 changes of distilled water (2 liters each). Based on analysis by a modified biuret method, 4 H P r is purified between 8000- and 10,000-fold by the procedure described above. The final yield is approximately 10-15 mg. Properties
Stability o] Enzymes I, II, and HPr. Purified enzyme I is stable for approximately 1 week when stored at 0 ° and is not stable to freezing and thawing; enzyme II is also inactivated by freezing and thawing and is stable for only 24-48 hours at 0% H P r is stable at 100° at neutral pH for at least 20 minutes, at pH 1 at room temperature for several hours, but is unstable in 0.1 M N a 0 H for 60 minutes at room temperature. It is completely resistant to prolonged digestion and dialysis with the following nucleases: purified venom phosphodiesterase, venom 5'-nucleotidase, polynucleotide phosphorylase, pancreatic RNAse and DNAse. On the other hand, it is very labile to the action of the following proteinases: chymotrypsin, trypsin, papain, pepsin, and pronase. H P r is either irreversibly adsorbed or inactivated by charcoal, but not by mixed-bed ion exchange resins. Properties o] the Complete System. The purified enzyme system is active only with P E P as the phosphate donor (Kin, 6 X 10-* M).~ The optimum divalent metal ion is Mg +÷, which can partially be replaced by Mn ÷÷, Zn +~, and Co ÷+. The following ions are strongly inhibitory: Cu% Fe ++, and Ca ÷÷. Other proteins cannot replace HPr. Preliminary experiments indicate that the components of the system ioslated from E . coli will substitute for the components of the system isolated from B. subtilis and other organisms.'-'
[73]
PHOSPHORAMIDATE-HEXOSE TRANSPHOSPHORYLASE
403
The sugar specificity of the system is confined to enzyme II, and there are at least three such enzymes present in the particulate fraction. The specificity of enzyme II is determined by the growth medium. For example, when a Todd-Hewitt glucose broth is used, active phosphate aeceptors are of the D-gluco and D-manno configuration (hexoses, hexitols, hexosamines, N-acylhexosamines, methyl a-D-glucopyranoside), while a number of pentoses, pentitols, other hexitols, fructose, galactose, and disaccharides are inactive. When the cells are grown in mineral medium containing the inactive compounds as the carbon source, then enzyme II activity is demonstrable with these substrates. Enzyme II from cells grown on a glycerol mineral salts medium shows a broad substrate specificity, and the activity toward galactose can be increased at least 4-fold when the nonmetabolized compound D-fucose is added to the medium at 0.001 M concentration. 2 When the following sugars are used as substrates, the corresponding 6-phosphate esters are formed in the reaction (all D-sugars): glucose, mannose, glucosamine, mannosamine, N-acetyl- and N-glycolylglucosamine, and the corresponding N-acyl mannosamines, galactose, and methyl fl-l-thiogalactoside. The pH optimum for the complete system is approximately 7.4 for hexoses of the gluco and manno configuration, and 8.0 for those of the galacto configuration.
[ 73 ] P h o s p h o r a m i d a t e - H e x o s e
Transphosphorylase
By ROBERTSA. SMITH and MYNA C. THEISEN KO3PN+H3 + glucose~ glucose 1-phosphate + NH~ Assay Method Principle. The most convenient and sensitive assay method is based on the formation of glucose 1-phosphate by phosphoryl transfer from 32P-NH2. The phosphorylated glucose is estimated after hydrolysis of the remaining ~2P-NH2 to 32p~ and its subsequent removal from solution by precipitation of the phosphomolybdate complex according to the procedure of Sugino and Miyoshi. 1 Phosphoramidate is so extremely acid labile ~ that it can be converted to P~ virtually quantitatively while glucose 1-phosphate remains unaltered. This conversion is accomplished by the addition of trichloroacetie acid to terminate the reaction.
'Y. Sugino and Y. Miyoshi, J. Biol. Chem. 239, 2360 (1964). ~"T. R'tlhlev anti T. Rosenberg, Arch. Biochim. Biophy.~. 65, 319 (1956).