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[127b] Threonine synthetase from Neurospora
[127b]
T H R E O N I N E SYNTHETASE FROM NEUROSPOR/k
951
in the mixing chamber was achieved by a magnetic stirrer. During the elution the flow rate was maintained at about 1.8 mh/min. Two main protein peaks usually resulted from the chromatography. The threonine dehydratase activity was associated with the second peak. The purification of the enzyme is summarized in Table II. Properties
Specificity. The enzyme also deaminates L-serine, and this appears to be an intrinsic property of the enzyme, since the ratio of activity on threonine and serine remains constant during purification. DL-Allothreonine is also attacked by the enzyme. Activators and Inhibitors. The coenzyme of this enzyme is pyridoxal phosphate. The latter can be dissociated from the apoenzyme by incubation with hydroxylamine or cysteine. The resolved enzyme is inactive, and activity can be restored by incubation with 4 to 8 X 10-' M pyridoxal phosphate after removal of the dissociating agent by dialysis. This reconstitution can be inhibited by the presence of 4-deoxypyridoxine phosphate. Whereas divalent cations are essentially inert in influencing the enzyme activity, monovalent ions have a marked influence. The activity is considerably enhanced by potassium and ammonium ions in comparison to sodium and lithium ions. Threonine is an SH enzyme, like other members of this group, and is inhibited by the usual SH reagents, except iodoacetate. This inhibition is partially reversed by glutathione, mercaptoethanol, and 2,3-dimercaptopropanol. Threonine deamination is also strongly inhibited by serine. Kinetic Properties. The Miehaelis constant for L-threonine was estimated to be 2.9 X 10-~ M.
[ 127b]
T h r e o n i n e S y n t h e t a s e f r o m Neurospora
O-Phosphohomoserine + H~O -~ L-Threonine + phosphate
By
MARTIN FLAVIN
Assay Method
Principle. The method is based on determination of the rate of threonine formation, under standardized incubation conditions, from O-phosphohomoserine. This substrate must be prepared with yeast
952
ENZYMES OF PROTEIN METABOLISM
[t27b]
homoserine kinase. 1,2 The acetaldehyde liberated by periodate oxidation of threonine may be rapidly measured in situ, after reduction of excess periodate with a mercaptan, by the amount of D P N H oxidized in the presence of alcohol dehydrogenase. 8 Reagents
Glycylglycine-K~)H buffer, 0.5 M, pH 7.3. Pyridoxal phosphate, 0.01 M. O-Phosphohomoserine, potassium salt, 0.01 M. Enzyme. An amount containing 0.001 to 0.02 unit is suitable for assay. M a y be diluted in distilled water, if necessary. Potassium phosphate buffer, 1 M , pH 7.5. Sodium metaperiodate, 4 ~ . fl-Mercaptopropionate (Eastman No. 6270), 10~ aqueous solution, by volume, adjusted to pH 6 with KOH. M a y be stored a week frozen without decline in sulfhydryl titer. D P N H , 0.0025 M, pH 7.5. Alcohol dehydrogenase. ~ Sufficient enzyme to bring acetaldehyde reduction to completion in 1 to 3 minutes should be used. In practice, commercial crystalline yeast alcohol dehydrogenase, provided as a suspension in (NH4)2S0~ of 60 mg./ml., specific activity 40,000, ~ has been diluted 1 to 100 for use in 0.1% bovine serum albumin, 0.01 M reduced neutral glutathione, and 0.02 M potassium pyrophosphate, pH 7.5. Procedure. A suitable aliquot of threonine synthetase is added to a stoppered centrifuge tube containing 0.06 ml. of glycylglycine buffer, 0.06 ml. of 0-phosphohomoserine solution, 0.01 ml. of pyridoxal phosphate, and enough water for a final volume of 0.6 ml. After incubation for 30 minutes at 30 ° , the reaction is stopped by placing the tube for 10 minutes in a boiling-water bath, and protein precipitate, if any, is removed by centrifugation. A 0.1- to 0.3-ml. aliquot is pipetted into a 1-ml. volume, 1-cm. light path silica cuvette, followed by 0.1 ml. of phosphate buffer and enough distilled water for a final volume of 1.0 ml. After addition of 0.02 ml. of periodate to the reaction cuvettes, the solutions are well mixed and allowed to react for 30 seconds. Then 0.03 ml. of mercaptopropionate is added, and the mixture is again stirred for 30 seconds. After addition of 0.04 or 0.05 ml. of D P N H , two successive optical density readings are 1y. Watanabe, S. Konishi, and K. Shimura, J. Biochem. 42, 887 (1955). 2y. Watanabe, S. Konishi, and K. Shimura, J. Biochem. 44, 299 (1957). s M. Flavin and C. Slaughter, Anal. Chem. 31, 1983 (1959). 'See Vol. I [79].
[127b]
THREONINE SYNTHETASE FROM NEUROSPORA
953
made at 340 m~, against a water blank. These will show no decline in absorbance, if the reduction of periodate to iodide by the mercaptan has been complete. Finally 0.02 or 0.03 ml. of the diluted alcohol dehydrogenase is added, and the oxidation of DPBIH by acetaldehyde is followed at 340 m~, until two successive readings at half-minute intervals show no further decline in absorbance. It is desirable to run an occasional reference cuvette with a known amount of threonine, since the absorbance change per 0.1 micromole of threonine has varied, with different batches of reagents, between limits of 0.40 and 0.47, after correction for dilution by the alcohol dehydrogenase. Control cuvettes without Ophosphohomoserine are not necessary, except with undialyzed crude extracts. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount catalyzing the formation of 1 micromole of threonine in 1 minute, under the above conditions. Specific activity is expressed as units per milligram of protein. Protein is measured turbidimetrically. 5 The protein sample is added to enough water for a final volume of 5 ml., followed by 2 ml. of 12% trichloroacetic acid, and the turbidity is promptly measured with a No. 42 filter in the Klett colorimeter. One milligram of protein corresponds to 130 klett units. For 4B enzyme fractions (Table II), the procedure is carried out in 1-ml. volume, in Beckman cuvettes at 420 m~. The method becomes nonproportional above 200 Klett units, due to flocculation, and is not very reproducible; however, it can be used with undialyzed extracts, and in the presence of glycylglycine. Application of Assay Method to Crude Extracts. Interfering enzymes result in slightly low assay values with crude extracts of yeast and Neurospora, as indicated by the recovery of units in step 2, Table II. In E. coli extracts, threonine is apparently rapidly destroyed through other reactions, e Preparation of Substra~e ~-Homoserine ~- ATP
Mg++ "O-Phosphohomoserine ~ ADP
(1)
O-Phosphohomoserine has been prepared according to Eq. (1) with yeast homoserine kinase, 5 by modifications of the method of Watanabe and Shimura. r It has not yet been possible to prepare this compound chemically, and kinase activity appears to be low or absent in Neuro5 M. Flavin and C. Slaughter, J. Biol. Chem. 235, 1103 (1960). e G. N. Cohen, M.-L. Hirsch, S. B. Wiesendanger, and M. B. Nisman, Compt, rend. acad. sci. 238, 1746 (1954). Y. Watanabe and K. Shimura, J. Biochem. 43, 283 (1956).
954
ENZYMES OF PROTEIN METABOLISM
[127b]
spora extracts2 The procedure for preparing substrate with yeast enzyme will be described briefly, since it has not yet been investigated extensively. Hamoserine Kinase Assay. The homoserine kinase assay is based on the determination of the amount of nonnucleotide, homoserine-dependent, acid-stable phosphate ester formed.5 Enzyme fractions are assayed in duplicate tubes, one of which contains 40 micromoles of D~-homoserine. In addition, both contain (in micromoles) : Tris-HC1, pH 7.4, 80; ATP, 10; MgS04, 20; NaF, 30; NaCN, 45. Samples are incubated for 30 minutes at 30 ° in 1.5-ml. volume. The reaction is stopped by adding 0.1 ml. of 2 N HCI04, followed by 0.4 ml. of 0.5 N potassium acetate, pH 4.5. Roughly 0.5 ml. of acid-washed Norit A is added with a calibrated scoop, and the stoppered tubes are shaken mechanically for 15 minutes and centrifuged? Aliquots of the filtered supernatants are used for the determination of total phosphate by wet-ashing,D and of acidlabile-~ inorganic phosphate by 15-minute hydrolysis in 1 N HCI at 100°. 1° The difference represents acid-stable phosphate, and the difference between the values for acid-stable phosphate obtained in the presence and in the absence of homoserine represents the amount of O-phosphohomoserine formed. The assay gives erroneously low results in crude extracts; interfering enzymes are inadequately suppressed by cyanide and fluoride. Homoserlne Kinase Purification. A simple salt fractionation of homoserine kinase gives an apparent fifteenfold purification. Fresh baker's yeast is disrupted with liquid nitrogen,11 thawed, and stirred for 2 hours at 0 ° with an equal volume of cold water, after the pH has been adjusted to 8.5 with NH4OH. After centrifugation, 1500 mh of the supernatant, containing 60 g. of protein, is adjusted to pH 7, and 60 ml. of M Tris-HC1, pH 7, is added. Solid (NH,)2SO~ is added, and the fraction precipitating between about 32 and 44% of saturation is redissolved in 300 ml. of 0.04M Tris-HC1, pH 7.4, containing 0.004M glutathione. Preparation of Phosphohomoserine. The fraction (10 g. of protein) is incubated for 90 minutes at 30 ° in 1200-ml. volume, with all components of the incubation mixture at the concentrations described above for the standard assay. After boiling and filtering, the solution contains 8 millimoles of acid-stable phosphate. Small-scale control incubations indicate that about 80% of this is O-phosphohomoserine. s M. Flavin, H. Castro-Mendoza, and S. Ochoa, J. Biol. Chem. 229, 981 (1957). ° M. Flavin, J. Biol. Chem. 210, 771 (1954). ~°C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). nSee Vol. I [81].
[127b]
THREONINE SYNTHETASE FROM NEUROSPORA
955
The filtrate is treated with excess barium acetate and 3 vol. of ethanol at pH 8.2. The precipitate which forms overnight at 0 ° is collected by centrifugation, washed with 75% cold ethanol, and extracted with 0.1 N HCl: The extract is adjusted to pH 4.5 and stirred for 1 hour at 25 ° with sufficient acid-washed Nuchar C to remove all absorbence at 260 m~. Barium-alcohol precipitation is-repeated twice, barium is removed from acid solution with Na2S04, the pH is adjusted to 11 with LiOH, and LisP04 is allowed to precipate overnight at 0 ° after addition of 2 vol. of ethanol. The supernatant is again treated with bariumethanol at pH 8.2, and the precipitate is dried in vacuo. Yield: 1.21 g. of amorphous white powder. Properties o/Phosphohomaserine. This preparation, converted to the potassium salt for use, was about 70% pure by phosphorus and ninhydrin analyses, and threonine synthetase assay. I t contained no 260-m~ absorbing material, orthophosphate (less than 1% of total phosphorus), homoserine, or threonine. Paper chromatography 9 revealed one major component reacting in both ninhydrin and phosphate tests, and two trace contaminants reacting only in one or the other. The contaminants can be removed by gradient elution from Dowex 1 acetate. Yeast homoserine kinase has been shown to be stereospecific for L-homoserine by Watanabe et al., 2 who have further characterized the reaction product by elementary analysis, identification of products of acid hydrolysis before and after treatment with nitrous acid, and preparation of the dinitrophenyl derivative. 7 Purification Procedure Growth conditions and ultraeentrifugation of extract, though they do not much affect initial specific activity, are the most critical factors for the success of the procedure, which has yielded 200- to 500-fold purification in all preparations where these conditions were carefully controlled, s Growth o] Neurospora. Neurospora wild-type 5297 is transferred from stock slant to a 25-cm.-diameter surface of nutrient agar 12 in a Fernbach flask, which is left at 30 ° until profuse aerial growth ensues (to 1 week), and may then be stored at -{-2°. Fifteen liters of basal medium TM in a carboy is inoculated from the Fernbach by shaking it with the sucrose ~ biotin solution which has been autoclaved separately, and incubated for 24" to 30 hours at 25 ° with rapid forced aeration. Yield: 150 to 300 g. wet weight. A carboy has also been used as inoculum for a 300-I. vat. Sixteen-hour growth at 30 ° with stirring and aeration at 250 1./min. yields 7000 g. Yields of 12000 g. may be obtained by longer N. H. Horowitz, J. Biol. Chem. 171, 255 (1947).
956
ENZYMES OF PROTEIN METABOLISM
[127b]
growth periods in the vat, but enzyme from these cell batches is fractionated poorly. Cells are harvested by Biichner filtrationthrough cheesecloth, washed with distilledwater, and frozen in thin "pancakes," which m a y be stored for months. Step 1. Preparation o] Extract. Portions of dry ice are added to a preehilled Waring blendor in a --15 ° room until the powder no longer sticks to the walls. Cakes of frozen Neurospora are then broken by hand into pieces which can be added to the blendor. Brief homogenization yields a fine frozen powder, which may also be stored. The frozen powder TABLE I PREPARATION OF
NeurosporaEXTRACT
Step
Absorbance,, 280/260 m~
Units
Protein, rag.
1A. Original extract lB. Ultracentrifuged 1C. RNAse-treated
0.72 0.95 1.21
7.1 5.0 4.7
1,520 950 930
Specific activity, units/rag. protein 0.0047 0.0053 0.0051
° After dialysis. is thawed, an equal volume of cold water added, and the viscous suspension returned to the blendor at -{-2° for 5 minutes. The pH is adjusted to 8.5 with N NH4OH. The suspension is stirred mechanically 2 hours at 0 ° and then centrifuged, for 20 minutes at 15,000 r.p.m. The supernatant is decanted through cheesecloth and adjusted to pH 7.3. Glycylglycine, pH 7.3, is added for a final concentration of 0.04 M. This crude extract may be frozen or at once centrifuged for 2 hours at 100,000 r.p.m. The supernatant is withdrawn by a hypodermic needle, to avoid surface pellicle. The enzyme remains in the supernatant, though there is some mechanical loss (Table I). The extract is now incubated for 30 minutes at 30 °, after the pH has been adjusted to 7.7, with crystalline pancreatic ribonuclease (3 mg. per 100 ml.). Step ~. Acetone Fraetionation. The extract is now adjusted to pH 8.5, and sufficient M Tris-HC1, pH 8.5, is added for a final concentration of 0.04 M, together with cold distilled water to give a protein concentration of 6.5 mg./ml. The solution is stirred in a large beaker in an ethanol-water bath (adjusted initially to 0 °, by addition of dry ice), while cold acetone is added gradually over 20 minutes to bring the concentration to 45% by volume. Simultaneously the temperature of the bath is gradually lowered to --10% After I0 minutes of further stirring,
[127b]
THREONINE SYNTHETASE FROM NEUROSPORA
957
the precipiate is centrifuged down for 7 minutes at 3000 r.p.m, at --10 °, and discarded. The PR2 International 850a angle head will accommodate six 250-ml. Nalgene plastic bottles (acetone-resistant). Addition of acetone to the supernatant is repeated in a --15 ° bath with lowering of the temperature from --10 ° to --15 °, to bring the concentration to 58~. After centrifugation at --15 °, the supernatant is discarded, and the precipitate is promptly dissolved in cold protein diluent: freshly prepared glycylglycine, pH 7.3, 0.02 M, and glutathione, 0.001 M. ,Step 3. Ammonium Sul]ate Fractionation. The acetone fraction is adjusted to pH 7.3 with N acetic acid, and protein diluent is added for a protein concentration of 8 mg./ml. Solid ammonium sulfate is gradually added during 20 minutes with stirring at 0 °, until the solution is 44~b saturated. After another 20 minutes of stirring, the precipitate is centrifuged down at 0 ° and discarded. The pH (Hydrion) is checked occasionally and maintained near 7.3 with NH~OH. Ammonium sulfate TABLE II SUMMARY OF PURIFICATION PROCEDURE
Step 1C. Extract • 2.
Acetone fraction
3. (NH4)zS04fraction 4A. DEAE eluate 4B. Concentrated eluate
Volume, ml.
Units
430 90 9.1 440 6.4
14.7 17.0 12.7 11.0 5.2
Protein, rag.
Specific activity, units/rag. protein
4000 830 158
0.0037 0.021 0.080
3
1.73
Yield, % 100 116 86 75 35
" From 700 g. of Neuro~ora;see Table I. is now again added to 57% saturation, and the centrifuged precipitate is dissolved in a small volume of diluent (see step 2). This fraction is dialyzed for several hours against 2 1. of distilled water, then overnight against 0.01 M glycylglycine, pH 7.3.
Step I~A. Elution from N,N-DiethylaminoethyIceUulose (DEAE). Eastman DEAE-eellulose, 300 g., is sieved through wire screens with vigorous mechanical shaking for 10 minutes, and the fraction between mesh 100 and 230 is washed successively with water (to remove fines), N NaOH (3 1. for 30 minutes X 2), water, N HC1 (3 1. for 20 minutes), NaOH (as above), and water extensively on a large Biichner funnel. It is then stirred twice for 30 minutes with 1.5 1. of M potassium formate, pH 7.3, washed extensively with water on the Biichner, stirred with 0.01 M glycylglycine, pH 7.3, and resuspended in this buffer; KOH is added until the pH of the supernatant is 7.3.
958
ENZYMES OF PROTEIN METABOLISM
[127b]
A 2 X 15-cm. column is prepared by packing DEAE-eellulose under 5 p.s.i., 13 and the dialyzed ammonium sulfate fraction (150 to 200 mg. of protein) is washed on and eluted with a logarithmic formate gradient 18 at -{-2° overnight, 15-ml. fractions being collected at 1 ml./min. The mixing vessel initially contains 550 ml. of 0.02 M glycylglycine and 0.08 M potassium formate, and the reservoir contains 800 ml. of 0.02 M glyeylglycine and 0.30 M potassium formate. Both are at pH 7.3 and contain pyridoxal phosphate, 7 X 10-5 M. The enzyme is eluted in the fractions between 500 and 800 ml., often some time after the appearance of the last detectable protein. It is located by first assaying 0.16 ml. of every third fraction, four at a time in one assay tube, then every fraction in the appropriate region. Step $B. Concentration o] D E A E Eluates. The fractions containing enzyme are at once concentrated by overnight ultra filtration. 14 Thirty to forty milliliters is placed in each filtration funnel and concentrated to 0.5 to 1 ml. with a vacuum of about 80 ram. Hg, with simultaneous dialysis against several changes of 500 ml. of magnetically stirred 0.02M glycylglycine or potassium dimethylglutarate, pH 7.3, in an 800-ml. beaker inside the desiccator used as a filtration vessel. TM The cellophane bags are extended to near the bottom of the beaker (10 era.), and a perforated a~-inch-thick Lucite plate with a brass vacuum take-off is used in place of a brass plate, to cover the desiccator. The purification procedure is summarized in Table II. An additional 2 5 ~ of the original units could be recovered, with 200-fold purification, from other ultrafiltered fractions. Properties Stability. Both apo- and holoenzyme preparations are stable for months when stored at --15 ° after dialysis, at all stages of purification except perhaps step 4A. The enzyme is precipitated and inactivated at pH 5.2 and below. Distribution. The enzyme is equally active in extracts of baker's yeast and Neurospara ~ and has also been studied in E. coli2 ,15 It is absent from a threonine-requiring Neurospora mutant; its activity in wild-type extract is not affected by inclusion of nL-homoserine or threonine in the culture medium2 Co]actors and Inhibitors. In contrast to yeast ~ and E. coli, ~5 Neurospora enzyme can readily be shown to require added pyridoxal phosphate for aetivity2 Some step 3 fractions are essentially completely dependSee Vol. V [1]. 1,H. A. Sober, F. J. Gutter, M. M. Wyckoff, and E. A. Peterson, J. Am. Chem. ~oe. 75, 756 (1956). mE. H. Wormser and A. B. Pardee, Arch. Bioehem. Biophye. 75, 416 (1958).
[128]
PYRROLINE-5-CARB OXYLATE
REDUCTASE
959
ent. Step 4 yields only holoenzyme when pyridoxal phosphate is included in eluting solutions. The enzymatic reaction is inhibited by hydroxylamine, cyanide, and orthophosphate, and the apoenzyme reaction is inhibited by preincubation with deoxypyridoxine phosphate, but not by dephosphorylated Be analogs2 Equilibrium. The equilibrium constant has not been determined, but the reaction is exergonic and proceeds essentially to completion, with the formation of equimolar amounts of phosphate and threonine. 5 Mechanism o/ the Reaction. Purified enzyme does not decompose O-phosphothreonine. 16 Studies with isotopic oxygen~6 and hydrogen17,1s have revealed that phosphate is removed by a non-hydrolytic, carbanion type of elimination, with C--O cleavage, followed by a shift of conjugated double bonds in the resultant enzyme-bound Schiff base of vinylglycine and pyridoxal phosphate, and addition of water. In the proposed mechanism/7 the coenzyme potentiates all 3 steps, and restricts the sequence and position of addition of water to yield threonine. 1~M. Flavin and T. Kono, J. Biol. Chem. 235, 1109 (1960). 1TM. Flavin and C. Slaughter, Y. Biol. Chem. 235, 1112 (1960). Is M. Flavin and C. Slaughter, Federation Proc. 19, 48 (1960).
[ 128]
Pyrroline-5-carboxylate Reductase
N H B y DAVID M. GRF_,ENBERG
This enzyme has a wide distribution. It occurs in many mammalian tissues, particularly in liver/, 2 brain and testis/ in the hepatopancreas of the terrestial slug, Ariolimax Cali]ornicus brachyphallus/ and in the microorganisms Neurospora crassa, • Escherichia cali, and Aerobacter aerogenes. 5 The enzymes from liver and the slug are quite similar except that the slug enzyme has a more acid pH optimum. This article presents 1A. Meister, A. M. Radhakrishnan, and S. D. Buckley, J. Biol. Chem. 229, 789 (1957). M. E. Smith and D. M. Greenberg, J. Biol. Chem. 226, 317 (1957). s p. Petrakis and D. M. Greenberg, unpublished data (1960). T. Yura and H. J. Vogel, J. Biol. Chem. 234, 335 (1959). H. J. Vogel and B. D. Davis, J. Am. Chem. Soc. 74, 109 (1952).
T H R E O N I N E SYNTHETASE FROM NEUROSPOR/k
951
in the mixing chamber was achieved by a magnetic stirrer. During the elution the flow rate was maintained at about 1.8 mh/min. Two main protein peaks usually resulted from the chromatography. The threonine dehydratase activity was associated with the second peak. The purification of the enzyme is summarized in Table II. Properties
Specificity. The enzyme also deaminates L-serine, and this appears to be an intrinsic property of the enzyme, since the ratio of activity on threonine and serine remains constant during purification. DL-Allothreonine is also attacked by the enzyme. Activators and Inhibitors. The coenzyme of this enzyme is pyridoxal phosphate. The latter can be dissociated from the apoenzyme by incubation with hydroxylamine or cysteine. The resolved enzyme is inactive, and activity can be restored by incubation with 4 to 8 X 10-' M pyridoxal phosphate after removal of the dissociating agent by dialysis. This reconstitution can be inhibited by the presence of 4-deoxypyridoxine phosphate. Whereas divalent cations are essentially inert in influencing the enzyme activity, monovalent ions have a marked influence. The activity is considerably enhanced by potassium and ammonium ions in comparison to sodium and lithium ions. Threonine is an SH enzyme, like other members of this group, and is inhibited by the usual SH reagents, except iodoacetate. This inhibition is partially reversed by glutathione, mercaptoethanol, and 2,3-dimercaptopropanol. Threonine deamination is also strongly inhibited by serine. Kinetic Properties. The Miehaelis constant for L-threonine was estimated to be 2.9 X 10-~ M.
[ 127b]
T h r e o n i n e S y n t h e t a s e f r o m Neurospora
O-Phosphohomoserine + H~O -~ L-Threonine + phosphate
By
MARTIN FLAVIN
Assay Method
Principle. The method is based on determination of the rate of threonine formation, under standardized incubation conditions, from O-phosphohomoserine. This substrate must be prepared with yeast
952
ENZYMES OF PROTEIN METABOLISM
[t27b]
homoserine kinase. 1,2 The acetaldehyde liberated by periodate oxidation of threonine may be rapidly measured in situ, after reduction of excess periodate with a mercaptan, by the amount of D P N H oxidized in the presence of alcohol dehydrogenase. 8 Reagents
Glycylglycine-K~)H buffer, 0.5 M, pH 7.3. Pyridoxal phosphate, 0.01 M. O-Phosphohomoserine, potassium salt, 0.01 M. Enzyme. An amount containing 0.001 to 0.02 unit is suitable for assay. M a y be diluted in distilled water, if necessary. Potassium phosphate buffer, 1 M , pH 7.5. Sodium metaperiodate, 4 ~ . fl-Mercaptopropionate (Eastman No. 6270), 10~ aqueous solution, by volume, adjusted to pH 6 with KOH. M a y be stored a week frozen without decline in sulfhydryl titer. D P N H , 0.0025 M, pH 7.5. Alcohol dehydrogenase. ~ Sufficient enzyme to bring acetaldehyde reduction to completion in 1 to 3 minutes should be used. In practice, commercial crystalline yeast alcohol dehydrogenase, provided as a suspension in (NH4)2S0~ of 60 mg./ml., specific activity 40,000, ~ has been diluted 1 to 100 for use in 0.1% bovine serum albumin, 0.01 M reduced neutral glutathione, and 0.02 M potassium pyrophosphate, pH 7.5. Procedure. A suitable aliquot of threonine synthetase is added to a stoppered centrifuge tube containing 0.06 ml. of glycylglycine buffer, 0.06 ml. of 0-phosphohomoserine solution, 0.01 ml. of pyridoxal phosphate, and enough water for a final volume of 0.6 ml. After incubation for 30 minutes at 30 ° , the reaction is stopped by placing the tube for 10 minutes in a boiling-water bath, and protein precipitate, if any, is removed by centrifugation. A 0.1- to 0.3-ml. aliquot is pipetted into a 1-ml. volume, 1-cm. light path silica cuvette, followed by 0.1 ml. of phosphate buffer and enough distilled water for a final volume of 1.0 ml. After addition of 0.02 ml. of periodate to the reaction cuvettes, the solutions are well mixed and allowed to react for 30 seconds. Then 0.03 ml. of mercaptopropionate is added, and the mixture is again stirred for 30 seconds. After addition of 0.04 or 0.05 ml. of D P N H , two successive optical density readings are 1y. Watanabe, S. Konishi, and K. Shimura, J. Biochem. 42, 887 (1955). 2y. Watanabe, S. Konishi, and K. Shimura, J. Biochem. 44, 299 (1957). s M. Flavin and C. Slaughter, Anal. Chem. 31, 1983 (1959). 'See Vol. I [79].
[127b]
THREONINE SYNTHETASE FROM NEUROSPORA
953
made at 340 m~, against a water blank. These will show no decline in absorbance, if the reduction of periodate to iodide by the mercaptan has been complete. Finally 0.02 or 0.03 ml. of the diluted alcohol dehydrogenase is added, and the oxidation of DPBIH by acetaldehyde is followed at 340 m~, until two successive readings at half-minute intervals show no further decline in absorbance. It is desirable to run an occasional reference cuvette with a known amount of threonine, since the absorbance change per 0.1 micromole of threonine has varied, with different batches of reagents, between limits of 0.40 and 0.47, after correction for dilution by the alcohol dehydrogenase. Control cuvettes without Ophosphohomoserine are not necessary, except with undialyzed crude extracts. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount catalyzing the formation of 1 micromole of threonine in 1 minute, under the above conditions. Specific activity is expressed as units per milligram of protein. Protein is measured turbidimetrically. 5 The protein sample is added to enough water for a final volume of 5 ml., followed by 2 ml. of 12% trichloroacetic acid, and the turbidity is promptly measured with a No. 42 filter in the Klett colorimeter. One milligram of protein corresponds to 130 klett units. For 4B enzyme fractions (Table II), the procedure is carried out in 1-ml. volume, in Beckman cuvettes at 420 m~. The method becomes nonproportional above 200 Klett units, due to flocculation, and is not very reproducible; however, it can be used with undialyzed extracts, and in the presence of glycylglycine. Application of Assay Method to Crude Extracts. Interfering enzymes result in slightly low assay values with crude extracts of yeast and Neurospora, as indicated by the recovery of units in step 2, Table II. In E. coli extracts, threonine is apparently rapidly destroyed through other reactions, e Preparation of Substra~e ~-Homoserine ~- ATP
Mg++ "O-Phosphohomoserine ~ ADP
(1)
O-Phosphohomoserine has been prepared according to Eq. (1) with yeast homoserine kinase, 5 by modifications of the method of Watanabe and Shimura. r It has not yet been possible to prepare this compound chemically, and kinase activity appears to be low or absent in Neuro5 M. Flavin and C. Slaughter, J. Biol. Chem. 235, 1103 (1960). e G. N. Cohen, M.-L. Hirsch, S. B. Wiesendanger, and M. B. Nisman, Compt, rend. acad. sci. 238, 1746 (1954). Y. Watanabe and K. Shimura, J. Biochem. 43, 283 (1956).
954
ENZYMES OF PROTEIN METABOLISM
[127b]
spora extracts2 The procedure for preparing substrate with yeast enzyme will be described briefly, since it has not yet been investigated extensively. Hamoserine Kinase Assay. The homoserine kinase assay is based on the determination of the amount of nonnucleotide, homoserine-dependent, acid-stable phosphate ester formed.5 Enzyme fractions are assayed in duplicate tubes, one of which contains 40 micromoles of D~-homoserine. In addition, both contain (in micromoles) : Tris-HC1, pH 7.4, 80; ATP, 10; MgS04, 20; NaF, 30; NaCN, 45. Samples are incubated for 30 minutes at 30 ° in 1.5-ml. volume. The reaction is stopped by adding 0.1 ml. of 2 N HCI04, followed by 0.4 ml. of 0.5 N potassium acetate, pH 4.5. Roughly 0.5 ml. of acid-washed Norit A is added with a calibrated scoop, and the stoppered tubes are shaken mechanically for 15 minutes and centrifuged? Aliquots of the filtered supernatants are used for the determination of total phosphate by wet-ashing,D and of acidlabile-~ inorganic phosphate by 15-minute hydrolysis in 1 N HCI at 100°. 1° The difference represents acid-stable phosphate, and the difference between the values for acid-stable phosphate obtained in the presence and in the absence of homoserine represents the amount of O-phosphohomoserine formed. The assay gives erroneously low results in crude extracts; interfering enzymes are inadequately suppressed by cyanide and fluoride. Homoserlne Kinase Purification. A simple salt fractionation of homoserine kinase gives an apparent fifteenfold purification. Fresh baker's yeast is disrupted with liquid nitrogen,11 thawed, and stirred for 2 hours at 0 ° with an equal volume of cold water, after the pH has been adjusted to 8.5 with NH4OH. After centrifugation, 1500 mh of the supernatant, containing 60 g. of protein, is adjusted to pH 7, and 60 ml. of M Tris-HC1, pH 7, is added. Solid (NH,)2SO~ is added, and the fraction precipitating between about 32 and 44% of saturation is redissolved in 300 ml. of 0.04M Tris-HC1, pH 7.4, containing 0.004M glutathione. Preparation of Phosphohomoserine. The fraction (10 g. of protein) is incubated for 90 minutes at 30 ° in 1200-ml. volume, with all components of the incubation mixture at the concentrations described above for the standard assay. After boiling and filtering, the solution contains 8 millimoles of acid-stable phosphate. Small-scale control incubations indicate that about 80% of this is O-phosphohomoserine. s M. Flavin, H. Castro-Mendoza, and S. Ochoa, J. Biol. Chem. 229, 981 (1957). ° M. Flavin, J. Biol. Chem. 210, 771 (1954). ~°C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375 (1925). nSee Vol. I [81].
[127b]
THREONINE SYNTHETASE FROM NEUROSPORA
955
The filtrate is treated with excess barium acetate and 3 vol. of ethanol at pH 8.2. The precipitate which forms overnight at 0 ° is collected by centrifugation, washed with 75% cold ethanol, and extracted with 0.1 N HCl: The extract is adjusted to pH 4.5 and stirred for 1 hour at 25 ° with sufficient acid-washed Nuchar C to remove all absorbence at 260 m~. Barium-alcohol precipitation is-repeated twice, barium is removed from acid solution with Na2S04, the pH is adjusted to 11 with LiOH, and LisP04 is allowed to precipate overnight at 0 ° after addition of 2 vol. of ethanol. The supernatant is again treated with bariumethanol at pH 8.2, and the precipitate is dried in vacuo. Yield: 1.21 g. of amorphous white powder. Properties o/Phosphohomaserine. This preparation, converted to the potassium salt for use, was about 70% pure by phosphorus and ninhydrin analyses, and threonine synthetase assay. I t contained no 260-m~ absorbing material, orthophosphate (less than 1% of total phosphorus), homoserine, or threonine. Paper chromatography 9 revealed one major component reacting in both ninhydrin and phosphate tests, and two trace contaminants reacting only in one or the other. The contaminants can be removed by gradient elution from Dowex 1 acetate. Yeast homoserine kinase has been shown to be stereospecific for L-homoserine by Watanabe et al., 2 who have further characterized the reaction product by elementary analysis, identification of products of acid hydrolysis before and after treatment with nitrous acid, and preparation of the dinitrophenyl derivative. 7 Purification Procedure Growth conditions and ultraeentrifugation of extract, though they do not much affect initial specific activity, are the most critical factors for the success of the procedure, which has yielded 200- to 500-fold purification in all preparations where these conditions were carefully controlled, s Growth o] Neurospora. Neurospora wild-type 5297 is transferred from stock slant to a 25-cm.-diameter surface of nutrient agar 12 in a Fernbach flask, which is left at 30 ° until profuse aerial growth ensues (to 1 week), and may then be stored at -{-2°. Fifteen liters of basal medium TM in a carboy is inoculated from the Fernbach by shaking it with the sucrose ~ biotin solution which has been autoclaved separately, and incubated for 24" to 30 hours at 25 ° with rapid forced aeration. Yield: 150 to 300 g. wet weight. A carboy has also been used as inoculum for a 300-I. vat. Sixteen-hour growth at 30 ° with stirring and aeration at 250 1./min. yields 7000 g. Yields of 12000 g. may be obtained by longer N. H. Horowitz, J. Biol. Chem. 171, 255 (1947).
956
ENZYMES OF PROTEIN METABOLISM
[127b]
growth periods in the vat, but enzyme from these cell batches is fractionated poorly. Cells are harvested by Biichner filtrationthrough cheesecloth, washed with distilledwater, and frozen in thin "pancakes," which m a y be stored for months. Step 1. Preparation o] Extract. Portions of dry ice are added to a preehilled Waring blendor in a --15 ° room until the powder no longer sticks to the walls. Cakes of frozen Neurospora are then broken by hand into pieces which can be added to the blendor. Brief homogenization yields a fine frozen powder, which may also be stored. The frozen powder TABLE I PREPARATION OF
NeurosporaEXTRACT
Step
Absorbance,, 280/260 m~
Units
Protein, rag.
1A. Original extract lB. Ultracentrifuged 1C. RNAse-treated
0.72 0.95 1.21
7.1 5.0 4.7
1,520 950 930
Specific activity, units/rag. protein 0.0047 0.0053 0.0051
° After dialysis. is thawed, an equal volume of cold water added, and the viscous suspension returned to the blendor at -{-2° for 5 minutes. The pH is adjusted to 8.5 with N NH4OH. The suspension is stirred mechanically 2 hours at 0 ° and then centrifuged, for 20 minutes at 15,000 r.p.m. The supernatant is decanted through cheesecloth and adjusted to pH 7.3. Glycylglycine, pH 7.3, is added for a final concentration of 0.04 M. This crude extract may be frozen or at once centrifuged for 2 hours at 100,000 r.p.m. The supernatant is withdrawn by a hypodermic needle, to avoid surface pellicle. The enzyme remains in the supernatant, though there is some mechanical loss (Table I). The extract is now incubated for 30 minutes at 30 °, after the pH has been adjusted to 7.7, with crystalline pancreatic ribonuclease (3 mg. per 100 ml.). Step ~. Acetone Fraetionation. The extract is now adjusted to pH 8.5, and sufficient M Tris-HC1, pH 8.5, is added for a final concentration of 0.04 M, together with cold distilled water to give a protein concentration of 6.5 mg./ml. The solution is stirred in a large beaker in an ethanol-water bath (adjusted initially to 0 °, by addition of dry ice), while cold acetone is added gradually over 20 minutes to bring the concentration to 45% by volume. Simultaneously the temperature of the bath is gradually lowered to --10% After I0 minutes of further stirring,
[127b]
THREONINE SYNTHETASE FROM NEUROSPORA
957
the precipiate is centrifuged down for 7 minutes at 3000 r.p.m, at --10 °, and discarded. The PR2 International 850a angle head will accommodate six 250-ml. Nalgene plastic bottles (acetone-resistant). Addition of acetone to the supernatant is repeated in a --15 ° bath with lowering of the temperature from --10 ° to --15 °, to bring the concentration to 58~. After centrifugation at --15 °, the supernatant is discarded, and the precipitate is promptly dissolved in cold protein diluent: freshly prepared glycylglycine, pH 7.3, 0.02 M, and glutathione, 0.001 M. ,Step 3. Ammonium Sul]ate Fractionation. The acetone fraction is adjusted to pH 7.3 with N acetic acid, and protein diluent is added for a protein concentration of 8 mg./ml. Solid ammonium sulfate is gradually added during 20 minutes with stirring at 0 °, until the solution is 44~b saturated. After another 20 minutes of stirring, the precipitate is centrifuged down at 0 ° and discarded. The pH (Hydrion) is checked occasionally and maintained near 7.3 with NH~OH. Ammonium sulfate TABLE II SUMMARY OF PURIFICATION PROCEDURE
Step 1C. Extract • 2.
Acetone fraction
3. (NH4)zS04fraction 4A. DEAE eluate 4B. Concentrated eluate
Volume, ml.
Units
430 90 9.1 440 6.4
14.7 17.0 12.7 11.0 5.2
Protein, rag.
Specific activity, units/rag. protein
4000 830 158
0.0037 0.021 0.080
3
1.73
Yield, % 100 116 86 75 35
" From 700 g. of Neuro~ora;see Table I. is now again added to 57% saturation, and the centrifuged precipitate is dissolved in a small volume of diluent (see step 2). This fraction is dialyzed for several hours against 2 1. of distilled water, then overnight against 0.01 M glycylglycine, pH 7.3.
Step I~A. Elution from N,N-DiethylaminoethyIceUulose (DEAE). Eastman DEAE-eellulose, 300 g., is sieved through wire screens with vigorous mechanical shaking for 10 minutes, and the fraction between mesh 100 and 230 is washed successively with water (to remove fines), N NaOH (3 1. for 30 minutes X 2), water, N HC1 (3 1. for 20 minutes), NaOH (as above), and water extensively on a large Biichner funnel. It is then stirred twice for 30 minutes with 1.5 1. of M potassium formate, pH 7.3, washed extensively with water on the Biichner, stirred with 0.01 M glycylglycine, pH 7.3, and resuspended in this buffer; KOH is added until the pH of the supernatant is 7.3.
958
ENZYMES OF PROTEIN METABOLISM
[127b]
A 2 X 15-cm. column is prepared by packing DEAE-eellulose under 5 p.s.i., 13 and the dialyzed ammonium sulfate fraction (150 to 200 mg. of protein) is washed on and eluted with a logarithmic formate gradient 18 at -{-2° overnight, 15-ml. fractions being collected at 1 ml./min. The mixing vessel initially contains 550 ml. of 0.02 M glycylglycine and 0.08 M potassium formate, and the reservoir contains 800 ml. of 0.02 M glyeylglycine and 0.30 M potassium formate. Both are at pH 7.3 and contain pyridoxal phosphate, 7 X 10-5 M. The enzyme is eluted in the fractions between 500 and 800 ml., often some time after the appearance of the last detectable protein. It is located by first assaying 0.16 ml. of every third fraction, four at a time in one assay tube, then every fraction in the appropriate region. Step $B. Concentration o] D E A E Eluates. The fractions containing enzyme are at once concentrated by overnight ultra filtration. 14 Thirty to forty milliliters is placed in each filtration funnel and concentrated to 0.5 to 1 ml. with a vacuum of about 80 ram. Hg, with simultaneous dialysis against several changes of 500 ml. of magnetically stirred 0.02M glycylglycine or potassium dimethylglutarate, pH 7.3, in an 800-ml. beaker inside the desiccator used as a filtration vessel. TM The cellophane bags are extended to near the bottom of the beaker (10 era.), and a perforated a~-inch-thick Lucite plate with a brass vacuum take-off is used in place of a brass plate, to cover the desiccator. The purification procedure is summarized in Table II. An additional 2 5 ~ of the original units could be recovered, with 200-fold purification, from other ultrafiltered fractions. Properties Stability. Both apo- and holoenzyme preparations are stable for months when stored at --15 ° after dialysis, at all stages of purification except perhaps step 4A. The enzyme is precipitated and inactivated at pH 5.2 and below. Distribution. The enzyme is equally active in extracts of baker's yeast and Neurospara ~ and has also been studied in E. coli2 ,15 It is absent from a threonine-requiring Neurospora mutant; its activity in wild-type extract is not affected by inclusion of nL-homoserine or threonine in the culture medium2 Co]actors and Inhibitors. In contrast to yeast ~ and E. coli, ~5 Neurospora enzyme can readily be shown to require added pyridoxal phosphate for aetivity2 Some step 3 fractions are essentially completely dependSee Vol. V [1]. 1,H. A. Sober, F. J. Gutter, M. M. Wyckoff, and E. A. Peterson, J. Am. Chem. ~oe. 75, 756 (1956). mE. H. Wormser and A. B. Pardee, Arch. Bioehem. Biophye. 75, 416 (1958).
[128]
PYRROLINE-5-CARB OXYLATE
REDUCTASE
959
ent. Step 4 yields only holoenzyme when pyridoxal phosphate is included in eluting solutions. The enzymatic reaction is inhibited by hydroxylamine, cyanide, and orthophosphate, and the apoenzyme reaction is inhibited by preincubation with deoxypyridoxine phosphate, but not by dephosphorylated Be analogs2 Equilibrium. The equilibrium constant has not been determined, but the reaction is exergonic and proceeds essentially to completion, with the formation of equimolar amounts of phosphate and threonine. 5 Mechanism o/ the Reaction. Purified enzyme does not decompose O-phosphothreonine. 16 Studies with isotopic oxygen~6 and hydrogen17,1s have revealed that phosphate is removed by a non-hydrolytic, carbanion type of elimination, with C--O cleavage, followed by a shift of conjugated double bonds in the resultant enzyme-bound Schiff base of vinylglycine and pyridoxal phosphate, and addition of water. In the proposed mechanism/7 the coenzyme potentiates all 3 steps, and restricts the sequence and position of addition of water to yield threonine. 1~M. Flavin and T. Kono, J. Biol. Chem. 235, 1109 (1960). 1TM. Flavin and C. Slaughter, Y. Biol. Chem. 235, 1112 (1960). Is M. Flavin and C. Slaughter, Federation Proc. 19, 48 (1960).
[ 128]
Pyrroline-5-carboxylate Reductase
N H B y DAVID M. GRF_,ENBERG
This enzyme has a wide distribution. It occurs in many mammalian tissues, particularly in liver/, 2 brain and testis/ in the hepatopancreas of the terrestial slug, Ariolimax Cali]ornicus brachyphallus/ and in the microorganisms Neurospora crassa, • Escherichia cali, and Aerobacter aerogenes. 5 The enzymes from liver and the slug are quite similar except that the slug enzyme has a more acid pH optimum. This article presents 1A. Meister, A. M. Radhakrishnan, and S. D. Buckley, J. Biol. Chem. 229, 789 (1957). M. E. Smith and D. M. Greenberg, J. Biol. Chem. 226, 317 (1957). s p. Petrakis and D. M. Greenberg, unpublished data (1960). T. Yura and H. J. Vogel, J. Biol. Chem. 234, 335 (1959). H. J. Vogel and B. D. Davis, J. Am. Chem. Soc. 74, 109 (1952).