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[46] Chorismate to tryptophan (Escherichia coli)—anthranilate synthetase, PR transferase, PRA isomerase, InGP synthetase, tryptophan synthetase
[46]
CHORISMATE
[46] C h o r i s m a t e
TRYPTOPHAN
to Tryptophan
Anthranilate PRA
TO
365
(Escherichia coli)-
Synthetase, PR Transferase,
Isomerase, InGP
Synthetase,
Tryptophan
Synthetase 1
By THOMAS
E. CREIGHTON a n d CHARLES YANOFSKY
Introduction The biosynthesis of tryptophan from chorismate in microorganisms is now believed to proceed as outlined in Fig. 1. Some of the characteristics of the enzymes and intermediates involved have been covered in previous volumes of this series (V [107]; VI [86]). The present article will not duplicate material in the previous volumes.
Preparation of Intermediates N-5'-Phosphoribosyl Anthranilate (PRA) This very unstable intermediate may be produced synthetically by mixing at room temperature equal volumes of 1.0 M ribose-5-P (sodium salt) in water and 1.0 M anthranilic acid dissolved in 95% ethanol. 2 Upon mixing the two reagents, PRA is formed very rapidly. However, formation of the Amadori rearrangement product, CdRP, proceeds at the same time, although at a slower rate. After 5 minutes' incubation at room temperature, a yield of PRA of at least 30% may be obtained, with no significant formation of CdRP. To stop the reaction after t h e 5 minutes, dilute the mixture with approximately 200 volumes of cold 0.1 M triethanolamine-HCl buffer, p H 8.6, and keep in ice. The PRA is stable for several hours under these conditions. However, after 2 hours at 37 ° approximately 90% of the PRA will have disappeared. It is therefore suggested that the PRA be synthesized immediately before use. PRA prepared in this manner is apparently contaminated with unreacted ribose f-phosphate and anthranilic acid and with any products of side reactions that may occur, in addition to the low levels of CdRP formed. Attempts to remove the contaminants have been unsuccessful due to the concomitant extensive loss of PRA. However, the PRA preWor related articles, see this volume [45], [47], [48], [48a]¢and [49]. 2T. E. Creighton, J. Biol. Chem. 243, 5605 (1968).
366 ....
AROMATIC AMINO ACIDS CO2H
[46]
CO2H
~O--C--CO~H OH
Anthranilicacid
Chorismicacid
C02H t
OH OH
O OH OH I
I
'~ I"
C
II"°-c--c"--CH--C"'°-~
I
N--CH--CH--CH-- CH--CH20-(P) N-5'-Phosphoribosyl-anthranilate (PR-anthranilate)
H
- H20 /
I -(O-Carboxyphenylamino)- 1deoxyribulose-5-P (CdRP)
OH OH
I
~
I
CH--CH--CH20-(~
+ g-scrine - glyceraldehyde-3-P A+B
~
CH2--CH--CChH
H
H
Indoleglycerol-P (InGP) ~ " N A
L-Tryptophan
-glyceraldehyde-3 - P ' ~
~
+
L-~rine
tt Indole
FIG. 1. T h e tryptophan biosynthetic pathway. T h e enzymatic activities catalyzing the various reactions are A, the tryptophan synthetase a subunit, and B, the fl, subunit; C, indole-3-glycerol phosphate synthetase and PR-anthranilate isomerase; D, anthranilatePR-transferase; E, anthranilic acid synthetase. This lettering system is consistent with the structural gene designations in Escherichia coll.
pared as described above has been found to be of direct use in enzyme assays. 1-(o-Carboxyphenylamino)-l-deoxyribulose 5-Phosphate (CdRP) The chemical synthesis of CdRP was presented in Vol. VI [86]. However, a slightly m o d i f e d procedure has been found which gives significantly greater yields: Add 1.5 millimoles of anthranilic acid dissolved sj. A. DeMoss, unpublished results.
[46]
CHORISMATE TO TRYPTOPHAN
367
in 2.5 ml of methanol to 0.5 millimole of ribose 5-phosphate (sodium salt) dissolved in 3.7 ml of water. Heat this mixture to 70 ° in a water bath and add 0.63 ml of ethyl malonate. Continue heating at 70'--80° for 30 minutes and then chill and store at 4 ° for several hours. Add 0.5 ml of water and remove the yellow-orange oil that has collected in the bottom of the reaction vessel. To the remainder add 1.0 N HCI in a dropwise manner to lower the p H to about 5. Extract this with three 7-ml portions of ethyl acetate and discard the extracts. Remove the remaining traces of ethyl acetate by bubbling nitrogen through the solution Until no ethyl acetate odor is detectable. The resulting preparation may be stored for weeks at --15 °. The CdRP will slowly break down, but the resulting anthranilic acid may be removed by repeating the extraction process. The initial yield of CdRP should be about 40%, based upon the limiting quantity of ribose-5-P. The resulting CdRP preparation is contaminated with unreacted ribose-5-P and side products. Several attempts to purify the CdRP have been unsuccessful. However, the above preparation is suitable for routine use in enzyme assays. A method reportedly capable of yielding the barium salt of CdRP at 95% purity has been reported from another laboratory. 4 Some of the properties of this intermediate have been described in Vol. VI [86]. Indole-3-glycerol Phosphate (InGP) The enzymatic synthesis of InGP from anthranilic acid and PRPP (Vol. VI [86]) or from indole and glucose 1,6-diphosphate plus aldolase 5 has been described previously. A more convenient method has been developed; 6 it involves enzymatic synthesis from anthranilic acid and ribose 5-phosphate via PRA and CdRP. To 1 ml of a 0.25 M solution'of ribose 5-phosphate at pH 6, an equimolar amount of solid anthranilic acid is added and the mixture is heated at 70-80 ° for a few minutes to dissolve all the anthranilic acid. The reaction mixture is immediately diluted 10-fold with 0.1 M TrisHCI buffer p H 7.8, brought to 37 °, and an appropriate amount of InGP synthetase (partially purified enzyme) is added. The amount of enzyme employed should be sufficient to convert all the PRA to InGP in approximately 60 minutes. The extent of the reaction can be followed readily by measuring the increase in absorbance at 280 m/~ of suitably diluted aliquots of the mixture. Upon completion of the reaction, the InGP is separated from the relatively small amount of protein in the re4C. H. Doy, Nature 211,736 (1966). 5j. K. Hardman and C. Yanofsky, J. Biol. Chem. 240, 725 (1965). 8K. Herrmann, unpublished results.
368
AROMATIC AMINO ACIDS
[46]
action mixture by passing the solution through a small column (1 cm × 5 cm) of Darco charcoal. To adsorb the InGP to the charcoal, the reaction mixture must be adjusted to pH 6.5. InGP is eluted from the charcoal with 40% ethanol containing 1 ml of conc. NH4OH per 100 ml. After elution the solvent is removed by flash evaporation, and the InGP is dissolved in 0.1 M Tris-HCl buffer pH 7.8. T h e overall yield is about 70%. InGP is quite stable and may be kept for months a t - 1 5 °. Some of the properties of this intermediate have been described (Vol. VI [86]).
Enzyme Assays Anthranilate Synthetase Chorismate + glutamine~ anthranilate + glutamate+ pyruvate Anthranilate synthetase activity is most conveniently measured by following anthranilate formation fluorometricallyY T h e reaction mixture contains the following in a total volume of 2.0 ml: 0.4 micromole of chorismic acid; 20 micromoles of ~.-glutamine; 8.0 micromoles of MgSO4; 20 micromoles of potassium phosphate buffer, pH 7.6, 2 micromoles of mercaptoethanol; enzyme. The reaction is initiated by the addition of enzyme to a mixture of the other components prewarmed to 37 °. T h e increase in fluorescence is followed using an activation wavelength of 314 m g and an emission wavelength of 390 m~. T h e assay can be quantitated by preparing standard curves with known concentrations of anthranilate. T h e presence of PR transferase in the enzyme preparation usually does not present any difficulties because anthranilate utilization is negligible in the absence of PRPP.
Anthranilate-PRPP Phosphoribosyltransferase (PR Transferase) Anthranilate + PRPP ---, PRA + H20 + PPt
A quantitative assay for this enzymatic activity has been presented s which depends upon the ability of the test extract to supplement a cellfree extract of a mutant strain of Neurospora crassa (tryp-4 mutant) lacking only PR transferase activity in catalyzing the overall conversion of anthranilate to InGP. Any enzyme preparation which has sufficient PRA isomerase and InGP synthetase activities, but lacks PR transferase activity, can be used instead of the N. crassa tryp-4 extract. The reaction 7M. T. Gibson and F~ Gibson, Biochem.J. 90, 248 (1964). 8j. Wegman and J. A. DeMoss, J. Biol. Chem. 240, 3781 (1965).
[46]
CHORISMATE TO TRYPTOPHAN
369
mixture contains the following in a total volume of 1.0 ml: 100 micromoles o f potassium phosphate buffer, pH 8.0; 0.3 micromole of anthranilate; 4.0 micromoles of MgSO4; 0.6 micromole of PRPP; excess N. crassa tryp-4 extract; enzyme. The above mixture is incubated at 37 °. At zero time and at the end of the incubation period (usually 15 minutes), 0.4-ml aliquots of the mixture are assayed for InGP, using the periodate method previously described (Vol. V [ 107]). A second assay method has been employed 9 which is somewhat less quantitative than the above method but considerably more convenient. The PR transferase reaction is followed by measuring the decrease in fluorescence that accompanies anthranilate disappearance. Since PRA, the product of the reaction, is slightly fluorescent, while CdRP, the next intermediate, is not fluorescent, the actual decrease in fluorescence will d e p e n d to some extent upon the ability of the enzyme preparation being assayed to convert the PRA to CdRP. However, this has not been found to be a serious obstacle to the use of this convenient assay. The reaction mixture contains the following in a final volume of 2.0 ml: 0.01 micromole of anthranilate; 0.5 micromole of PRPP; 4.0 micromoles of MgSO4; 50 micromoles of Tris-HC1 buffer, pH 7.8; enzyme. The reaction is followed at 37 ° by observing the decrease in fluorescence at 390 m/z (activation at 314 m/z) as compared to a control without added enzyme.
N-5'-Phosphoribosylanthranilate Isomerase (PRA Isomerase) PRA --* CdRP
PRA isomerase activity is best assayed by following InGP formation spectrophotometrically in the presence of excess InGP synthetase. 2 T h e ultraviolet spectra of CdRP and PRA are very similar, while differing considerably from that of InGP. 1° T h e source of excess InGP synthetase has been the enzyme purified from mutant 9830 of Escherichia coli, a strain which lacks PRA isomerase activity. A freshly prepared solution of PRA (see above) may be diluted a further 10-fold with 0.10 M triethanolamine-HCl~ buffer, pH 8.6; it is then warmed to 37 ° and placed in a spectrophotometer cuvette. While monitoring the absorbance at 280 m/z, the excess InGP synthetase is added. The absorbance may increase rapidly to a new level, due to conversion of any contaminating CdRP to InGP. When the absorbance reaches a constant value again, the enzyme preparation to be assayed is added. The rate of increase in absorbance that is thus observed is pro9j. Ito and I. P. Crawford, Genetics 52, 1303 (1965). leT. E. Creighton and C. Yanofsky, J; Biol. Chem. 241, 4616 (1966).
370
AROMATIC AMINO ACIDS
[46]
portional to the amount of PRA isomerase activity added. Under these conditions the conversion of 0.10 mM PRA to CdRP causes an increase in absorbance at 280 m/~ of 0.43. Alternatively, InGP formation may be measured with the periodate assay (Vol. V [107]). Other assays for this activity have been described which make use of PR transferase to enzymatically synthesize PRA. T h e disappearance of the PRA n ' n or its conversion to InGP TM is then followed.
Indole-3-glycerol-P Synthetase (InGP Synthetase) CdRP --~ InGP + CO~+ H20 The rate of increase in absorbance at 280 mt~ upon conversion of CdRP to InGP is used routinely to measure InGP synthetase activity,t° Enzyme is normally added to a 0.1 mM solution of CdRP (see above) in 0.10 M Tris-HCl buffer, pH 7.8, at 37 °. T h e complete conversion of 0.10 mM CdRP to InGP results in an increase in absorbance at 280 m~ of 0.448 under these conditions. With crude extracts containing very low levels of activity, a spurious slow increase in absorbance is often found which is not dependent upon the presence of CdRP. With such extracts it is best to use the periodate assay for InGP formation (Vol. V [107]).
Tryptophan Synthetase InGP + L-serine~ L-tryptophan+ glyceraldehyde-3-P InGP + H~O ~=~indole + glyceraldehyde-3-P Indole + L-serine---, L-tryptophan+ HzO The standard enzyme assays for tryptophan synthetase have been described in Vol. V [107]. However, a spectrophotometric assay procedure convenient for kinetic studies has been developed. 14a5 T h e glyceraldehyde-3-P formed in the conversion of InGP to indole or tryptophan is measured by coupling with glyceraldehyde-3-P dehydrogenase. The complete reaction mixture for the conversion of InGP to tryptophan contains the following in a final volume of 1.0 ml: 0.1 micromole of InGP; 60 micromoles of VL-serine; 0.04 micromole of pyridoxalP; 180 micromoles of NaCi; 12 micromoles of Na2HAsO4; 1.0 micromole of NAD; 100 micromoles of Tris-HCl buffer, pH 7.8; excess crystalline nj. A. DeMoss,R. W. Jackson, and J. H. Chalmers,Jr., Genetics 56, 413 (1967). nO. H. Smith, Genetics 57, 95 (1967). 13j. A. DeMoss,Biochem. Biophys. Res. Commun. 18, 850 (1965). 14I. P. Crawford,Biochira. Biophys. Acta 45, 405 (1960). nT. E. Creighton and C. Yanofsky,J. Biol. Chem. 241,980 (1966).
[46]
CHORISMATE TO TRYPTOPHAN
371
glyceraldehyde-3-P dehydrogenase; enzyme. For the enzymatic conversion of InGP to indole, the serine, pyridoxal-P, and NaCI are omitted. The enzyme is added to start the reaction, and NAD reduction is followed spectrophotometrically at 340 m/.~. A very rapid qualitative spot test for indole to tryptophan activity has been devised 16which is extremely useful during preparative procedures. The substrate mixture is the same as that of the normal indole to tryptophan assay (Vol. V [107]) and consists of the following mixture: 0.4 mM indole; 0.04 mM pyridoxal-P; 60 mM DL-serine; 180 mM NaCI; 0.1 M Tris-HCl, pH 7.8. Aliquots (0.3 ml) o f this mixture are placed into the depression of a polyvinylchloride spot test plate. Approximately one drop o f the preparation to be tested is added to each depression, and the plate is incubated for 10 minutes by floating it in a water bath at 37 °. Ehrlich's indole reagent (3-4 drops) is then added to each depression and the presence or absence of indole noted. The conditions of this assay, such as the relative proportion of reaction mixture and enzyme and the length of the incubation period, may be varied to produce a spot assay of the desired sensitivity.
Preparation of Enzymes Growth of Bacteria Bacteria are grown in single-strength Vogel and Bonnet 17 minimal medium supplemented with 0.05% acid-hydrolyzed casein, 2.5/.~g/ml indole, and 0.5% glucose. When large cultures are grown in a Biogen (American Sterilizer Co.) or Fermacell (New Brunswick Scientific Co.) fermenter, the indole supplement can be increased to 5/zg/ml and derepression will still be observed. The cells are generally harvested after 16-18 hours of growth at 37 ° and disrupted by sonic oscillation. Sonic extracts are employed in the purification procedures without prior centrifugation.
Anthranilate Synthetase-PR Transferase These first two enzymes of tryptophan biosynthesis occur in E. coil as a complex of two proteins, tentatively designated as components I and II. is Component I is the product of the E gene and has an estimated s20.w of 4.3 S. This protein, when not in the complex, has anthranilate IsU. Henning, D. R. Helinski, F. C. Chao, and C. Yanofsky,J.Biol. Chem. 237, 1523 (1962). 17H. J. Vogel and D. M. Bonner,J. Biol. Chem. 218, 97 (1956); see this volume [1], Section 3. lsj. lto and C. Yanofsky,J. Biol. Chem. 241, 4112 (1966); J. Ito, E. C. Cox, and C. Yanofsky, J. Bacteriol. 97, 723 (1969); J. Ito and C. Yanofsky, ibid., p. 734.
372
AROMATIC AMINO ACIDS
[46]
synthetase activity only in the presence of high concentrations of N H + ion. Component II is the product of the D gene, with an s20,wof 4.4 S. It possesses full PR transferase activity whether or not it is in the complex. When mixed together or when both are synthesized in vivo, the two components form a complex of unknown structure with an szo,w of 7.5-9 S. This complex has full anthranilate synthetase and PR transferase activities. Components I and II may be obtained individually in cell-free extracts of mutant strains containing nonsense mutations in the D and E genes, respectively. TM A protein containing anthranilate synthetase activity has been purified from E. coli and its catalytic properties studied, TM but its relationship to components I and II is not clear. T h e feedback inhibition of anthranilate synthetase activity by tryptophan has been studied by several investigators, xs-2x The two-component nature of the enzyme from Salmonella typhimurium 22 and Aerobacter aerogenes z° has also been demonstrated. The anthranilate synthase from Neurospora crassa has been purified 90-fold z3 and the transferase 64-fold. s P R A Isomerase-InGP
Synthetase
In E. coil these two activities are catalyzed by a single polypeptide chain of approximately 45,000 molecular weight, x° T h e enzyme is best isolated from mutant trp A2, a particularly good source being a strain (trp A2/F' trp A2) with the mutation on both the chromosome and an F' episome. The enzyme is most easily detected by assay of its InGP synthetase activity. In the following isolation procedure, all operations are carried out at 0-4 ° . Unless otherwise stated, all potassium phosphate buffers are at p H 7.0 and contain 1.0 mM EDTA and 0.1 mM dithiothreitol. T h e cellfree extract is supplemented with EDTA and dithiothreitol to 1 raM, and anthranilic acid 24 (dissolved in the minimum of 95% ethanol) to 10mM. Step 1. Treatment with Streptomycin Sulfate. To each 100 ml of crude extract is added 5 ml of a 20% solution of streptomycin sulfate. T h e l q ' . I. Baker and I. P. Crawford,J. Biol. Chem. 242, 5577 (1967). 2°A. F. Egan and F. Gibson, Biochim. Biophys. Acta 130, 276 (1966); see also this volume [47]. SIR. L. Somerville and E. Eiford, Biochem. Biophys. Res. Commun. 28, 437 (1967). S2R. H. Bauerle and P. Margolin, C0/d spring Harbor Symp. Quant. Biol. 31,203 (1966). uj. A. DeMoss and J. Wegman, Proc. Natl. Acad. Sci. U.S. 54, 241 (1965); see also this volume [481. ~Preliminary experiments indicated that anthranilic acid protected the enzyme during the acetic acid step to follow, but this point has not been investigated further.
[46]
CHORISMATE TO TRYPTOPHAN
373
mixture is stirred for 15 minutes and then centrifuged for 50 minutes at 16,000 g. The precipitate is discarded. Step 2. Treatment with Acetic Acid. To the supernatant is added slowly, with stirring, 1.0 M sodium acetate buffer, pH 3.2, to lower the pH of the extract to 4.90--+ 0.05 (determined with a glass electrode after 10-fold dilution with water). Approximately 9-10 ml of acetate buffer is required for 100 ml of streptomycin sulfate supernatant. Immediately the suspension is centrifuged at 16,000 g for 25 minutes. The precipitate is discarded. As soon as possible after centrifugation, the pH of the supernatant is raised to 7.0 -+ 0.2 (narrow range pH paper, pH 6.0-8.0) by addition of concentrated NH4OH. Step 3. Ammonium Sulfate Precipitation. To each 100 ml of acetic acid supernatant slowly add 28.0 g of solid ammonium sulfate with stirring. After further stirring for 15 minutes, centrifuge the suspension for 25 minutes at 16,000 g. The precipitate is suspended in a small volume of 0.1 M potassium phosphate buffer; the supernatant is discarded. Step 4. Sephadex G-IO0 Filtration. After dialysis of the suspended ammonium sulfate precipitate against 0.1 M potassium phosphate buffer for 3-6 hours, the slightly turbid and brown solution is applied to the top of a Sephadex G-100 column (5 × 60 cm) equilibrated wih 0.1 M potassium phosphate buffer. The protein is eluted with the same buffer at the maximum flow rate (80 ml per hour, 12-minute fractions). Those fractions containing greater than 1000 units 25 of InGP synthetase activity per milliliter (these fractions usually comprise 130-170 ml) are combined and the protein precipitated by the addition of 35 g of solid ammonium sulfate for each 100 ml of solution. Collect the resulting precipitate by centrifugation at 16,000 g for 25 minutes. The precipitate is suspended in a small quantity of 0.1 M potassium phosphate buffer; the supernatant is discarded. Step 5. DEAE-Sephadex Chromatography. After dialysis of the suspended precipitate from step 4 against 0.1 M potassium phosphate buffer for 12-16 hours, the clear yellow solution is applied to a column (3 × 60 cm) of DEAE-Sephadex A50 equilibrated with the same buffer. The protein is washed on with a small amount (50-100 ml) of buffer and eluted with a 2000-ml linear gradient of 0.1-0.7 M potassium phosphate buffer. The flow rate should be approximately 80 ml per hour; 15-minute fractions are collected. Those fractions containing greater than 1000 units 25 of InGP synthetase activity per milliliter are pooled, and the protein is precipitated by addition of solid ammonium sulfate (40 g/100 ml). These fractions generally comprise 200-250 ml. After collection of the ~One unit o f InGP synthetase activity is defined as that activity which causes the formation o f 0.1 micromole o f InGP in 20 minutes at 37*.
374
AROMATIC AMINO ACreS
[46]
precipitate by centrifugation at 16;000 g for 25 minutes, the precipitate is suspended in a small volume of 0.1 M KC1 containing 0.01 M potassium phosphate buffer. Step 6. Hydroxytapatite Chromatography. The suspended precipitate from step 5 is dialyzed 12-16 hours against 0.1 M KC1 in 0.01 M potassium phosphate buffer (without EDTA). The clear, slightly yellow solution is then placed on a column (4.5 × 20 cm) of hydroxylapatite (BioGel HT, Bio-Rad) equilibrated with the same buffer. The protein is washed on with approximately 100 ml of buffer and eluted with a 1000-ml linear gradient of 0.01-0.08 M potassium phosphate buffer in 0.1 M KCI (without EDTA). The flow rate should be approximately 20 ml per hour, and 1-hour fractions should be collected. The InGP synthetase activity emerges as the first major peak of protein. The active fractions may be stored directly at--15 ° for several weeks without appreciable losses of activity. The purification procedure is summarized in Table I. The purified enzyme is homogeneous upon sedimentation and electrophoresis. It may be readily crystallized by the following procedure, with no change in specific activity. Raise the pH of a saturated solution of ammonium sulfate to 7.0 by addition of concentrated NH4OH. Add this solution dropwise to a concentrated (greater than I0 mg/ml) soluTABLE
Ia
PURIFICATION OF I N G P SYNTHETASE OF Escherichia coli ~
Fraction C r u d e extract Streptomycin sulfate supernatant Acetic acid s u p e r n a t a n t Dialyzed a m m o n i u m sulfate precipitate S e p h a d e x G-100 eluate e DEAE-Sephadex eluate e Hydroxylapatite eluate t
Total volume (ml)
Total enzyme (units c × 10 -6)
Total protein (g)
865 760
2.85 2.62
79.5 _ d
640 71
1.90 1.86
_ a 8.52
28 21
1.35 1.30
14
0.48
Specific activity
Yield (%)
Purification
92
--
_ 218
67 65
5.9
2.44 1.12
554 1160
48 45
15 31.5
0.66
1380
23
37.5
35.9 _
a T a k e n f r o m T. E. Creighton a n d C. Yanofsky,J. Biol. Chem. 241, 4616 (1966). OTryptophan a n x o t r o p h with the A2 point mutation on both the c h r o m o s o m e a n d an F' episome. c O n e unit o f InGP synthetase activity is defined as that activity which causes the formation o f 0.1 micromole o f InGP in 20 minutes at 37*. nNot d e t e r m i n e d because o f the p r e s e n c e o f streptomycin sulfate. e Dialyzed a m m o n i u m sulfate precipitate.
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CHORISMATE TO TRYPTOPHAN
375
tion of the purified enzyme preparation until a distinct turbidity is observed. U p o n storage of this preparation at 4 ° for several days, crystallization should be nearly complete. T h e hydroxylapatite step may be omitted and InGP synthetase crystallized directly from the final fraction of step 5 (with the addition of seed crystals). T h e crystals so obtained generally have the same specific activity as the crystalline material obtained after the hydroxylapatite step. ~ T h e purified enzyme is very susceptible to oxidation and heavy metals, but the constant presence of dithiothreitol or mercaptoethanol will alleviate this problem. T h e amino acid composition has been determined, and some of its physical properties have been studied. TM T h e Kr~ for CdRP in the InGP synthetase reaction is approximately 5 × 10-~ M. T h e enzyme from N. crassa has been purified approximately 83-fold, and some of its properties have been examined, s T r y p t o p h a n Synthetase 2aa T h e tryptophan synthetase of E. coli is composed of two protein subunits, the a and//2 subunits (formerly designated as the A and B proteins, respectively), in the form of an a2 /32 complex. 27 T h e catalytic properties of the subunits and the complex have been described in Vol. V [107]. Subunit
T h e ot subunit of E. coli tryptophan synthetase is a single polypeptide chain 267 residues in length, with a molecular weight of 28,700. 28 Pure preparations of the ot subunit can catalyze the InGP to indole + 3-phosphoglyceraldehyde reaction, but the reaction rate is only about 1% of the rate observed with the c~2/32 complex. T h c~ subunit is most conveniently assayed in the conversion of indole to tryptophan in the presence of an excess of f12 subunits (Vol. V [107]). Assay procedures for the InGP reactions are described earlier in this article and in Vol. V [107]. Mutant B8 is the best source of the c~ subunit. T h e following purification procedure is a modification of the procedure described by H e n n i n g et al. TM All operations are carried out at 2-4 °. ~SE. Orias, unpublished results. ~saEC 4.2.1.90; L-serine hydro-lyase (adding indole). 27M. Goldberg, T. E. Creighton, R. L. Baldwin, and C. Yanofsky,J. Mol. Biol. 21, 71 (1966). 2sc. Yanofsky, G. R. Drapeau, J. R. Guest, and B. C. Carlton, Proc. Natl. Acad. Sci. U.S. 57, 296 (1967).
376
AROMATIC AMINO ACIDS
[46]
Step I. Treatment with Manganous Chloride. For each 100 ml of crude extract, 7 ml of 1 M MnCI, is added slowly with stirring. T h e mixture is stirred for an additional 10 minutes, and the suspension is then centrifuged for 30 minutes at 15,000 g. The precipitate is suspended in cold water (6-8 ml per 100 ml of starting volume), agitated in a Waring blendor, and centrifuged. The wash is combined with the supernatant solution. Step 2. Acid Treatment. To the supernatant from step 1, 1 M sodium acetate buffer, pH 3.2, is added slowly, with stirring, until the pH is lowered to about 4 (determined with a bromocresol green solution on 2-drop aliquots in the depressions of a spot plate). Approximately 6-8 ml of acetate buffer are required for each 100 ml of supernatant. T h e suspension is centrifuged immediately at 15,000 g for 15 minutes, and the precipitate is rapidly washed with a small amount of cold water as described above. Step 3. Ammonium Sulfate Precipitation. To each 100 ml of the combined supernatants from step 2, 31 g of solid ammonium sulfate is added slowly with stirring. After an additional 15 minutes of stirring the precipitate is collected by centrifugation for 15 minutes at 15,000 g. T h e precipitate is suspended in 3 ml of 1 M Tris-HCl buffer, pH 7.8, for each 100 ml of starting extract. This fraction can be stored a t - 1 5 ° indefinitely without loss of activity. Step 4. Sephadex G-IO0 Fractionation. The suspension from step 3 is dialyzed against 2 liters of 0.05 M potassium phosphate buffer, pH 7.0, for 2 hours. The dialyzing solution is replaced, and dialysis is continued for an additional 2 hours. The slightly turbid solution is then applied to a 5.5 x 50 cm column of Sephadex G-100 equilibrated with 0.05 M potassium phosphate buffer, pH 7.0. T h e same buffer is used for elution, and 20-ml fractions are collected. T h e fractions are tested for a subunit activity by the spot test procedure described earlier in this article (the spot test mixture is supplemented with a suitable excess of a /32 subunit preparation). The active fractions are combined, and solid ammonium sulfate is slowly added (44 g for each 100 ml of combined fractions) with stirring. After additional stirring for 15 minutes the precipitate is collected by centrifugation at 15,000 g for 15 minutes. The precipitate is dissolved in 0.1 M Tris-HCl buffer, pH 7.8, using approximately 1 ml of buffer for each 100 ml of starting extract. Step 5. DEAE-Cellulose Chromatography. DEAE-Selectacel (0.9 meq/g) equilibrated with 0.01 M potassium phosphate buffer, pH 7.0, is poured into a column (2.5 x 125 cm) to a height o f 110 cm of cellulose, after settling. The dissolved precipitate from step 4 is dialyzed against 1 liter of 0.01 M potassium phosphate buffer, pH 7.0, for 3 hours. T h e dialyz-
[46]
CHORISMATE TO TRYPTOPHAN
377
ing fluid is replaced and dialysis is continued for an additiDnal 3 hours. T h e sample is occasionally,.sua:l~d and requir.es clarification by centrifugation, T h e p r o t e i n sample is applied to the DEAE-cellulose column, and the a subunit is eluted with a linear gradient of potassium phosphate b u f f e r , pH 7.0. (The mixing flask contains 600 ml of 0.01 M phosphate while the second flask contains 600 ml of 0.3 M phosphate buffer.) A flow rate of 30-40 ml/hour is employed, and fractions of 20 ml are collected. T h e a subunit elutes at about 700 ml. All fractions which give a positive spot test for enzymatic activity are assayed quantitatively for.protein-and ot subunit activity. A typical purification procedure is summarized in Table II. TABLE II PURIFICATION OF THE Ot SUBUNIT OF Escherichia coli a TRYPTOPHAN SYNTHETASE
Fraction
Total volume (ml)
Total enzyme (units ~ × 10-5)
Crude extract MnCl~ supernatant Dialyzed a m m o n i u m sulfate fraction Combined G-100 fractions DEAE-cellulose peak tubes
700 660 80 15 60
33.5 30 21 14 8
Total Specific protein activity Yield (g) (units/rag) (%) 36 32 5.1 0.94 0.16
93 94 410 1500 5000
90 63 42 24
aExtract,of tryptophan auxotroph B8. bActivity measured in the standard indole to tryptophan assay (Voi. V [107]) in the presence of a 3-fold excess of f12 subunit activity. One unit of activity is defined as that which causes the disappearance o f 0.1 micromole of indole in 20 minutes at 37 ° in the standard enzyme assay.
Step 6. Crystallization. T h e high specific activity fractions from step 5 are combined, and the protein is precipitated by adding a m m o n i u m sulfate (4.4 g/10 ml) slowly, with stirring. T h e suspension is stirred for an additional 15 minutes and the precipitate is collected by centrifugation at 20,000 g for 10 minutes. T h e precipitate is dissolved in cold water to give a protein concentration of 2-4%. If slight turbidity is evident, the insoluble material is removed by a brief centrifugation. T o the clear protein solution a saturated a m m o n i u m sulfate solution (adjusted to pH 6.0-6.8 with 2 N ammonia, pH measurements made on a 50-fold dilution with water) is added dropwise with stirring until a faint but distinct turbidity appears. A small a m o u n t of seed crystals is added, and the mixture is stored at 2-4 °. Crystals are evident within 24 hours. Crystallization is generally observed ,without seed crystals in 2-4 days. Crystalline suspensions retain full activity for at least five years.
378
AROMATIC AMINO ACIDS
[46]
[3~ Subunit The/3~ subunit of E. coli is a dimer with a molecular weight of approximately 100,000Y a9 Two moles of pyridoxal phosphate are bound per mole of the dimer; the cofactor is essential for the intrinsic enzymatic activity of the subunit in converting indole plus serine to tryptophan 3° and for the overall conversion of InGP to tryptophan with the a subunit. The/3~ subunit is most readily assayed by measuring the conversion of indole to tryptophan in the presence of an excess of the ot subunit (Vol. V [107]). T h e following purification procedure is adapted from that described by Wilson and Crawford. s° Unless otherwise stated, all operations are carried out at 0-4 ° and all buffers contain 40/zM (10/zg/ml) pyridoxal phosphate and 10 mM mercaptoethanol. The best sources of the subunit are mutant strains lacking ~ subunit, which would interfere with the purification procedure by combining with the /32 subunit. A particularly good source of the/3~ subunit is the mutant trp A2, described as a source of InGP synthetase. Step 1. First Heat Step. To each 100 ml o f crude extract are added I0 mg of pyridoxal phosphate and 1 millimole of mercaptoethanol. The extract is then heated to 56 ° for 3 minutes, quickly cooled to 5°, and centrifuged for 30 minutes at 16,000 g. The precipitate may be washed twice by resuspending in 20 ml of 0.1 M potassium phosphate buffer, pH 7.0, for each 100 ml of original crude extract. After centrifugation for 40 minutes at 16,000 g, the supernatant solutions are combined. Step 2. Protamine Sulfate Treatment. For each 100 ml of supernatant solution, add 2 ml of a 5% suspension of protamine sulfate prewarmed to 37 °. After 10 minutes of further stirring, the mixture is centrifuged at 16,000 g for 20 minutes; the precipitate is discarded. Step 3. Ammonium Sulfate Precipitation. For each 100 ml of supernatant from the protamine sulfate step, 24.5 g of solid ammonium sulfate is added slowly with stirring. After stirring for a further 10 minutes, the suspension is centrifuged at 16,000 g for 20 minutes. T h e precipitate is suspended in 0.5 M potassium phosphate buffer, pH 7.5 (approximately 2 ml for each 100 ml of protamine sulfate supernatant). This
~D. A. Wilson and I. P. Crawford,Bacteriol. Proc.,p. 92 (1964); G. M. Hathaway,S. Kida, and I. P. Crawford,Biochemistry8, 989 (1969). SOD. A. Wilson and I. P. Crawford, J. Biol. Chem. 240, 4801 (1965).
[46]
CHORISMATE TO TRYPTOPHAN
379
fraction may be stored a t - 1 5 ° for up to 2 weeks without loss of activity. Step 4. DEAE-Sephadex Chromatography. Prepare a 4.5 x 40 cm column of DEAE-Sephadex A50 equilibrated with 0.1 M potassium phosphate buffer, pH 7.0, without the usual additives. Pass through the column approximately 100 ml of the same buffer containing the usual additives. Dialyze the ammonium sulfate preparation against this buffer for 6 hours. Apply the dialyzed protein solution (containing up to 2 g of protein) to the column and wash it on with approximately 10 ml of buffer. Elution is carried out with a 3000-ml linear gradient of 0.1 to 0.7 M potassium phosphate buffer, pH 7.0. T h e flow rate should be 20-30 ml per hour; fractions of 15 ml are collected. The/32 subunit activity emerges from the column after approximately 2000 ml of eluent has been collected. T h e peak fractions are combined, and the protein is precipitated by the addition of solid ammonium sulfate (28 g/100 ml). These fractions generally comprise about 300 ml. After centrifugation at 16,000 g for 30 minutes, the precipitate is suspended at a concentration of approximately 15 mg/ml in 0.5 M potassium phosphate buffer, pH 7.5. Step 5. Second Heat Step. T h e product of the previous step is dialyzed against 500 ml of 0.6 M potassium phosphate buffer, pH 7.5, for 8 hours. The buffer is replaced twice during the dialysis. The clear yellow solution is heated in a 13-mm diameter thick-walled glass centrifuge tube in an 88 ° water bath. The protein solution is allowed to reach 82 ° , gently stirred at this temperature for 2 minutes, and then immediately cooled to 0°. The heat-denatured protein is removed by centrifugation, followed by filtration, if necessary. A purification procedure is summarized in Table III. The purified /35 subunit is relatively unstable and is best stored at - 1 5 ° as a suspension after adding 2.8 g of solid ammonium sulfate to each 10 ml of solution.
as/35 Complex The as/32 tryptophan synthetase complex is formed by the reversible binding of individual ot subunits to two identical and independent sites of the/32 subunit. 15 Pyridoxal-P and L-serine together markedly increase the association of the two subunits. Apparent association constants for the subunits have been estimated enzymatically and found to range from 4 × 10 ° to 2.6 × 109 M -1. The complex may be formed simply by mixing appropriate quantities of the ot and/3~ subunits, either in the purified form or in crude extracts. Equilibrium is reached essentially instantaneously.
380
[47]
AROMATIC AMINO ACIDS
TABLE Ili a PURIFICATIONOF THE ~2 SUBUNITOF Escherichia coilb TRYPTOPHANSYNTHETASE Total volume (ml)
Fraction Crude extract First heat step combined supernatants Protamine sulfate supernatant Dialyzed ammonium sulfate supernatant DEAE-Sephadex eluate Second heat step supernatant
Total enzyme (unitsc x 10-5)
Total Specific protein activity Yield (g) (units/rag) (%)
884 978
8.40 8.07
32.7 17.6
26 46
96
990 27
7.90 4.68
16.8 1.3
47 360
94 56
1652 2700
45 30
11.5 8
3.80 2.48
0.23 0.092
aTaken from D. A. Wilson and I. P. Crawford,J. Biol. Chem. 240, 4801 (1965). nTryptophan auxotroph containing the A2 point mutation. CAcuvity measured in the standard indole to tryptophan assay (Vol. V [107]) in the presence of a 3-fold excess of a subunit. See Table I I for the definition of a unit of activity.
The tryptophan synthetase of N. crassa has also been purified and characterized.31 31M. Carsiods, E. Appella, P. Provost, J. Gemershausen, and S. R. Suskind, Biochem. Biophys, Res. Commun. ,18, 877 (1965); see also this volume [49].
[47] Anthranilate Synthase and Anthranilate-5'phosphoribosyl- 1-pyrophosphate Phosphoribosyl Transferase (PR Transferase) Aggregate from Aerobacter aerogenes 1 By
A. F. EGAN and
F. GIBSON
T h e first two e n z y m a t i c activities r e l a t e d t o t r y p t o p h a n b i o s y n t h e s i s i n Aerobacter aerogenes, a n t h r a n i l a t e s y n t h e t a s e a n d a n t h r a n i l a t e - 5 ' p h o s p h o r i b o s y i - ~ p y r o p h o s p h a t e p h o s p h o f i b o s y l t r a n s f e r a s e (PR t r a n s ferase), p u r i f y t o g e t h e r as a n e n z y m e a g g r e g a t e J a 1For the preparation of related enzymes from other sources, see Vol. V [107], Vol. VI [86], and ~this~volume[46], [48] and [48a]. I"A~F. Egan, and F. Gibson, Biochira Biophys. Aeta 130,276 ('1966).
CHORISMATE
[46] C h o r i s m a t e
TRYPTOPHAN
to Tryptophan
Anthranilate PRA
TO
365
(Escherichia coli)-
Synthetase, PR Transferase,
Isomerase, InGP
Synthetase,
Tryptophan
Synthetase 1
By THOMAS
E. CREIGHTON a n d CHARLES YANOFSKY
Introduction The biosynthesis of tryptophan from chorismate in microorganisms is now believed to proceed as outlined in Fig. 1. Some of the characteristics of the enzymes and intermediates involved have been covered in previous volumes of this series (V [107]; VI [86]). The present article will not duplicate material in the previous volumes.
Preparation of Intermediates N-5'-Phosphoribosyl Anthranilate (PRA) This very unstable intermediate may be produced synthetically by mixing at room temperature equal volumes of 1.0 M ribose-5-P (sodium salt) in water and 1.0 M anthranilic acid dissolved in 95% ethanol. 2 Upon mixing the two reagents, PRA is formed very rapidly. However, formation of the Amadori rearrangement product, CdRP, proceeds at the same time, although at a slower rate. After 5 minutes' incubation at room temperature, a yield of PRA of at least 30% may be obtained, with no significant formation of CdRP. To stop the reaction after t h e 5 minutes, dilute the mixture with approximately 200 volumes of cold 0.1 M triethanolamine-HCl buffer, p H 8.6, and keep in ice. The PRA is stable for several hours under these conditions. However, after 2 hours at 37 ° approximately 90% of the PRA will have disappeared. It is therefore suggested that the PRA be synthesized immediately before use. PRA prepared in this manner is apparently contaminated with unreacted ribose f-phosphate and anthranilic acid and with any products of side reactions that may occur, in addition to the low levels of CdRP formed. Attempts to remove the contaminants have been unsuccessful due to the concomitant extensive loss of PRA. However, the PRA preWor related articles, see this volume [45], [47], [48], [48a]¢and [49]. 2T. E. Creighton, J. Biol. Chem. 243, 5605 (1968).
366 ....
AROMATIC AMINO ACIDS CO2H
[46]
CO2H
~O--C--CO~H OH
Anthranilicacid
Chorismicacid
C02H t
OH OH
O OH OH I
I
'~ I"
C
II"°-c--c"--CH--C"'°-~
I
N--CH--CH--CH-- CH--CH20-(P) N-5'-Phosphoribosyl-anthranilate (PR-anthranilate)
H
- H20 /
I -(O-Carboxyphenylamino)- 1deoxyribulose-5-P (CdRP)
OH OH
I
~
I
CH--CH--CH20-(~
+ g-scrine - glyceraldehyde-3-P A+B
~
CH2--CH--CChH
H
H
Indoleglycerol-P (InGP) ~ " N A
L-Tryptophan
-glyceraldehyde-3 - P ' ~
~
+
L-~rine
tt Indole
FIG. 1. T h e tryptophan biosynthetic pathway. T h e enzymatic activities catalyzing the various reactions are A, the tryptophan synthetase a subunit, and B, the fl, subunit; C, indole-3-glycerol phosphate synthetase and PR-anthranilate isomerase; D, anthranilatePR-transferase; E, anthranilic acid synthetase. This lettering system is consistent with the structural gene designations in Escherichia coll.
pared as described above has been found to be of direct use in enzyme assays. 1-(o-Carboxyphenylamino)-l-deoxyribulose 5-Phosphate (CdRP) The chemical synthesis of CdRP was presented in Vol. VI [86]. However, a slightly m o d i f e d procedure has been found which gives significantly greater yields: Add 1.5 millimoles of anthranilic acid dissolved sj. A. DeMoss, unpublished results.
[46]
CHORISMATE TO TRYPTOPHAN
367
in 2.5 ml of methanol to 0.5 millimole of ribose 5-phosphate (sodium salt) dissolved in 3.7 ml of water. Heat this mixture to 70 ° in a water bath and add 0.63 ml of ethyl malonate. Continue heating at 70'--80° for 30 minutes and then chill and store at 4 ° for several hours. Add 0.5 ml of water and remove the yellow-orange oil that has collected in the bottom of the reaction vessel. To the remainder add 1.0 N HCI in a dropwise manner to lower the p H to about 5. Extract this with three 7-ml portions of ethyl acetate and discard the extracts. Remove the remaining traces of ethyl acetate by bubbling nitrogen through the solution Until no ethyl acetate odor is detectable. The resulting preparation may be stored for weeks at --15 °. The CdRP will slowly break down, but the resulting anthranilic acid may be removed by repeating the extraction process. The initial yield of CdRP should be about 40%, based upon the limiting quantity of ribose-5-P. The resulting CdRP preparation is contaminated with unreacted ribose-5-P and side products. Several attempts to purify the CdRP have been unsuccessful. However, the above preparation is suitable for routine use in enzyme assays. A method reportedly capable of yielding the barium salt of CdRP at 95% purity has been reported from another laboratory. 4 Some of the properties of this intermediate have been described in Vol. VI [86]. Indole-3-glycerol Phosphate (InGP) The enzymatic synthesis of InGP from anthranilic acid and PRPP (Vol. VI [86]) or from indole and glucose 1,6-diphosphate plus aldolase 5 has been described previously. A more convenient method has been developed; 6 it involves enzymatic synthesis from anthranilic acid and ribose 5-phosphate via PRA and CdRP. To 1 ml of a 0.25 M solution'of ribose 5-phosphate at pH 6, an equimolar amount of solid anthranilic acid is added and the mixture is heated at 70-80 ° for a few minutes to dissolve all the anthranilic acid. The reaction mixture is immediately diluted 10-fold with 0.1 M TrisHCI buffer p H 7.8, brought to 37 °, and an appropriate amount of InGP synthetase (partially purified enzyme) is added. The amount of enzyme employed should be sufficient to convert all the PRA to InGP in approximately 60 minutes. The extent of the reaction can be followed readily by measuring the increase in absorbance at 280 m/~ of suitably diluted aliquots of the mixture. Upon completion of the reaction, the InGP is separated from the relatively small amount of protein in the re4C. H. Doy, Nature 211,736 (1966). 5j. K. Hardman and C. Yanofsky, J. Biol. Chem. 240, 725 (1965). 8K. Herrmann, unpublished results.
368
AROMATIC AMINO ACIDS
[46]
action mixture by passing the solution through a small column (1 cm × 5 cm) of Darco charcoal. To adsorb the InGP to the charcoal, the reaction mixture must be adjusted to pH 6.5. InGP is eluted from the charcoal with 40% ethanol containing 1 ml of conc. NH4OH per 100 ml. After elution the solvent is removed by flash evaporation, and the InGP is dissolved in 0.1 M Tris-HCl buffer pH 7.8. T h e overall yield is about 70%. InGP is quite stable and may be kept for months a t - 1 5 °. Some of the properties of this intermediate have been described (Vol. VI [86]).
Enzyme Assays Anthranilate Synthetase Chorismate + glutamine~ anthranilate + glutamate+ pyruvate Anthranilate synthetase activity is most conveniently measured by following anthranilate formation fluorometricallyY T h e reaction mixture contains the following in a total volume of 2.0 ml: 0.4 micromole of chorismic acid; 20 micromoles of ~.-glutamine; 8.0 micromoles of MgSO4; 20 micromoles of potassium phosphate buffer, pH 7.6, 2 micromoles of mercaptoethanol; enzyme. The reaction is initiated by the addition of enzyme to a mixture of the other components prewarmed to 37 °. T h e increase in fluorescence is followed using an activation wavelength of 314 m g and an emission wavelength of 390 m~. T h e assay can be quantitated by preparing standard curves with known concentrations of anthranilate. T h e presence of PR transferase in the enzyme preparation usually does not present any difficulties because anthranilate utilization is negligible in the absence of PRPP.
Anthranilate-PRPP Phosphoribosyltransferase (PR Transferase) Anthranilate + PRPP ---, PRA + H20 + PPt
A quantitative assay for this enzymatic activity has been presented s which depends upon the ability of the test extract to supplement a cellfree extract of a mutant strain of Neurospora crassa (tryp-4 mutant) lacking only PR transferase activity in catalyzing the overall conversion of anthranilate to InGP. Any enzyme preparation which has sufficient PRA isomerase and InGP synthetase activities, but lacks PR transferase activity, can be used instead of the N. crassa tryp-4 extract. The reaction 7M. T. Gibson and F~ Gibson, Biochem.J. 90, 248 (1964). 8j. Wegman and J. A. DeMoss, J. Biol. Chem. 240, 3781 (1965).
[46]
CHORISMATE TO TRYPTOPHAN
369
mixture contains the following in a total volume of 1.0 ml: 100 micromoles o f potassium phosphate buffer, pH 8.0; 0.3 micromole of anthranilate; 4.0 micromoles of MgSO4; 0.6 micromole of PRPP; excess N. crassa tryp-4 extract; enzyme. The above mixture is incubated at 37 °. At zero time and at the end of the incubation period (usually 15 minutes), 0.4-ml aliquots of the mixture are assayed for InGP, using the periodate method previously described (Vol. V [ 107]). A second assay method has been employed 9 which is somewhat less quantitative than the above method but considerably more convenient. The PR transferase reaction is followed by measuring the decrease in fluorescence that accompanies anthranilate disappearance. Since PRA, the product of the reaction, is slightly fluorescent, while CdRP, the next intermediate, is not fluorescent, the actual decrease in fluorescence will d e p e n d to some extent upon the ability of the enzyme preparation being assayed to convert the PRA to CdRP. However, this has not been found to be a serious obstacle to the use of this convenient assay. The reaction mixture contains the following in a final volume of 2.0 ml: 0.01 micromole of anthranilate; 0.5 micromole of PRPP; 4.0 micromoles of MgSO4; 50 micromoles of Tris-HC1 buffer, pH 7.8; enzyme. The reaction is followed at 37 ° by observing the decrease in fluorescence at 390 m/z (activation at 314 m/z) as compared to a control without added enzyme.
N-5'-Phosphoribosylanthranilate Isomerase (PRA Isomerase) PRA --* CdRP
PRA isomerase activity is best assayed by following InGP formation spectrophotometrically in the presence of excess InGP synthetase. 2 T h e ultraviolet spectra of CdRP and PRA are very similar, while differing considerably from that of InGP. 1° T h e source of excess InGP synthetase has been the enzyme purified from mutant 9830 of Escherichia coli, a strain which lacks PRA isomerase activity. A freshly prepared solution of PRA (see above) may be diluted a further 10-fold with 0.10 M triethanolamine-HCl~ buffer, pH 8.6; it is then warmed to 37 ° and placed in a spectrophotometer cuvette. While monitoring the absorbance at 280 m/z, the excess InGP synthetase is added. The absorbance may increase rapidly to a new level, due to conversion of any contaminating CdRP to InGP. When the absorbance reaches a constant value again, the enzyme preparation to be assayed is added. The rate of increase in absorbance that is thus observed is pro9j. Ito and I. P. Crawford, Genetics 52, 1303 (1965). leT. E. Creighton and C. Yanofsky, J; Biol. Chem. 241, 4616 (1966).
370
AROMATIC AMINO ACIDS
[46]
portional to the amount of PRA isomerase activity added. Under these conditions the conversion of 0.10 mM PRA to CdRP causes an increase in absorbance at 280 m/~ of 0.43. Alternatively, InGP formation may be measured with the periodate assay (Vol. V [107]). Other assays for this activity have been described which make use of PR transferase to enzymatically synthesize PRA. T h e disappearance of the PRA n ' n or its conversion to InGP TM is then followed.
Indole-3-glycerol-P Synthetase (InGP Synthetase) CdRP --~ InGP + CO~+ H20 The rate of increase in absorbance at 280 mt~ upon conversion of CdRP to InGP is used routinely to measure InGP synthetase activity,t° Enzyme is normally added to a 0.1 mM solution of CdRP (see above) in 0.10 M Tris-HCl buffer, pH 7.8, at 37 °. T h e complete conversion of 0.10 mM CdRP to InGP results in an increase in absorbance at 280 m~ of 0.448 under these conditions. With crude extracts containing very low levels of activity, a spurious slow increase in absorbance is often found which is not dependent upon the presence of CdRP. With such extracts it is best to use the periodate assay for InGP formation (Vol. V [107]).
Tryptophan Synthetase InGP + L-serine~ L-tryptophan+ glyceraldehyde-3-P InGP + H~O ~=~indole + glyceraldehyde-3-P Indole + L-serine---, L-tryptophan+ HzO The standard enzyme assays for tryptophan synthetase have been described in Vol. V [107]. However, a spectrophotometric assay procedure convenient for kinetic studies has been developed. 14a5 T h e glyceraldehyde-3-P formed in the conversion of InGP to indole or tryptophan is measured by coupling with glyceraldehyde-3-P dehydrogenase. The complete reaction mixture for the conversion of InGP to tryptophan contains the following in a final volume of 1.0 ml: 0.1 micromole of InGP; 60 micromoles of VL-serine; 0.04 micromole of pyridoxalP; 180 micromoles of NaCi; 12 micromoles of Na2HAsO4; 1.0 micromole of NAD; 100 micromoles of Tris-HCl buffer, pH 7.8; excess crystalline nj. A. DeMoss,R. W. Jackson, and J. H. Chalmers,Jr., Genetics 56, 413 (1967). nO. H. Smith, Genetics 57, 95 (1967). 13j. A. DeMoss,Biochem. Biophys. Res. Commun. 18, 850 (1965). 14I. P. Crawford,Biochira. Biophys. Acta 45, 405 (1960). nT. E. Creighton and C. Yanofsky,J. Biol. Chem. 241,980 (1966).
[46]
CHORISMATE TO TRYPTOPHAN
371
glyceraldehyde-3-P dehydrogenase; enzyme. For the enzymatic conversion of InGP to indole, the serine, pyridoxal-P, and NaCI are omitted. The enzyme is added to start the reaction, and NAD reduction is followed spectrophotometrically at 340 m/.~. A very rapid qualitative spot test for indole to tryptophan activity has been devised 16which is extremely useful during preparative procedures. The substrate mixture is the same as that of the normal indole to tryptophan assay (Vol. V [107]) and consists of the following mixture: 0.4 mM indole; 0.04 mM pyridoxal-P; 60 mM DL-serine; 180 mM NaCI; 0.1 M Tris-HCl, pH 7.8. Aliquots (0.3 ml) o f this mixture are placed into the depression of a polyvinylchloride spot test plate. Approximately one drop o f the preparation to be tested is added to each depression, and the plate is incubated for 10 minutes by floating it in a water bath at 37 °. Ehrlich's indole reagent (3-4 drops) is then added to each depression and the presence or absence of indole noted. The conditions of this assay, such as the relative proportion of reaction mixture and enzyme and the length of the incubation period, may be varied to produce a spot assay of the desired sensitivity.
Preparation of Enzymes Growth of Bacteria Bacteria are grown in single-strength Vogel and Bonnet 17 minimal medium supplemented with 0.05% acid-hydrolyzed casein, 2.5/.~g/ml indole, and 0.5% glucose. When large cultures are grown in a Biogen (American Sterilizer Co.) or Fermacell (New Brunswick Scientific Co.) fermenter, the indole supplement can be increased to 5/zg/ml and derepression will still be observed. The cells are generally harvested after 16-18 hours of growth at 37 ° and disrupted by sonic oscillation. Sonic extracts are employed in the purification procedures without prior centrifugation.
Anthranilate Synthetase-PR Transferase These first two enzymes of tryptophan biosynthesis occur in E. coil as a complex of two proteins, tentatively designated as components I and II. is Component I is the product of the E gene and has an estimated s20.w of 4.3 S. This protein, when not in the complex, has anthranilate IsU. Henning, D. R. Helinski, F. C. Chao, and C. Yanofsky,J.Biol. Chem. 237, 1523 (1962). 17H. J. Vogel and D. M. Bonner,J. Biol. Chem. 218, 97 (1956); see this volume [1], Section 3. lsj. lto and C. Yanofsky,J. Biol. Chem. 241, 4112 (1966); J. Ito, E. C. Cox, and C. Yanofsky, J. Bacteriol. 97, 723 (1969); J. Ito and C. Yanofsky, ibid., p. 734.
372
AROMATIC AMINO ACIDS
[46]
synthetase activity only in the presence of high concentrations of N H + ion. Component II is the product of the D gene, with an s20,wof 4.4 S. It possesses full PR transferase activity whether or not it is in the complex. When mixed together or when both are synthesized in vivo, the two components form a complex of unknown structure with an szo,w of 7.5-9 S. This complex has full anthranilate synthetase and PR transferase activities. Components I and II may be obtained individually in cell-free extracts of mutant strains containing nonsense mutations in the D and E genes, respectively. TM A protein containing anthranilate synthetase activity has been purified from E. coli and its catalytic properties studied, TM but its relationship to components I and II is not clear. T h e feedback inhibition of anthranilate synthetase activity by tryptophan has been studied by several investigators, xs-2x The two-component nature of the enzyme from Salmonella typhimurium 22 and Aerobacter aerogenes z° has also been demonstrated. The anthranilate synthase from Neurospora crassa has been purified 90-fold z3 and the transferase 64-fold. s P R A Isomerase-InGP
Synthetase
In E. coil these two activities are catalyzed by a single polypeptide chain of approximately 45,000 molecular weight, x° T h e enzyme is best isolated from mutant trp A2, a particularly good source being a strain (trp A2/F' trp A2) with the mutation on both the chromosome and an F' episome. The enzyme is most easily detected by assay of its InGP synthetase activity. In the following isolation procedure, all operations are carried out at 0-4 ° . Unless otherwise stated, all potassium phosphate buffers are at p H 7.0 and contain 1.0 mM EDTA and 0.1 mM dithiothreitol. T h e cellfree extract is supplemented with EDTA and dithiothreitol to 1 raM, and anthranilic acid 24 (dissolved in the minimum of 95% ethanol) to 10mM. Step 1. Treatment with Streptomycin Sulfate. To each 100 ml of crude extract is added 5 ml of a 20% solution of streptomycin sulfate. T h e l q ' . I. Baker and I. P. Crawford,J. Biol. Chem. 242, 5577 (1967). 2°A. F. Egan and F. Gibson, Biochim. Biophys. Acta 130, 276 (1966); see also this volume [47]. SIR. L. Somerville and E. Eiford, Biochem. Biophys. Res. Commun. 28, 437 (1967). S2R. H. Bauerle and P. Margolin, C0/d spring Harbor Symp. Quant. Biol. 31,203 (1966). uj. A. DeMoss and J. Wegman, Proc. Natl. Acad. Sci. U.S. 54, 241 (1965); see also this volume [481. ~Preliminary experiments indicated that anthranilic acid protected the enzyme during the acetic acid step to follow, but this point has not been investigated further.
[46]
CHORISMATE TO TRYPTOPHAN
373
mixture is stirred for 15 minutes and then centrifuged for 50 minutes at 16,000 g. The precipitate is discarded. Step 2. Treatment with Acetic Acid. To the supernatant is added slowly, with stirring, 1.0 M sodium acetate buffer, pH 3.2, to lower the pH of the extract to 4.90--+ 0.05 (determined with a glass electrode after 10-fold dilution with water). Approximately 9-10 ml of acetate buffer is required for 100 ml of streptomycin sulfate supernatant. Immediately the suspension is centrifuged at 16,000 g for 25 minutes. The precipitate is discarded. As soon as possible after centrifugation, the pH of the supernatant is raised to 7.0 -+ 0.2 (narrow range pH paper, pH 6.0-8.0) by addition of concentrated NH4OH. Step 3. Ammonium Sulfate Precipitation. To each 100 ml of acetic acid supernatant slowly add 28.0 g of solid ammonium sulfate with stirring. After further stirring for 15 minutes, centrifuge the suspension for 25 minutes at 16,000 g. The precipitate is suspended in a small volume of 0.1 M potassium phosphate buffer; the supernatant is discarded. Step 4. Sephadex G-IO0 Filtration. After dialysis of the suspended ammonium sulfate precipitate against 0.1 M potassium phosphate buffer for 3-6 hours, the slightly turbid and brown solution is applied to the top of a Sephadex G-100 column (5 × 60 cm) equilibrated wih 0.1 M potassium phosphate buffer. The protein is eluted with the same buffer at the maximum flow rate (80 ml per hour, 12-minute fractions). Those fractions containing greater than 1000 units 25 of InGP synthetase activity per milliliter (these fractions usually comprise 130-170 ml) are combined and the protein precipitated by the addition of 35 g of solid ammonium sulfate for each 100 ml of solution. Collect the resulting precipitate by centrifugation at 16,000 g for 25 minutes. The precipitate is suspended in a small quantity of 0.1 M potassium phosphate buffer; the supernatant is discarded. Step 5. DEAE-Sephadex Chromatography. After dialysis of the suspended precipitate from step 4 against 0.1 M potassium phosphate buffer for 12-16 hours, the clear yellow solution is applied to a column (3 × 60 cm) of DEAE-Sephadex A50 equilibrated with the same buffer. The protein is washed on with a small amount (50-100 ml) of buffer and eluted with a 2000-ml linear gradient of 0.1-0.7 M potassium phosphate buffer. The flow rate should be approximately 80 ml per hour; 15-minute fractions are collected. Those fractions containing greater than 1000 units 25 of InGP synthetase activity per milliliter are pooled, and the protein is precipitated by addition of solid ammonium sulfate (40 g/100 ml). These fractions generally comprise 200-250 ml. After collection of the ~One unit o f InGP synthetase activity is defined as that activity which causes the formation o f 0.1 micromole o f InGP in 20 minutes at 37*.
374
AROMATIC AMINO ACreS
[46]
precipitate by centrifugation at 16;000 g for 25 minutes, the precipitate is suspended in a small volume of 0.1 M KC1 containing 0.01 M potassium phosphate buffer. Step 6. Hydroxytapatite Chromatography. The suspended precipitate from step 5 is dialyzed 12-16 hours against 0.1 M KC1 in 0.01 M potassium phosphate buffer (without EDTA). The clear, slightly yellow solution is then placed on a column (4.5 × 20 cm) of hydroxylapatite (BioGel HT, Bio-Rad) equilibrated with the same buffer. The protein is washed on with approximately 100 ml of buffer and eluted with a 1000-ml linear gradient of 0.01-0.08 M potassium phosphate buffer in 0.1 M KCI (without EDTA). The flow rate should be approximately 20 ml per hour, and 1-hour fractions should be collected. The InGP synthetase activity emerges as the first major peak of protein. The active fractions may be stored directly at--15 ° for several weeks without appreciable losses of activity. The purification procedure is summarized in Table I. The purified enzyme is homogeneous upon sedimentation and electrophoresis. It may be readily crystallized by the following procedure, with no change in specific activity. Raise the pH of a saturated solution of ammonium sulfate to 7.0 by addition of concentrated NH4OH. Add this solution dropwise to a concentrated (greater than I0 mg/ml) soluTABLE
Ia
PURIFICATION OF I N G P SYNTHETASE OF Escherichia coli ~
Fraction C r u d e extract Streptomycin sulfate supernatant Acetic acid s u p e r n a t a n t Dialyzed a m m o n i u m sulfate precipitate S e p h a d e x G-100 eluate e DEAE-Sephadex eluate e Hydroxylapatite eluate t
Total volume (ml)
Total enzyme (units c × 10 -6)
Total protein (g)
865 760
2.85 2.62
79.5 _ d
640 71
1.90 1.86
_ a 8.52
28 21
1.35 1.30
14
0.48
Specific activity
Yield (%)
Purification
92
--
_ 218
67 65
5.9
2.44 1.12
554 1160
48 45
15 31.5
0.66
1380
23
37.5
35.9 _
a T a k e n f r o m T. E. Creighton a n d C. Yanofsky,J. Biol. Chem. 241, 4616 (1966). OTryptophan a n x o t r o p h with the A2 point mutation on both the c h r o m o s o m e a n d an F' episome. c O n e unit o f InGP synthetase activity is defined as that activity which causes the formation o f 0.1 micromole o f InGP in 20 minutes at 37*. nNot d e t e r m i n e d because o f the p r e s e n c e o f streptomycin sulfate. e Dialyzed a m m o n i u m sulfate precipitate.
[46]
CHORISMATE TO TRYPTOPHAN
375
tion of the purified enzyme preparation until a distinct turbidity is observed. U p o n storage of this preparation at 4 ° for several days, crystallization should be nearly complete. T h e hydroxylapatite step may be omitted and InGP synthetase crystallized directly from the final fraction of step 5 (with the addition of seed crystals). T h e crystals so obtained generally have the same specific activity as the crystalline material obtained after the hydroxylapatite step. ~ T h e purified enzyme is very susceptible to oxidation and heavy metals, but the constant presence of dithiothreitol or mercaptoethanol will alleviate this problem. T h e amino acid composition has been determined, and some of its physical properties have been studied. TM T h e Kr~ for CdRP in the InGP synthetase reaction is approximately 5 × 10-~ M. T h e enzyme from N. crassa has been purified approximately 83-fold, and some of its properties have been examined, s T r y p t o p h a n Synthetase 2aa T h e tryptophan synthetase of E. coli is composed of two protein subunits, the a and//2 subunits (formerly designated as the A and B proteins, respectively), in the form of an a2 /32 complex. 27 T h e catalytic properties of the subunits and the complex have been described in Vol. V [107]. Subunit
T h e ot subunit of E. coli tryptophan synthetase is a single polypeptide chain 267 residues in length, with a molecular weight of 28,700. 28 Pure preparations of the ot subunit can catalyze the InGP to indole + 3-phosphoglyceraldehyde reaction, but the reaction rate is only about 1% of the rate observed with the c~2/32 complex. T h c~ subunit is most conveniently assayed in the conversion of indole to tryptophan in the presence of an excess of f12 subunits (Vol. V [107]). Assay procedures for the InGP reactions are described earlier in this article and in Vol. V [107]. Mutant B8 is the best source of the c~ subunit. T h e following purification procedure is a modification of the procedure described by H e n n i n g et al. TM All operations are carried out at 2-4 °. ~SE. Orias, unpublished results. ~saEC 4.2.1.90; L-serine hydro-lyase (adding indole). 27M. Goldberg, T. E. Creighton, R. L. Baldwin, and C. Yanofsky,J. Mol. Biol. 21, 71 (1966). 2sc. Yanofsky, G. R. Drapeau, J. R. Guest, and B. C. Carlton, Proc. Natl. Acad. Sci. U.S. 57, 296 (1967).
376
AROMATIC AMINO ACIDS
[46]
Step I. Treatment with Manganous Chloride. For each 100 ml of crude extract, 7 ml of 1 M MnCI, is added slowly with stirring. T h e mixture is stirred for an additional 10 minutes, and the suspension is then centrifuged for 30 minutes at 15,000 g. The precipitate is suspended in cold water (6-8 ml per 100 ml of starting volume), agitated in a Waring blendor, and centrifuged. The wash is combined with the supernatant solution. Step 2. Acid Treatment. To the supernatant from step 1, 1 M sodium acetate buffer, pH 3.2, is added slowly, with stirring, until the pH is lowered to about 4 (determined with a bromocresol green solution on 2-drop aliquots in the depressions of a spot plate). Approximately 6-8 ml of acetate buffer are required for each 100 ml of supernatant. T h e suspension is centrifuged immediately at 15,000 g for 15 minutes, and the precipitate is rapidly washed with a small amount of cold water as described above. Step 3. Ammonium Sulfate Precipitation. To each 100 ml of the combined supernatants from step 2, 31 g of solid ammonium sulfate is added slowly with stirring. After an additional 15 minutes of stirring the precipitate is collected by centrifugation for 15 minutes at 15,000 g. T h e precipitate is suspended in 3 ml of 1 M Tris-HCl buffer, pH 7.8, for each 100 ml of starting extract. This fraction can be stored a t - 1 5 ° indefinitely without loss of activity. Step 4. Sephadex G-IO0 Fractionation. The suspension from step 3 is dialyzed against 2 liters of 0.05 M potassium phosphate buffer, pH 7.0, for 2 hours. The dialyzing solution is replaced, and dialysis is continued for an additional 2 hours. The slightly turbid solution is then applied to a 5.5 x 50 cm column of Sephadex G-100 equilibrated with 0.05 M potassium phosphate buffer, pH 7.0. T h e same buffer is used for elution, and 20-ml fractions are collected. T h e fractions are tested for a subunit activity by the spot test procedure described earlier in this article (the spot test mixture is supplemented with a suitable excess of a /32 subunit preparation). The active fractions are combined, and solid ammonium sulfate is slowly added (44 g for each 100 ml of combined fractions) with stirring. After additional stirring for 15 minutes the precipitate is collected by centrifugation at 15,000 g for 15 minutes. The precipitate is dissolved in 0.1 M Tris-HCl buffer, pH 7.8, using approximately 1 ml of buffer for each 100 ml of starting extract. Step 5. DEAE-Cellulose Chromatography. DEAE-Selectacel (0.9 meq/g) equilibrated with 0.01 M potassium phosphate buffer, pH 7.0, is poured into a column (2.5 x 125 cm) to a height o f 110 cm of cellulose, after settling. The dissolved precipitate from step 4 is dialyzed against 1 liter of 0.01 M potassium phosphate buffer, pH 7.0, for 3 hours. T h e dialyz-
[46]
CHORISMATE TO TRYPTOPHAN
377
ing fluid is replaced and dialysis is continued for an additiDnal 3 hours. T h e sample is occasionally,.sua:l~d and requir.es clarification by centrifugation, T h e p r o t e i n sample is applied to the DEAE-cellulose column, and the a subunit is eluted with a linear gradient of potassium phosphate b u f f e r , pH 7.0. (The mixing flask contains 600 ml of 0.01 M phosphate while the second flask contains 600 ml of 0.3 M phosphate buffer.) A flow rate of 30-40 ml/hour is employed, and fractions of 20 ml are collected. T h e a subunit elutes at about 700 ml. All fractions which give a positive spot test for enzymatic activity are assayed quantitatively for.protein-and ot subunit activity. A typical purification procedure is summarized in Table II. TABLE II PURIFICATION OF THE Ot SUBUNIT OF Escherichia coli a TRYPTOPHAN SYNTHETASE
Fraction
Total volume (ml)
Total enzyme (units ~ × 10-5)
Crude extract MnCl~ supernatant Dialyzed a m m o n i u m sulfate fraction Combined G-100 fractions DEAE-cellulose peak tubes
700 660 80 15 60
33.5 30 21 14 8
Total Specific protein activity Yield (g) (units/rag) (%) 36 32 5.1 0.94 0.16
93 94 410 1500 5000
90 63 42 24
aExtract,of tryptophan auxotroph B8. bActivity measured in the standard indole to tryptophan assay (Voi. V [107]) in the presence of a 3-fold excess of f12 subunit activity. One unit of activity is defined as that which causes the disappearance o f 0.1 micromole of indole in 20 minutes at 37 ° in the standard enzyme assay.
Step 6. Crystallization. T h e high specific activity fractions from step 5 are combined, and the protein is precipitated by adding a m m o n i u m sulfate (4.4 g/10 ml) slowly, with stirring. T h e suspension is stirred for an additional 15 minutes and the precipitate is collected by centrifugation at 20,000 g for 10 minutes. T h e precipitate is dissolved in cold water to give a protein concentration of 2-4%. If slight turbidity is evident, the insoluble material is removed by a brief centrifugation. T o the clear protein solution a saturated a m m o n i u m sulfate solution (adjusted to pH 6.0-6.8 with 2 N ammonia, pH measurements made on a 50-fold dilution with water) is added dropwise with stirring until a faint but distinct turbidity appears. A small a m o u n t of seed crystals is added, and the mixture is stored at 2-4 °. Crystals are evident within 24 hours. Crystallization is generally observed ,without seed crystals in 2-4 days. Crystalline suspensions retain full activity for at least five years.
378
AROMATIC AMINO ACIDS
[46]
[3~ Subunit The/3~ subunit of E. coli is a dimer with a molecular weight of approximately 100,000Y a9 Two moles of pyridoxal phosphate are bound per mole of the dimer; the cofactor is essential for the intrinsic enzymatic activity of the subunit in converting indole plus serine to tryptophan 3° and for the overall conversion of InGP to tryptophan with the a subunit. The/3~ subunit is most readily assayed by measuring the conversion of indole to tryptophan in the presence of an excess of the ot subunit (Vol. V [107]). T h e following purification procedure is adapted from that described by Wilson and Crawford. s° Unless otherwise stated, all operations are carried out at 0-4 ° and all buffers contain 40/zM (10/zg/ml) pyridoxal phosphate and 10 mM mercaptoethanol. The best sources of the subunit are mutant strains lacking ~ subunit, which would interfere with the purification procedure by combining with the /32 subunit. A particularly good source of the/3~ subunit is the mutant trp A2, described as a source of InGP synthetase. Step 1. First Heat Step. To each 100 ml o f crude extract are added I0 mg of pyridoxal phosphate and 1 millimole of mercaptoethanol. The extract is then heated to 56 ° for 3 minutes, quickly cooled to 5°, and centrifuged for 30 minutes at 16,000 g. The precipitate may be washed twice by resuspending in 20 ml of 0.1 M potassium phosphate buffer, pH 7.0, for each 100 ml of original crude extract. After centrifugation for 40 minutes at 16,000 g, the supernatant solutions are combined. Step 2. Protamine Sulfate Treatment. For each 100 ml of supernatant solution, add 2 ml of a 5% suspension of protamine sulfate prewarmed to 37 °. After 10 minutes of further stirring, the mixture is centrifuged at 16,000 g for 20 minutes; the precipitate is discarded. Step 3. Ammonium Sulfate Precipitation. For each 100 ml of supernatant from the protamine sulfate step, 24.5 g of solid ammonium sulfate is added slowly with stirring. After stirring for a further 10 minutes, the suspension is centrifuged at 16,000 g for 20 minutes. T h e precipitate is suspended in 0.5 M potassium phosphate buffer, pH 7.5 (approximately 2 ml for each 100 ml of protamine sulfate supernatant). This
~D. A. Wilson and I. P. Crawford,Bacteriol. Proc.,p. 92 (1964); G. M. Hathaway,S. Kida, and I. P. Crawford,Biochemistry8, 989 (1969). SOD. A. Wilson and I. P. Crawford, J. Biol. Chem. 240, 4801 (1965).
[46]
CHORISMATE TO TRYPTOPHAN
379
fraction may be stored a t - 1 5 ° for up to 2 weeks without loss of activity. Step 4. DEAE-Sephadex Chromatography. Prepare a 4.5 x 40 cm column of DEAE-Sephadex A50 equilibrated with 0.1 M potassium phosphate buffer, pH 7.0, without the usual additives. Pass through the column approximately 100 ml of the same buffer containing the usual additives. Dialyze the ammonium sulfate preparation against this buffer for 6 hours. Apply the dialyzed protein solution (containing up to 2 g of protein) to the column and wash it on with approximately 10 ml of buffer. Elution is carried out with a 3000-ml linear gradient of 0.1 to 0.7 M potassium phosphate buffer, pH 7.0. T h e flow rate should be 20-30 ml per hour; fractions of 15 ml are collected. The/32 subunit activity emerges from the column after approximately 2000 ml of eluent has been collected. T h e peak fractions are combined, and the protein is precipitated by the addition of solid ammonium sulfate (28 g/100 ml). These fractions generally comprise about 300 ml. After centrifugation at 16,000 g for 30 minutes, the precipitate is suspended at a concentration of approximately 15 mg/ml in 0.5 M potassium phosphate buffer, pH 7.5. Step 5. Second Heat Step. T h e product of the previous step is dialyzed against 500 ml of 0.6 M potassium phosphate buffer, pH 7.5, for 8 hours. The buffer is replaced twice during the dialysis. The clear yellow solution is heated in a 13-mm diameter thick-walled glass centrifuge tube in an 88 ° water bath. The protein solution is allowed to reach 82 ° , gently stirred at this temperature for 2 minutes, and then immediately cooled to 0°. The heat-denatured protein is removed by centrifugation, followed by filtration, if necessary. A purification procedure is summarized in Table III. The purified /35 subunit is relatively unstable and is best stored at - 1 5 ° as a suspension after adding 2.8 g of solid ammonium sulfate to each 10 ml of solution.
as/35 Complex The as/32 tryptophan synthetase complex is formed by the reversible binding of individual ot subunits to two identical and independent sites of the/32 subunit. 15 Pyridoxal-P and L-serine together markedly increase the association of the two subunits. Apparent association constants for the subunits have been estimated enzymatically and found to range from 4 × 10 ° to 2.6 × 109 M -1. The complex may be formed simply by mixing appropriate quantities of the ot and/3~ subunits, either in the purified form or in crude extracts. Equilibrium is reached essentially instantaneously.
380
[47]
AROMATIC AMINO ACIDS
TABLE Ili a PURIFICATIONOF THE ~2 SUBUNITOF Escherichia coilb TRYPTOPHANSYNTHETASE Total volume (ml)
Fraction Crude extract First heat step combined supernatants Protamine sulfate supernatant Dialyzed ammonium sulfate supernatant DEAE-Sephadex eluate Second heat step supernatant
Total enzyme (unitsc x 10-5)
Total Specific protein activity Yield (g) (units/rag) (%)
884 978
8.40 8.07
32.7 17.6
26 46
96
990 27
7.90 4.68
16.8 1.3
47 360
94 56
1652 2700
45 30
11.5 8
3.80 2.48
0.23 0.092
aTaken from D. A. Wilson and I. P. Crawford,J. Biol. Chem. 240, 4801 (1965). nTryptophan auxotroph containing the A2 point mutation. CAcuvity measured in the standard indole to tryptophan assay (Vol. V [107]) in the presence of a 3-fold excess of a subunit. See Table I I for the definition of a unit of activity.
The tryptophan synthetase of N. crassa has also been purified and characterized.31 31M. Carsiods, E. Appella, P. Provost, J. Gemershausen, and S. R. Suskind, Biochem. Biophys, Res. Commun. ,18, 877 (1965); see also this volume [49].
[47] Anthranilate Synthase and Anthranilate-5'phosphoribosyl- 1-pyrophosphate Phosphoribosyl Transferase (PR Transferase) Aggregate from Aerobacter aerogenes 1 By
A. F. EGAN and
F. GIBSON
T h e first two e n z y m a t i c activities r e l a t e d t o t r y p t o p h a n b i o s y n t h e s i s i n Aerobacter aerogenes, a n t h r a n i l a t e s y n t h e t a s e a n d a n t h r a n i l a t e - 5 ' p h o s p h o r i b o s y i - ~ p y r o p h o s p h a t e p h o s p h o f i b o s y l t r a n s f e r a s e (PR t r a n s ferase), p u r i f y t o g e t h e r as a n e n z y m e a g g r e g a t e J a 1For the preparation of related enzymes from other sources, see Vol. V [107], Vol. VI [86], and ~this~volume[46], [48] and [48a]. I"A~F. Egan, and F. Gibson, Biochira Biophys. Aeta 130,276 ('1966).