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[47] Anthranilate synthase—Anthranilate phosphoribosyltransferase complex and subunits of Salmonella typhimurium
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BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[47]
tase has been identified as a separate enzyme that is required for the activity of chorismate synthase from this source? Physical Properties. Sucrose density gradient analyses of chorismate synthase indicate that the enzyme can exist in at least two active forms with sedimentation values of approximately 8 S and 10 S. 5 From SDS gel electrophoresis of the purified enzyme a single band with a molecular weight of 55,000 is obtained.l These results are consistent with the idea that the 8 S form of chorismate synthase is a dimer of the 55,000 molecular weight subunit and that the 10 S form is a tetramer. Since both the flavin reductase and the chorismate synthase activity appear to be catalyzed by a single protein, it is likely that in N. crassa chorismate synthase exists as a bifunctional enzyme. In contrast in E. coh~ and B. subtilis, 3 as has already been noted, flavin reductase and chorismate synthase are separable enzymatic activities.
[47] A n t h r a n i l a t e S y n t h a s e - A n t h r a n i l a t e Phosphoribosyltransferase Complex and Subunits of Salmonella t y p h i m u r i u m B y R O N A L D B A U E R L E , J O H N H E S S , and SARAH FRENCH
Introduction In Salmonella typhimurium and a number of other enteric bacteria, including Escherichia coli and Enterobacter aerogenes, the first two specific steps of tryptophan biosynthesis are catalyzed by a multifunctional allosteric enzyme, the anthranilate synthase-anthranilate 5-phosphorylribose-l-pyrophosphate phosphoribosyltransferase (AS-PRT) complex (Volume XVIIA [46,47,48a]). The A S - P R T complex is a tetramer made up of two molecules each of subunits component I and component II, the products of trpE and trpD, the first two genes of the trp oper0n.I The intact A S - P R T complex, 2 an AS partial complex derived by proteolytic digestion of the intact complex,3,4 and the uncomplexed subunits obtained from trpE and trpD mutant strains 5,6 have been purified and well characI R. Bauerle and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 31, 203 (1966). E. J. H e n d e r s o n and H. Zalkin, J. Biol. Chem. 246, 6891 (1971). 3 L. H. H w a n g and H. Zalkin, J. Biol. Chem. 246, 2338 (1971). 4 H. T a m i r and P. R. Srinivasan, J. Biol. Chem. 244, 6507 (1969). 5 H. Zalkin and D. Kling, Biochemistry 7, 3566 (1968). 6 M. G r i e s h a b e r and R. Bauerle, Biochemistry 13, 373 (1974).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987by AcademicPress, Inc.
All rights of reproduction in any form reserved.
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Salmonella ANTHRANILATE SYNTHASE--PR TRANSFERASE
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terized. The properties of the enzymes have been comprehensively rev i e w e d . 7-9
The A S - P R T complex and the AS partial complex, which is a tetramer made up of intact component I and an amino-terminal fragment of component II ~° (component II*), catalyze the formation of anthranilate from chorismate and L-glutamine 2a [Reaction (l), AS-GIn activity]. Both enzymes also catalyze what can be considered partial reactions, namely, the deamidation of L-glutamine 11 [Reaction (2), glutaminase activity] and the synthesis of anthranilate from chorismate and ammonia 2-4 [Reaction (3), AS-NH3 activity]. The AS partial complex is homologous in structure and function to the monofunctional anthranilate synthase of Serratia marc e s c e n s (this series, Vol. 113 [37]). Uncomplexed component I has A S NH3 activity but lacks AS-GIn activity) Neither the component I nor component II subunit has glutaminase activity. When mixed together in vitro the two subunits spontaneously assemble to form an A S - P R T complex indistinguishable from the in vivo complex. 12The ability to utilize Lglutamine derives from a composite active site created by the aggregation of component I with the amino-terminal domain of component II. In the AS-GIn reaction, glutamine is bound to residue Cys-84 of component II, and its amide group is removed and transferred to a site on component I which uses it in the amination of chorismate. 9 The AS-GIn, AS-NH3, and glutaminase activities of the A S - P R T complex and the AS partial complex as well as the AS-NH3 activity of the component I subunit are feedback inhibited by tryptophan, z-5 Inhibition is competitive with respect to chorismate and noncompetitive with respect to glutamine and ammonia. The A S - P R T complex and component II subunit catalyze the formation of N-(5'-phosphoribosyl)anthranilate from anthranilate and PRPP (5phosphorylribose-l-pyrophosphate) [Reaction (4), PRT activity]. 6,13 The PRT activities of the two are equivalent. Uncomplexed component II forms a stable dimeric form in solution which retains full PRT activity but has lost the ability to assemble in vitro with component 1.6 PRT activity resides in the carboxy-terminal domain of the bifunctional component II 7 H. Zalkin, Adv. Enzymol. 38, 1 (1973). 8 H. Zalkin, in "The Enzymes of Glutamine Metabolism" (S. Prusiner and E. R. Stadtman, ed.), p. 523. Academic Press, New York, 1973. 9 H. Zalkin, in "Multifunctional Proteins" (H. Biswanger and E. Schmincke-Ott, eds.), p. 123. Wiley, New York, 1980. 10 M. Grieshaber and R. Bauerle, Nature (London), New Biol. 236, 232 (1972). II H. Nagano, H. Zalkin, and E. J. Henderson, J. Biol. Chem. 245, 3810 (1970). 12 D. Smith and R. Bauerle, Biochemistry 8, 1451 (1969). 13 E. J. Henderson, H. Zalkin, and L. H. Hwang, J. Biol. Chem. 245, 1424 (1970).
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BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
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molecule. 1°,14,15 T h e P R T activity of the A S - P R T complex is subject to f e e d b a c k inhibition b y t r y p t o p h a n 6,~3 mediated by its binding to the component I subunit. Most studies o f the A S - P R T c o m p l e x and c o m p o n e n t II subunit have b e e n carried out with e n z y m e s isolated from the LT2 strain of S. typhimurium, while those of the c o m p o n e n t I subunit with e n z y m e from the LT7 strain. N o major differences in properties of the two c o m p o n e n t I subunits h a v e b e e n noted; h o w e v e r , the LT7 c o m p o n e n t II has only 25% the P R T activity of the L T 2 c o m p o n e n t II. 16The amino acid sequences of the LT2 c o m p o n e n t I and c o m p o n e n t II polypeptides are k n o w n f r o m the D N A sequences o f the trpE 17 and trpD TM genes. The nucleotide sequence of the LT7 trpE is identical to that of the LT2 gene.16 The nucleotide sequence of the LT7 trpD gene has not yet been determined. E n z y m a t i c Activities G l u t a m i n e - d e p e n d e n t anthranilate synthase (AS-GIn): Chorismate + L-glutamine
Mg2+
~ anthranilate + L-glutamate + pyruvate + H_,O (1)
Glutaminase: L-Glutamine + H20
chorismate
~ L-glutamate + NH3
(2)
A m m o n i a - d e p e n d e n t anthranilate synthase ( A S - N H 3 ) : Chorismate + (NH4hSO4
Mg2+
) anthranilate + pyruvate + H20
Anthranilate 5 - p h o s p h o r y l r i b o s e - l - p y r o p h o s p h a t e transferase (PRT):
(3)
phosphoribosyl-
Anthranilate + PRPP Mg:+)N-(5'-phosphoribosyl)anthranilate + PP~ + H20
(4)
Assay Methods Anthranilate S y n t h a s e Principle. Anthranilate synthase activity ( A S - G I n and A S - N H 3 ) is a s s a y e d fluorometrically by measuring the rate of a p p e a r a n c e of anthranilate. 14L. J. LaScolea, Jr., and E. Balbinder, J. Bacteriol. 112, 877 (1972). 15M. Grieshaber, Z. Naturforsch. 33¢, 235 (1978). 16R. Bauerle, unpublished observations. 17C. Yanofsky and M. van Cleemput, J. Mol. Biol. 155, 235 (1982). 18H. Horowitz, J. van Arsdell, and T. Platt, J. Mol. Biol. 169, 775 (1983).
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Salmonella ANTHRANILATESYNTHASE-PRTRANSFERASE
369
Reaction Mixtures 1. AS-GIn Activity. The reaction mixture of 1.0 ml contains 0.25 mM chorismic acid (see volume XVIIA [45]), 20 mM e-glutamine, 10 mM MgCI2, 100 mM potassium phosphate buffer, pH 7.0, and enzyme. 2. AS-NH3 Activity. The reaction mixture of 1.0 ml contains 0.25 mM chorismic acid, 50 mM (NH4)2SO4, 10 mM MgCl2, 50 mM Tricine buffer, pH 8.0, and enzyme. 3. Col- and Coil-Complementing Activity. The reaction mixture is identical to that of the AS-Gln assay but also contains either 5 complementing units of purified component I subunit (for the assay of uncomplexed component II subunit) or 5 complementing units of purified component II subunit (for the assay of uncomplexed component I subunit). Procedure. The reaction mixture is placed in a round glass cuvette (8 mm i.d.) and equilibrated to 22°. The reaction is started by the addition of enzyme. The rate of formation of anthranilate is monitored continuously in a Farrand model A4 fluorometer equipped with a strip recorder. The excitation wavelength of 313 nm (half band width, _+ 5 nm) is selected by means of an interference filter; emitted fluorescence (peak wavelength of 390 nm) is selected by a combination of Coming 7-59 and 3-74 glass filters. The amount of anthranilate synthesized is quantified using a standard curve obtained with solutions of crystalline anthranilic acid prepared in appropriate reaction mixture buffer. Definition o f Unit and Specific Activity. One unit of AS-Gln, A S NH3, CoI-complementing, or CoII-complementing activity is defined as the amount of enzyme catalyzing the formation of 1 nmol of anthranilate per min at 22°. Specific activity is expressed as units per milligram of protein. Comments on the Assay. The AS-PRT complex and the AS partial complex can be assayed with either the AS-GIn or the AS-NH3 procedure. Uncomplexed component I subunit can be assayed with either the AS-NH3 or CoII-complementation procedure. Uncomplexed component II monomer can be measured by CoI-complementation or by assay of its PRT activity (see below). Component II dimer can only be assayed in the PRT reaction since it does not assemble with component I. Thus, the complementation assays are useful for determining the amount of uncomplexed component I subunit in a mixture containing intact complex and for estimating the relative abundance of the monomeric and dimeric forms of component II in a preparation. A comparison of the specific activities and the apparent KmS of the different enzyme forms in the various reactions is presented in Table I. AS-GIn activity is linear until chorismate is nearly exhausted. In the complementation assays, subunit assembly begins immediately and the
370
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
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TABLE I PROPERTIES OF ANTHRANILATE SYNTHASE--PHOSPHORIBOSYLTRANSFERASE COMPLEX AND SUBUN1TS
Property
Composition Molecular weight ~ Enzymatic activity c (units/mg) AS-GIn AS-NH3 Col-complementing Coil-complementing PRT Michaelis constant (Kin) Chorismate Glutamine (NH4)2SO4 Anthranilate PRPP
AS-PRT complex
AS partial complex
Component I
Component II monomer
Component II dimer
Coi2 ColI2 228,000
CoI 2 ColI* 156,000 b
CoI 57,000
Coil 56,900
ColI2 113,800
1000 950 --1400
1700 1700 ----
-300 -1800 --
~ m 1900 -3100
-__ 60 -3800
4 I~Md 0.7 m M d 1.0 m M d 8.3/zM e 6.7/zM e
3.3 I~M: 0.5 m M r 0.9 m M f ---
0.1 mMg -24 mMg ---
---6 tzM h 10/xM h
---4 Id,M h 10 lxM h
a Calculated from amino acid compositions as deduced from the D N A sequence of the trpE and trpD genes. b Calculated for the partial complex obtained by trypsin digestion of purified A S - P R T complex, assuming the carboxy-terminus of the digested component II subunit (Coil*) is Lys-193. Accordingly, the molecular weight of Coil* is 20,850. c Determined with enzymes purified and assayed as described herein. a F r o m E. Henderson, H. Nagano, H. Zalkin, and L. Hwang, J. Biol. Chem. 245, 1416 (1970); and D. Smith and R. Bauerle, Biochemistry 8, 1451 (1969). e F r o m E. Henderson, H. Zalkin, and L. Hwang, J. Biol. Chem. 245, 1424 (1970). YF r o m H. Tamir and P. Srinivasan, J. Biol. Chem. 244, 6507 (1969). g F r o m H. Zalkin and D. Kling, Biochemistry 7, 3566 (1968). h F r o m M. Grieshaber and R. Bauerle, Biochemistry 13, 373 (1974).
emerging AS-GIn activity usually attains a maximum linear rate in 3-5 min. The Tricine buffer system of the AS-NH3 assay is superior to others in that it provides an extended period (5-10 min) of linear activity in assays of the component I subunit. The use of large amounts of crude extract in the AS assays can be troublesome because of fluorescence quenching and depletion of chorismate by competing enzymes. The removal of anthranilate during the assay of preparations having PRT activity is not a problem because PRPP is not present. The continuous assay method described is rapid and convenient. A spectrofluorometer is not required. The use of round glass cuvettes is recommended for ease and economy and does not greatly compromise the
[47]
Salmonella ANTHRANILATESYNTHASE--PRTRANSFERASE
371
sensitivity of the assay, which can be extended to the picomole range. AS activity can also be determined by a discontinuous method using ethyl acetate extraction of acidified reaction mixture (see Volume XVIIA [47]).
Phosphoribosyltransferase Principle. PRT activity is assayed fluorometrically by measuring the rate of disappearance of anthranilate. Reaction Mixture. The reaction mixture of 1.0 ml contains 10 /zM anthranilic acid, 0.3 mM PRPP, I0 mM MgCI2, 100 mM Tricine buffer, pH 7.6, and enzyme. Procedure. The procedure is identical to that described above for the assay of AS activity except that the rate of disappearance of anthranilate is measured. Definition of Unit and Specific Activity. One unit of PRT activity is defined as the amount of enzyme catalyzing the removal of 1 nmol of anthranilate per min at 22 °. Specific activity is expressed as units per milligram of protein. Comments on the Assay. In assays of purified PRT preparations and of crude extracts lacking PRA isomerase-indoleglycerolphosphate synthase (PRI-InGPS), the product of the trpC gene (see Volume XVIIA [46]), the reaction proceeds at the maximum linear rate for only a limited period because of product inhibition by PRA. An extended period of linearity can be achieved by including in the reaction mixture 5 units of purified PRI-InGPS. PRI-InGPS converts the PRA product to InGP, preventing inhibition. Glutaminase Principle. Glutaminase activity is assayed spectrophotometrically by measuring the formation of glutamate in a coupled reaction with glutamate dehydrogenaseJ 9 Reaction Mixture. The reaction mixture of 1.0 ml contains 5 mM Lglutamine, 0.1 mM chorismate, 5 mM EDTA, 1 mM acetylpyridine NAD ÷, 100 mM tricine buffer, pH 8.0, 0.37 mg bovine liver glutamate dehydrogenase, and enzyme. Procedure. The reaction mixture is placed in a cuvette and equilibrated to 22 °. The reaction is started by the addition of enzyme. The rate of reduction of acetylpyridine NAD ÷ is monitored continuously in a recording spectrophotometer by measuring the increase in absorbance of the solution at 365 nm. The amount of glutamate formed is calculated I9 H. Nagano, H. Zalkin, and E. J. Henderson, J. Biol. Chem. 245, 3810 (1970).
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B I O S Y N T H E S I S O F T H E A R O M A T I C A M I N O ACIDS
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using a molar extinction coefficient of 9100 for acetylpyridine NADH at 365 nm. Definition of Unit and Specific Activity. One unit of glutaminase activity is defined as the amount of enzyme catalyzing the formation of 1 nmol of glutamate per min at 22°. Specific activity is expressed as units per milligram of protein. Comments on the Assay. The assay can be used for the A S - P R T complex and the AS partial complex. Glutaminase activity (units/mg) is equivalent to the AS-GIn activity of the preparation. Chorismate is required in the reaction as an activator. The presence of EDTA prevents the synthesis of anthranilate by the enzyme by chelating divalent cations required for the amination of chorismate. A second assay method is available in which the formation of glutamyl hydroxamic acid is measured using hydroxylamine as acceptor instead of H20.~9 Purification of Enzymes Purification of the enzymes is facilitated by three factors: (1) the use of strains carrying the trp operons of appropriate mutants cloned on multicopy plasmids; (2) the growth of these auxotrophs with limiting supplements of tryptophan to effect derepression of operon expression in the terminal stages of growth; and (3) the application of dye-ligand chromatography as a fractionation method. In those cases where these approaches can be applied together, it is possible to isolate substantial amounts of homogeneous enzyme in high yield from small volumes of culture in a minimum of time.
Bacterial Strains Although specific strains are listed here, many others are available that are just as appropriate as sources of the various enzymes. The A S PRT complex was purified from S. typhimurium strain TB 1409/pSTP89. Plasmid pSTP89 contains the entire trp operon of LT2 auxotroph TB79, which has an amber nonsense mutation (trpA71) in the trpA gene. The AS partial complex was purified from strain TB1409/pSTP38. Plasmid pSTP38 contains the trp operon of LT2 auxotroph TB37, which has an ochre nonsense mutation (trpD479) at the beginning of the PRT-coding region of trpD.l° Component I subunit was purified from strain TB 1409/ pSTP66. Plasmid pSTP66 contains the trp operon of LT7 strain TB8, which has a deletion (AtrpDCBA43) that removes all structural genes but trpE. The component II subunit was purified from the single copy LT2 strain TB41, which has a UGA nonsense mutation (trpE703) in the trpE
[47]
Salmonella ANTHRANILATESYNTHASE-PRTRANSFERASE
373
gene. All recombinant plasmids are derivatives of plasmid pBR322 that were constructed by an in viuo cloning procedure, z° Host strain TB 1409 has a deletion of the entire trp operon (AtrpEDCBAI67) and is RecA(recA1). Plasmid subclones with smaller inserts have been constructed from those described above, including several that may be used for the production of the component II subunit/6 These are recommended for future applications.
Growth of Bacteria Bacteria are grown in minimal salts medium (KzHPO4, 10.5 g/liter; KHzPO4, 4.5 g/liter; ( N H 4 ) z S O 4 , 1.0 g/liter; MgSO4, 0.1 g/liter) supplemented at the time of inoculation with glucose (2.5 g/liter), Difco casamino acids (2.0 g/liter), and a growth limiting amount of L-tryptophan (4 or 5 mg/liter). Small preparations (5-10 liters) are grown in 1 liter volumes in 2-liter baffled flasks. Larger cultures (150-300 liters) are grown in a batch fermentor. The medium is inoculated with 50 ~l/liter of an overnight broth culture of the strain containing, in the case of the plasmidbearing strains, ampicillin (50 mg/liter). The culture is incubated with vigorous aeration at 37°. The tryptophan supplement becomes exhausted after about 10 hr (OD550 nm ~ 1.5-1.7) at which time derepression of trp operon expression ensues. The culture is incubated for an additional 1-2 hr before the cells are harvested by centrifugation.
Binding Interactions of the Enzymes with Dye-Conjugated Agaroses The A S - P R T and AS partial complexes and the component I and component II subunits exhibit distinct patterns of binding to the five dyeconjugated M~ttrex Gel chromatographic media manufactured by Amicon Corp. zl (Table II). While salt elution uniformly displaces the bound enzymes from the gel media, low concentrations of tryptophan and, to a lesser extent, chorismate, are effective in selectively eluting the AS-PRT complex from Orange A gel, the AS partial complex from the Orange A and Red A gels, and the component I subunit from the Blue A and Red A gels. There is no similar elution of the bound component II subunit by its ligands, whether applied singly or in combinations. The specificities of adsorption of the enzymes to the various gel media suggest differences in the surface structures of the complexed and uncomplexed subunits. The effectiveness of L-tryptophan and chorismate in 2o T. Patterson and R. Bauerle, Plasmid 12, 149 (1984). 2~ In " D y e - L i g a n d C h r o m a t o g r a p h y . " Amicon Corporation, Lexington, M a s s a c h u s e t t s , 1980.
374
[47]
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
T A B L E II BINDING AND LIGAND ELUTION OF ANTHRANILATE SYNTHASE-PHOSPHORIBOSYLTRANSFERASE COMPLEX AND SUBUNITS TO AMICON M.~TREX AGAROSE GELS" Percentage of applied activity
Enzyme AS-PRT complex
AS partial c o m p l e x
Component I subunit
C o m p o n e n t II subunit
Bound/eluted Bound Eluted by L-Tryptophan Chorismate L-Glutamine Bound Eluted by L-Tryptophan Chorismate L-Glutamine Bound Eluted by L-Tryptophan Chorismate Bound Eluted by Anthranilate L-Glutamine PRPP L-Tryptophan
M~trex Blue A
M~trex Orange A
Mhtrex Red A
M~ttrex Green A
Mfitrex Blue B
97
92
99
97
8
0 0 0 38
80 8 2 78
0 0 0 100
0 0 0 17
---4
---97
67 16 7 6
91 34 0 89
---28
---13
92 29 97
--27
61 24 97
--93
--20
0 6
---
5 5 5 7
0 5 7 5
-----
6
--
6
--
a T h e results are from e x p e r i m e n t s using crude extracts o f derepressed cultures of appropriate m u t a n t strains o f S. typhimurium. Essentially identical results were obtained using purified e n z y m e s . C o l u m n s (2 ml) o f each M~ttrex gel were regenerated at r o o m temperature with 10 ml o f 8 M urea a n d w a s h e d with 30 ml of buffer B. After this, all steps were p e r f o r m e d at 4 °. C r u d e extract (0.5 ml) containing approximately 1.5 m g protein was applied to the gel and w a s h e d in with 0.3 ml of buffer B. T h e c o l u m n was closed off for 30 rain, w a s h e d with 10 ml o f buffer B, and then eluted with 10 ml of buffer B containing the indicated ligand. Ligand concentrations were as follows: L-tryptophan, 0.5 m M ; c h o r i s m a t e , 0.5 m M ; L-glutamine, l0 m M ; anthranilate, 0. l raM; PRPP, 0.5 m M . A S - P R T c o m p l e x a n d AS partial c o m p l e x were a s s a y e d by A S - G l n activity, compon e n t I s u b u n i t by A S - N H 3 activity, and c o m p o n e n t II subunit (a mixture of m o n o m e r a n d dimer) by P R T activity. T h e percentage of activity indicated as " b o u n d " is the difference b e t w e e n the activity applied to the column and that present in the wash.
SalmonellaANTHRANILATESYNTHASE-PRTRANSFERASE
[47]
375
eluting the bound enzymes containing the component I subunit indicates that, in both the complexed and uncomplexed state, significant conformational changes accompany the binding of these two ligands to the subunit. Further analysis has established that when coupled with the specificity of tryptophan elution, M~ttrex Gel Orange A is a superior chromatographic medium for the purification of both the AS-PRT complex and the AS partial complex, while M~trex Gel Blue A is best for the preparation of the component I subunit. The critical concentration of tryptophan in the column buffer, as determined by gradient analysis, is about 5/zM in all cases. Mhtrex Gel Red A is not as useful as the Orange A gel for the preparation of the AS partial complex since there is a significant amount of uncomplexed component I subunit in the trpD mutant extract which is coeluted by tryptophan. The Blue A gel yields a more highly purified preparation of component I subunit than does the Red A gel. Although specific ligand elution of the component II subunit bound to the Blue A, Red A, and Green A gels does not occur, these gels are very effective chromatographic media for the purification of the subunit when used with salt gradient elution.
Buffers Buffer A: Potassium phosphate, 100 raM, pH 7.0, containing glycerol, 1 mM dithiothreitol, and 0.1 mM EDTA Buffer B: Potassium phosphate, 100 mM, pH 7.5, containing glycerol, 1 mM dithiothreitol, and 0.1 mM EDTA Buffer C: Potassium phosphate, 50 mM, pH 7.5, containing glycerol, 0. I mM dithiothreitol, and 0.1 mM EDTA Buffer D: Potassium phosphate, 10 mM, pH 7.5, containing glycerol, 0.1 mM dithiothreitol, and 0.1 mM EDTA
10% 10% 10% 10%
Preparation of Crude Extracts After harvest, the cells are washed once with cold saline (0.9% NaCI) and resuspended in either buffer A or B, as indicated, using 4 ml per gram packed cells. Small preparations are sonified for 45 sec in 10-12 aliquots in an ice-jacketed 50 ml stainless-steel beaker using a Branson model W185D sonifier operated at 75 W. Large preparations are sonified at 100 W in a 60 ml water-jacketed (0°) continuous-flow Rosette cell using an exchange rate of 60 ml of cell suspension per min. The sonicate is centrifuged at 48,000 g for 60 min (27,000 g for large preparations) and the supernatant collected. All subsequent steps are carried out at 0-4 °.
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B I O S Y N T H E S I S O F T H E A R O M A T I C A M I N O ACIDS
[47]
Preparation of Mgttrex Gel Columns for Enzyme Purification Immediately before use, gel columns are cleared of unconjugated dye and other adsorbed material by washing with at least one column volume of 8.0 M urea and then equilibrated with 2-3 column volumes of buffer. Buffer A is used for M~ttrex Gel Orange A and buffer B for M~ttrex Gel Blue A. These preliminary steps are done at room temperature because of the solubility limits of urea. After use, the gel columns can be regenerated for reuse by first washing with buffer containing 1.5 M KC1 and then with urea and buffer as described above.
Purification of Anthranilate Synthase-Phosphoribosyltransferase Complex Step 1. Preparation of Crude Extract. Cells (22 g) obtained from 5 liters of culture of S. typhimurium strain TB1409/pSTP89 limited with 5 mg/liter of L-tryptophan are resuspended in buffer A and crude extract is prepared as described above. Step 2. Ammonium Sulfate Fractionation. Crude extract (96 ml) is brought to 40% saturation by the addition of 23.3 g solid ultrapure ammonium sulfate with constant stirring. The solution is stirred for an additional hour and then centrifuged at 12,000 g for 30 min. The precipitate is dissolved in 10 ml buffer A and dialyzed against 500 ml of the same buffer for 12 hr with 3 changes. Step 3. Mgttrex Gel Orange A Chromatography. The dialyzed preparation is diluted to 35 ml with buffer A and applied to a 2.0 × 22 cm column of Mhtrex Gel Orange A (70 ml bed volume). After the enzyme solution is loaded into the gel bed, the column is closed off and allowed to stand for 2 hr. The column is washed with 100 ml buffer A and then eluted with buffer A containing 50/xM L-tryptophan at a flow rate of 10 ml/hr. Fractions of 5 ml are collected. The bulk of the A S - P R T activity (75% of that applied) emerges in 6 fractions beginning approximately 70 ml after elution with tryptophan is begun. A minor portion of the activity (about 15%) appears in the wash. This is presumably the membrane-bound particulate form of the enzyme present in crude cell-free preparations, since its PRT activity is masked relative to its AS activity) The results of the purification are presented in Table III. The isolated A S - P R T complex is essentially homogeneous as judged by SDS-polyacrylamide gel electrophoresis (SDS PAGE) which resolves the component I and component II subunits (Fig. 1, lane B). The enzyme is stable when stored as a solution at - 2 0 ° in buffer A containing 50% glycerol, losing less than 10% of its activity per year.
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377
TABLE 111 PURIFICATION OF ANTHRANILATE SYNTHASE--PHOSPHORIBOSYLTRANSFERASECOMPLEX
Total activity
Fraction 1. Crude extract 2. Ammoniumsulfate (0-40% saturation) 3. M~ttrexGel Orange A
Recovery Specificactivity
Total Total AS-GIn PRT volume protein~ AS-GIn PRT AS-GIn PRT (units/ (units/ (ml) (mg) (units) (units) (%) (%) mg) rag) 96 35
1277 363
63,900 100,800 47,000 69,750
100 74
100 69
50 130
79 192
28
31
31,500 43,700
52
44
1015
1410
" Determinedby the methodofM. M. Bradford[Anal.Biochem.72, 248 (1976)] usingbovine serum albumin as standard.
Comments on the Purification Procedure. The affinity of the AS-PRT complex, and of proteins in general, for the M~trex Gel Orange A is weak. The use of buffer A (pH 7.0) instead of buffer B (pH 7.5), which was used in the preliminary binding experiments of Table II, greatly enhances the performance of the Orange A gel as an affinity adsorbent. It is also important to adjust the sample load to a volume equivalent to the void volume of the gel bed (about 50% of the total bed volume) and to interrupt the column flow for several hours immediately after the sample is applied. Under the conditions described here, about 15% of the protein applied to the gel is tightly bound. More than half of this is the AS-PRT complex. The bulk of the applied protein (about 65%) and the nucleic acid fraction appears in the first half of the column wash. A smaller protein fraction (about 20%) is retarded by the gel and emerges in the second half of the wash, immediately before the tryptophan-eluted protein peak. This method of purification of the AS-PRT complex effectively replaces procedures used previously. 2,~z Purification of Anthranilate Synthase Partial Complex Step 1. Preparation of Crude Extract. Cells (28 g) obtained from 5 liters of culture of S. typhimurium stain TB 1409/pSTP38 limited with 5 mg/liter of L-tryptophan are resuspended in buffer A and crude extract is prepared as described above. Step 2. Ammonium Sulfate Fractionation. Crude extract (117 ml) is brought to 38% saturation by the addition of 26.9 g solid ultrapure ammo-
378
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
A
B
C
D
[47]
E
kD
92.5 66.2
45
31
21.5 14.4 FIG. 1. SDS-polyacrylamide gel electrophoresis of AS-PRT complex, AS partial complex, and component I subunit purified by dye ligand chromatography. Lane A, molecular weight standards (Bio-Rad Labs.); lane B, AS-PRT complex (6 /zg); lane C, AS-PRT complex (6/xg) digested with trypsin; lane D, AS partial complex (4/~g) isolated from the trpD479 ochre mutant; lane E, component I subunit (3/zg). Electrophoresis was in a 10% polyacrylamide slab gel using the discontinuous buffer system of Laemmli (see this series, Vol. 104 [12]).
nium sulfate with constant stirring. The solution is stirred for an additional hour and then centrifuged at 12,000 g for 30 min. The precipitate is dissolved in 10 ml buffer A and dialyzed against 500 ml of the same buffer for 12 hr with 3 changes.
[47]
Salmonella ANTHRANILATE SYNTHASE-PR TRANSFERASE
379
TABLE IV PURIFICATION OF ANTHRANILATE SYNTHASE PARTIAL COMPLEX
Fraction 1. Crude extract 2. Ammonium sulfate (0-38% saturation) 3. Mhtrex Gel Orange A
Total Total Totalactivity Recovery Specificactivity volume proteina AS-GIn AS-GIn AS-GIn (ml) (mg) (units) (%) (units/rag) 117 35
1100 425
149,000 130,000
100 87
135 306
37
59
102,000
68
1730
Determined by the method of M. M. Bradford [Anal. Biochem. 72, 248 (1976)] using bovine serum albumin as standard.
Step 3. Mdtrex Gel Orange A Chromatography. The dialyzed preparation is diluted to 35 ml with buffer A and applied to a 2.0 × 22 cm column of M~trex Gel Orange A (70 ml bed volume). The column is developed as described in step 3 of the previous section. Nearly 80% of the applied activity is recovered in the tryptophan-eluted peak; only 4% is present in the unbound wash fraction. The results of the purification are presented in Table IV. The isolated AS partial complex is essentially homogeneous as judged by SDS-PAGE (Fig. 1, lane D). The component II* fragment of this mutant complex has an estimated molecular weight of 25,000, consistent with the location of the trpD479 ochre mutation. It is slightly larger than the component II* fragment generated by trypsin digestion of the purified AS-PRT complex (see below). The component I subunit is intact, as expected. Comments on the Purification Procedure. Strain TB1409/pSTP38 is ideal as a source of a mutationally generated AS partial complex since its nonsense mutation lies very close if not within the hinge region between the amino-terminal glutaminase domain and the carboxy-terminal PRT domain of the component II subunit. Unlike other trpD nonsense mutants of the PRT domain, its AS-GIn activity is near the wild-type level and no activity is found in the particulate form in crude extracts. However, as determined by Coil complementation, about 25% of the component I of TB1409/pSTP38 is uncomplexed. Uncomplexed component I molecules are completely resolved from the AS partial complex by Mhtrex Gel Orange A chromatography, as they emerge in the retarded fraction of column wash. All comments included in the previous section on the purification of the A S - P R T complex apply here as well.
380
B I O S Y N T H E S OF I S THE AROMATIC AMINO ACIDS
[47]
Preparation of AS Partial Complex by Tryptic Digestion of AS-PR T Complex An alternate method for the preparation of AS partial complex is by proteolytic removal of the PRT domain of the intact AS-PRT complex. 22 Trypsin digestion of homogeneous AS-PRT complex is followed by Sephadex G-200 gel filtration of the digest mixture to remove the proteolyzed fragments, any undigested complex molecules, and the trypsin. It is also possible to treat crude extract containing the AS-PRT complex with trypsin 3 and then to purify the AS partial complex by M~trex Gel Orange A chromatography as described in the previous section. In either case, 40/~g of trypsin is added per mg protein, the mixture incubated at 20 ° for 1-2 hr, and then an amount of soybean trypsin inhibitor equal to the amount of trypsin used is added. PRT activity is completely destroyed by the treatment while AS-GIn activity remains unchanged. The component II* fragment of the AS partial complex produced by trypsin digestion is slightly smaller (estimated molecular weight of 24,000) than that of the partial complex isolated from the TB1409/pSTP38 trpD ochre mutant strain (Fig. 1, lanes C and D). Other than this, the two enzymes are indistinguishable in properties. Considering the estimated molecular weights and the specific origins of the two component II* fragments, it can be speculated that the carboxy-terminus of the trypsingenerated fragment is Lys-193 and that of the ochre fragment is Ala-210. The component I subunit is not "nicked" or significantly degraded by trypsinolysis of the AS-PRT complex (Fig. 1, Lane C).
Purification of Component I Subunit Step 1. Preparation of Crude Extract. Cells (38 g) obtained from 10 liters of culture of S. typhimurium strain TB1409/pSTP66 limited with 4 mg/liter of L-tryptophan are resuspended in 76 ml of buffer B and crude extract is prepared as described above. Step 2. Ammonium Sulfate Fractionation. Crude extract (93 ml) is brought to 40% saturation by the addition of 22.6 g solid ultrapure ammonium sulfate with constant stirring. The solution is stirred for an additional hour and then centrifuged at 12,000 g for 30 min. The precipitate is dissolved in I0 ml buffer B and dialzed against 500 ml of the same buffer for 12 hr with 3 changes. ,_2The previous report in this series on the anthranilate synthase of S. typhimurium (Volume XVIIA [48a]) dealt unknowingly with the AS partial complex. Proteolytic enzyme contaminants present in the crude intestinal lipase used in an early step of the purification procedure to solubilize the particulate enzyme fraction digested the AS-PRT complex to the AS partial complex (see Ref. 3).
[47]
Salmonella ANTHRANILATESYNTHASE-PRTRANSFERASE
381
TABLE V PURIFICATION OF COMPONENT 1 SUBUN1T OF ANTHRANILATE SYNTHASE--PHOSPHORIBOSYLTRANSFERASE COMPLEX
Fraction 1. Crude extract 2. Ammoniumsulfate (0-40% saturation) 3. M~trexGel Blue A
Total Total Totalactivity Recovery Specificactivity volume proteina AS-NH3 A S - N H 3 AS-NH3 (ml) (mg) (units) (%) (units/rag) 93 30
1620 460
28,300 23,500
100 83
17 51
24
63
19,000
67
300
" Determinedby the method of M. M. Bradford [Anal. Biochem. 72, 248 (1976)] using bovine serum albuminas standard.
Step 3. M~trex Gel Blue A Chromatography. The dialyzed preparation from step 2 is brought to 30 ml with buffer B and is applied to a 2.0 x 19 cm column of M~trex Gel Blue A (60 ml bed volume) equilibrated with buffer B. The column is developed as described in step 3 of the section on the purification of the AS-PRT complex, with the exception that buffer B is used throughout. Nearly 70% of the applied activity is recovered in 5 fractions beginning about 60 ml after the start of the tryptophan elution. The results of the purification are presented in Table V. The isolated component I subunit is essentially homogeneous when analyzed by SDSPAGE (Fig. 1, lane E) and by HPLC gel filtration. The enzyme is stable when stored at -20 ° in buffer B containing 50% glycerol. Comments on the Purification Procedure. M~trex Gel Blue A has an excellent binding capacity for proteins. Loading experiments indicate that, under the conditions described here, 35 to 40 mg of crude cell extract can be applied per ml of gel bed before the efficiency of binding of component I subunit begins to be compromised. In the purification procedure summarized in Table V, about 25% of the protein applied to the gel is tightly bound, as a major portion (75%) is present in the column wash along with the nucleic acid fraction of the preparation. About half of the bound protein is the component I subunit which is specifically eluted by the tryptophan-containing buffer. This method of purification of the component I subunit effectively replaces earlier procedures. 5 Purification o f Component H Subunit 6 Step 1. Preparation o f Crude Extract. Cells (700 g) obtained from 300 liters of culture of S. typhimurium strain TB41 grown in a batch fermentor
382
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[47]
with tryptophan limitation at 4 mg/liter are resuspended in buffer C and crude extract is prepared as described above. Step 2. Treatment with Streptomycin Sulfate. Nucleic acids are precipitated from the crude extract (1200 ml) by the addition, with constant stirring, of 120 ml of 20% streptomycin sulfate in buffer C. After 30 min the precipitate is removed by centrifugation at 27,000 g for 30 min. Step 3. Ammonium Sulfate Fractionation. The supernatant from step 2 (1340 ml) is brought to 40% saturation by the addition of 325 g solid ultrapure ammonium sulfate with constant stirring. After 30 min the precipitate is collected by centrifugation at 24,000 g for 30 min and redissolved in 90 ml buffer C containing 1 mM dithiothreitol. The preparation is dialyzed against 4 liters of the same buffer for 8 hr with two changes. Step 4. Ultracentrifugation. The dialyzed extract is centrifuged at 135,000 g for 3 hr and the pellet discarded. Step 5. DEAE-Sephadex Chromatography. The supernatant is applied to a column (7.5 x 80 cm) of DEAE-Sephadex A50 equilibrated with buffer D. The gel column is washed with 6 liters of buffer D and then eluted with a 4 liter KCI gradient (0-0.5 M) prepared in buffer D, at a flow rate of I00 ml/hr. Fractions of 100 ml are collected. Peak fractions containing PRT activity are pooled and brought to 60% saturation by the addition of solid ammonium sulfate (312 g/800 ml of pooled material). The precipitate is collected by centrifugation at 24,000 g for 30 min, dissolved in 20 ml of buffer C, and dialyzed against 4 liters of buffer C for 6 hr with one change. Step 6. Sephadex G-150 Gel Filtration. The dialyzed preparation is applied to the bottom of a column (10 x 115 cm) of Sephadex G-150 equilibrated with buffer C. The column is developed by upward flow with buffer C at a flow rate of 100 ml/hr. Fractions of 150 ml are collected. Those containing the PRT activity (between 5.8 and 7.0 liters of effluent) are pooled and concentrated to 30 ml by ultrafiltration. Step 7. Hydroxylapatite Fractionation. The enzyme concentrate is dialyzed against 1 liter of buffer D for 12 hr with 3 changes and applied to a column (1.5 x 27 cm) of hydroxylapatite (BioGel HT) equilibrated with buffer D. The column is washed with 80 ml of buffer D and then eluted with 100 ml of buffer C at a flow rate of 10 ml/hr. Fractions of 4 ml are collected throughout the wash and elution. Component II dimer does not adsorb to the hydroxylapatite and is recovered in the wash in 8 fractions; component II monomer elutes as a sharp peak beginning about 50 ml after the change to buffer C. Both component I monomer and dimer are stable when stored at - 2 0 ° in buffer C containing 50% glycerol, each losing about 5% of its PRT activity per year.
[47]
Salmonella ANTHRANILATESYNTHASE-PR TRANSFERASE
383
TABLE VI PURIFICATION OF COMPONENT I I SUBUNIT OF ANTHRANILATE SYNTHASE--PHOSPHORIBOSYLTRANSFERASE COMPLEX a
Fraction 1. Crude extract 2. Streptomycin sulfate 3. Ammonium sulfate (0-40% saturation) 4. 135,000 g supernatant 5. DEAE-Sephadex 6. Sephadex G-150 7. Hydroxylapatite Wash (ColI Dimer) Eluate (ColI Monomer)
Total volume (mi)
Total Total activity proteinh PRT (mg) (units)
1200 1340 120
17,700
Recovery PRT (%)
Specific activity PRT (units/rag)
100 93 78
28
7,000
498,000 466,000 398,000
117 40 1300
6,700 560 200
402,000 316,000 217,000
81 63 44
59 590 1085
30 15
17 23
65,000 7 i ,000
13 14
3800 3100
56
a Taken from M. Grieshaber and R. Bauerle, Biochemistry 13, 373 (1974). b Determined by the method of Lowry (see this series, Vol. 3 [73]) using bovine serum albumin as standard.
C o m m e n t s on the Procedure. C o m p o n e n t II m o n o m e r and dimer copurify in steps 2 - 5 and are only partially resolved by Sephadex G-150 fractionation, as evidenced b y the disproportionate level of CoI complementing activity in the trailing fractions of the PRT activity peak. 6 The c o m p o n e n t II d i m e r is believed to be an artifact of preparations having high concentrations of u n c o m p l e x e d c o m p o n e n t II subunit, arising by the f o r m a t i o n of a disulfide linkage b e t w e e n the active site cysteines of two subunit molecules. 9 The final hydroxylapatite treatment effectively resolves the physiologically important c o m p o n e n t II m o n o m e r from the artifactual c o m p o n e n t II dimer. It is possible to increase the yield of m o n o m e r b y dissociating dimer molecules present in the preparation by raising the concentration o f dithiothreitol in the e n z y m e concentrate of step 6 to 10 m M and then incubating the preparation at 41 ° for 60 min immediately before the hydroxylapatite fractionation. The purified c o m p o n e n t I I subunits (T~,ble VI) are estimated to be 9 0 95% h o m o g e n e o u s b y S D S - P A G E . 6 Strains with the trpD gene cloned on a multicopy plasmid p r o d u c e 5 - 1 0 times as m u c h e n z y m e as the single c o p y strain. The use of these should m a k e it possible to simplify the purification p r o c e d u r e and to obtain e n z y m e of increased purity.
384
[47]
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS 1.0-~ern~_
~
=
?
w
0.8
~
0.7
~
_~0.5~~ )" p-
0.6
.¢
0.4
&
Complex
7
Co II D
v
~
0.3 D u) "'
Complex 0.2
~ 0.1
~t~
Co II M
~Co I I
0
10
I
20
--
I
I
I
30
40
50
I
60
MINUTES
FIG. 2. Effect of trypsin treatment on the enzymatic activities of the AS-PRT complex and subunits. Digest mixtures were prepared in buffer C and contained the following in a final volume of I mh (1) 500/zg A S - P R T complex + 20/xg trypsin; (2) 210/zg component I subunit + 0.3/xg trypsin; (3) 500/zg component II monomer + 7/xg trypsin; (4) 400 p.g component II dimer + 7 /zg trypsin. Incubation was at 25 °. Samples were removed to enzyme reaction mixtures at the indicated times for the assay of residual AS (closed symbols) and PRT (open symbols) activity. AS-PRT complex was assayed in the AS-GIn and PRT reactions; component I subunit in the AS-NH3 reaction; component II monomer and dimer in the PRT reaction.
Domain Structure of Component I and Component H Subunits The existence of independent functional domains for glutaminase and PRT activity in the component II subnit has been demonstrated by mutational analysis of the trpD gene and by in vitro proteolytic digestion of the trpD polypeptide. Monofunctional mutant polypeptides of both types exist as stable enzyme forms in various nonsense and deletion mutant strains. 1°,14-16The carboxy-terminal PRT domain of both complexed 3 and uncomplexed 6 component II is destroyed by in vitro trypsinolysis (Fig. 1), while the amino-terminal glutaminase domain remains structurally and functionally intact. The proteolytic attack entails the sequential removal of peptides from the carboxy-terminus, 1° rather than the nicking of an interdomain hinge. The uncomplexed monomeric subunit is much more susceptible to proteolysis than the complexed subunit, as indicated by the relative rates of inactivation of PRT activity upon trypsin treatment (Fig. 2). The dimeric subunit, however, is highly resistant to proteolytic attack under the same conditions.
[47]
SalmonellaANTHRANILATESYNTHASE-PRTRANSFERASE
385
MINUTES in
kD 92.5 66.2 45 31
21.5 14.4. FIG. 3. SDS-polyacrylamide gel electrophoresis of tryptic digest of component I subunit. Component I (140 p~g) in 700/zl of TE buffer (50 mM Tris-HCl, 2 mM EDTA, pH 7.6) was digested with 0.2/zg trypsin (TPCK treated) at 25°. Samples of 25/zl were removed at the indicated times to 35 /~1 of sample denaturing buffer (see this series, Vol. 104 [12]) and immediately heated at 100° for 3 min. The entire sample was then analyzed by SDS-PAGE as described in Fig. 1.
Mutational analysis and physical mapping of the trpE gene indicate that all amino acid residues critical for catalytic activity of the component I subunit are found within its carboxy-terminal 200 residues. 16 Critical residues identified by mutation include Thr-329, Glu-358, His-398, Thr425, Gly-485, and Glu-495. Chemical modification studies indicate that Cys-377 is also an essential residue. 23 The results of limited proteolysis experiments suggest the possibility of domain substructures in the polypeptide. While the complexed component I subunit is remarkably resistant to trypsin digestion, the uncomplexed subunit is extremely susceptible (Figs. 1 and 2). Trypsin cleaves the component I subunit into several metastable fragments which appear in sequence (Fig. 3). Initially, a doublet of large fragments with molecular 23 H. Zalkin, J. Paluk, M. van Cleemput, S. Moye, and C. Yanofsky, J. Biol. Chem. 259, 3985 (1984).
386
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[48]
weights of 45,000 and 42,000 is formed from the 58-kDa polypeptide. The loss of catalytic activity is congruent with the initial cleavage as determined by enzymatic assay and gel densitometry. The initial doublet is then reduced to a doublet of fragments with molecular weights of 33,000 and 30,000, respectively. Several smaller fragments appear which may be the products of the initial cleavages. The presence of ligands of the subunit in the digest mixture (i.e., tryptophan, chorismate, Mg2+, and NH~) affects the rate of digestion in some cases, but does not alter the pattern of the proteolytic fragments.
[48] P h o s p h o r i b o s y l a n t h r a n i l a t e Isomerase-Indoleglycerol-Phosphate Synthase from Escherichia coli By KASPER KIRSCHNER, HALINA SZADKOWSKI, THEODORE S. JARDETZKY,
and V R E N I H A G E R
Introduction Phosphoribosylanthranilate (PRA) isomerase catalyzes the fourth step of tryptophan biosynthesis PRA --* I-(2-carboxyphenylamino)- I-deoxyribulose 5'-phosphate (CdRP)
( 1)
This reaction is a practically irreversible Amadori rearrangement. Indoleglycerol-phosphate (InGP) synthase catalyzes the fifth step CdRP ~ InGP + HCO~ + H20
(2)
in which ring closure to the indole nucleus occurs, again a practically irreversible reaction. These two reactions have been shown to occur in every organism capable of synthesizing tryptophan. In enteric bacteria (Escherichia coli, Salmonella typhimurium, Serratia marcescens) a monomeric protein with a single polypeptide chain of Mr 49,400 catalyzes both reactions. ~-4 In other gram-negative ( P s e u d o m o n a s putida, ~,2Acinei I. P. Crawford, CRC Crit. Rev. Biochem. 8, 175 (1980). 2 I. P. Crawford, in "MultifunctionalProteins" (H. Bisswanger and E. Schmincke-Ott, eds.), p. 151. Wiley, New York, 1980. 3j. F. McQuadeIII and T. E. Creighton,Eur. J. Biochem. 16, 199 (1970). 4j. M. Potts and G. Drapeau, J. Bacteriol. 111, 334 (1972).
METHODS IN ENZYMOLOGY,VOL. 142
Copyright © 1987by AcademicPress, Inc. All rights of reproductionin any form reserved.
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[47]
tase has been identified as a separate enzyme that is required for the activity of chorismate synthase from this source? Physical Properties. Sucrose density gradient analyses of chorismate synthase indicate that the enzyme can exist in at least two active forms with sedimentation values of approximately 8 S and 10 S. 5 From SDS gel electrophoresis of the purified enzyme a single band with a molecular weight of 55,000 is obtained.l These results are consistent with the idea that the 8 S form of chorismate synthase is a dimer of the 55,000 molecular weight subunit and that the 10 S form is a tetramer. Since both the flavin reductase and the chorismate synthase activity appear to be catalyzed by a single protein, it is likely that in N. crassa chorismate synthase exists as a bifunctional enzyme. In contrast in E. coh~ and B. subtilis, 3 as has already been noted, flavin reductase and chorismate synthase are separable enzymatic activities.
[47] A n t h r a n i l a t e S y n t h a s e - A n t h r a n i l a t e Phosphoribosyltransferase Complex and Subunits of Salmonella t y p h i m u r i u m B y R O N A L D B A U E R L E , J O H N H E S S , and SARAH FRENCH
Introduction In Salmonella typhimurium and a number of other enteric bacteria, including Escherichia coli and Enterobacter aerogenes, the first two specific steps of tryptophan biosynthesis are catalyzed by a multifunctional allosteric enzyme, the anthranilate synthase-anthranilate 5-phosphorylribose-l-pyrophosphate phosphoribosyltransferase (AS-PRT) complex (Volume XVIIA [46,47,48a]). The A S - P R T complex is a tetramer made up of two molecules each of subunits component I and component II, the products of trpE and trpD, the first two genes of the trp oper0n.I The intact A S - P R T complex, 2 an AS partial complex derived by proteolytic digestion of the intact complex,3,4 and the uncomplexed subunits obtained from trpE and trpD mutant strains 5,6 have been purified and well characI R. Bauerle and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 31, 203 (1966). E. J. H e n d e r s o n and H. Zalkin, J. Biol. Chem. 246, 6891 (1971). 3 L. H. H w a n g and H. Zalkin, J. Biol. Chem. 246, 2338 (1971). 4 H. T a m i r and P. R. Srinivasan, J. Biol. Chem. 244, 6507 (1969). 5 H. Zalkin and D. Kling, Biochemistry 7, 3566 (1968). 6 M. G r i e s h a b e r and R. Bauerle, Biochemistry 13, 373 (1974).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987by AcademicPress, Inc.
All rights of reproduction in any form reserved.
[47]
Salmonella ANTHRANILATE SYNTHASE--PR TRANSFERASE
367
terized. The properties of the enzymes have been comprehensively rev i e w e d . 7-9
The A S - P R T complex and the AS partial complex, which is a tetramer made up of intact component I and an amino-terminal fragment of component II ~° (component II*), catalyze the formation of anthranilate from chorismate and L-glutamine 2a [Reaction (l), AS-GIn activity]. Both enzymes also catalyze what can be considered partial reactions, namely, the deamidation of L-glutamine 11 [Reaction (2), glutaminase activity] and the synthesis of anthranilate from chorismate and ammonia 2-4 [Reaction (3), AS-NH3 activity]. The AS partial complex is homologous in structure and function to the monofunctional anthranilate synthase of Serratia marc e s c e n s (this series, Vol. 113 [37]). Uncomplexed component I has A S NH3 activity but lacks AS-GIn activity) Neither the component I nor component II subunit has glutaminase activity. When mixed together in vitro the two subunits spontaneously assemble to form an A S - P R T complex indistinguishable from the in vivo complex. 12The ability to utilize Lglutamine derives from a composite active site created by the aggregation of component I with the amino-terminal domain of component II. In the AS-GIn reaction, glutamine is bound to residue Cys-84 of component II, and its amide group is removed and transferred to a site on component I which uses it in the amination of chorismate. 9 The AS-GIn, AS-NH3, and glutaminase activities of the A S - P R T complex and the AS partial complex as well as the AS-NH3 activity of the component I subunit are feedback inhibited by tryptophan, z-5 Inhibition is competitive with respect to chorismate and noncompetitive with respect to glutamine and ammonia. The A S - P R T complex and component II subunit catalyze the formation of N-(5'-phosphoribosyl)anthranilate from anthranilate and PRPP (5phosphorylribose-l-pyrophosphate) [Reaction (4), PRT activity]. 6,13 The PRT activities of the two are equivalent. Uncomplexed component II forms a stable dimeric form in solution which retains full PRT activity but has lost the ability to assemble in vitro with component 1.6 PRT activity resides in the carboxy-terminal domain of the bifunctional component II 7 H. Zalkin, Adv. Enzymol. 38, 1 (1973). 8 H. Zalkin, in "The Enzymes of Glutamine Metabolism" (S. Prusiner and E. R. Stadtman, ed.), p. 523. Academic Press, New York, 1973. 9 H. Zalkin, in "Multifunctional Proteins" (H. Biswanger and E. Schmincke-Ott, eds.), p. 123. Wiley, New York, 1980. 10 M. Grieshaber and R. Bauerle, Nature (London), New Biol. 236, 232 (1972). II H. Nagano, H. Zalkin, and E. J. Henderson, J. Biol. Chem. 245, 3810 (1970). 12 D. Smith and R. Bauerle, Biochemistry 8, 1451 (1969). 13 E. J. Henderson, H. Zalkin, and L. H. Hwang, J. Biol. Chem. 245, 1424 (1970).
368
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[47]
molecule. 1°,14,15 T h e P R T activity of the A S - P R T complex is subject to f e e d b a c k inhibition b y t r y p t o p h a n 6,~3 mediated by its binding to the component I subunit. Most studies o f the A S - P R T c o m p l e x and c o m p o n e n t II subunit have b e e n carried out with e n z y m e s isolated from the LT2 strain of S. typhimurium, while those of the c o m p o n e n t I subunit with e n z y m e from the LT7 strain. N o major differences in properties of the two c o m p o n e n t I subunits h a v e b e e n noted; h o w e v e r , the LT7 c o m p o n e n t II has only 25% the P R T activity of the L T 2 c o m p o n e n t II. 16The amino acid sequences of the LT2 c o m p o n e n t I and c o m p o n e n t II polypeptides are k n o w n f r o m the D N A sequences o f the trpE 17 and trpD TM genes. The nucleotide sequence of the LT7 trpE is identical to that of the LT2 gene.16 The nucleotide sequence of the LT7 trpD gene has not yet been determined. E n z y m a t i c Activities G l u t a m i n e - d e p e n d e n t anthranilate synthase (AS-GIn): Chorismate + L-glutamine
Mg2+
~ anthranilate + L-glutamate + pyruvate + H_,O (1)
Glutaminase: L-Glutamine + H20
chorismate
~ L-glutamate + NH3
(2)
A m m o n i a - d e p e n d e n t anthranilate synthase ( A S - N H 3 ) : Chorismate + (NH4hSO4
Mg2+
) anthranilate + pyruvate + H20
Anthranilate 5 - p h o s p h o r y l r i b o s e - l - p y r o p h o s p h a t e transferase (PRT):
(3)
phosphoribosyl-
Anthranilate + PRPP Mg:+)N-(5'-phosphoribosyl)anthranilate + PP~ + H20
(4)
Assay Methods Anthranilate S y n t h a s e Principle. Anthranilate synthase activity ( A S - G I n and A S - N H 3 ) is a s s a y e d fluorometrically by measuring the rate of a p p e a r a n c e of anthranilate. 14L. J. LaScolea, Jr., and E. Balbinder, J. Bacteriol. 112, 877 (1972). 15M. Grieshaber, Z. Naturforsch. 33¢, 235 (1978). 16R. Bauerle, unpublished observations. 17C. Yanofsky and M. van Cleemput, J. Mol. Biol. 155, 235 (1982). 18H. Horowitz, J. van Arsdell, and T. Platt, J. Mol. Biol. 169, 775 (1983).
[47]
Salmonella ANTHRANILATESYNTHASE-PRTRANSFERASE
369
Reaction Mixtures 1. AS-GIn Activity. The reaction mixture of 1.0 ml contains 0.25 mM chorismic acid (see volume XVIIA [45]), 20 mM e-glutamine, 10 mM MgCI2, 100 mM potassium phosphate buffer, pH 7.0, and enzyme. 2. AS-NH3 Activity. The reaction mixture of 1.0 ml contains 0.25 mM chorismic acid, 50 mM (NH4)2SO4, 10 mM MgCl2, 50 mM Tricine buffer, pH 8.0, and enzyme. 3. Col- and Coil-Complementing Activity. The reaction mixture is identical to that of the AS-Gln assay but also contains either 5 complementing units of purified component I subunit (for the assay of uncomplexed component II subunit) or 5 complementing units of purified component II subunit (for the assay of uncomplexed component I subunit). Procedure. The reaction mixture is placed in a round glass cuvette (8 mm i.d.) and equilibrated to 22°. The reaction is started by the addition of enzyme. The rate of formation of anthranilate is monitored continuously in a Farrand model A4 fluorometer equipped with a strip recorder. The excitation wavelength of 313 nm (half band width, _+ 5 nm) is selected by means of an interference filter; emitted fluorescence (peak wavelength of 390 nm) is selected by a combination of Coming 7-59 and 3-74 glass filters. The amount of anthranilate synthesized is quantified using a standard curve obtained with solutions of crystalline anthranilic acid prepared in appropriate reaction mixture buffer. Definition o f Unit and Specific Activity. One unit of AS-Gln, A S NH3, CoI-complementing, or CoII-complementing activity is defined as the amount of enzyme catalyzing the formation of 1 nmol of anthranilate per min at 22°. Specific activity is expressed as units per milligram of protein. Comments on the Assay. The AS-PRT complex and the AS partial complex can be assayed with either the AS-GIn or the AS-NH3 procedure. Uncomplexed component I subunit can be assayed with either the AS-NH3 or CoII-complementation procedure. Uncomplexed component II monomer can be measured by CoI-complementation or by assay of its PRT activity (see below). Component II dimer can only be assayed in the PRT reaction since it does not assemble with component I. Thus, the complementation assays are useful for determining the amount of uncomplexed component I subunit in a mixture containing intact complex and for estimating the relative abundance of the monomeric and dimeric forms of component II in a preparation. A comparison of the specific activities and the apparent KmS of the different enzyme forms in the various reactions is presented in Table I. AS-GIn activity is linear until chorismate is nearly exhausted. In the complementation assays, subunit assembly begins immediately and the
370
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
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TABLE I PROPERTIES OF ANTHRANILATE SYNTHASE--PHOSPHORIBOSYLTRANSFERASE COMPLEX AND SUBUN1TS
Property
Composition Molecular weight ~ Enzymatic activity c (units/mg) AS-GIn AS-NH3 Col-complementing Coil-complementing PRT Michaelis constant (Kin) Chorismate Glutamine (NH4)2SO4 Anthranilate PRPP
AS-PRT complex
AS partial complex
Component I
Component II monomer
Component II dimer
Coi2 ColI2 228,000
CoI 2 ColI* 156,000 b
CoI 57,000
Coil 56,900
ColI2 113,800
1000 950 --1400
1700 1700 ----
-300 -1800 --
~ m 1900 -3100
-__ 60 -3800
4 I~Md 0.7 m M d 1.0 m M d 8.3/zM e 6.7/zM e
3.3 I~M: 0.5 m M r 0.9 m M f ---
0.1 mMg -24 mMg ---
---6 tzM h 10/xM h
---4 Id,M h 10 lxM h
a Calculated from amino acid compositions as deduced from the D N A sequence of the trpE and trpD genes. b Calculated for the partial complex obtained by trypsin digestion of purified A S - P R T complex, assuming the carboxy-terminus of the digested component II subunit (Coil*) is Lys-193. Accordingly, the molecular weight of Coil* is 20,850. c Determined with enzymes purified and assayed as described herein. a F r o m E. Henderson, H. Nagano, H. Zalkin, and L. Hwang, J. Biol. Chem. 245, 1416 (1970); and D. Smith and R. Bauerle, Biochemistry 8, 1451 (1969). e F r o m E. Henderson, H. Zalkin, and L. Hwang, J. Biol. Chem. 245, 1424 (1970). YF r o m H. Tamir and P. Srinivasan, J. Biol. Chem. 244, 6507 (1969). g F r o m H. Zalkin and D. Kling, Biochemistry 7, 3566 (1968). h F r o m M. Grieshaber and R. Bauerle, Biochemistry 13, 373 (1974).
emerging AS-GIn activity usually attains a maximum linear rate in 3-5 min. The Tricine buffer system of the AS-NH3 assay is superior to others in that it provides an extended period (5-10 min) of linear activity in assays of the component I subunit. The use of large amounts of crude extract in the AS assays can be troublesome because of fluorescence quenching and depletion of chorismate by competing enzymes. The removal of anthranilate during the assay of preparations having PRT activity is not a problem because PRPP is not present. The continuous assay method described is rapid and convenient. A spectrofluorometer is not required. The use of round glass cuvettes is recommended for ease and economy and does not greatly compromise the
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sensitivity of the assay, which can be extended to the picomole range. AS activity can also be determined by a discontinuous method using ethyl acetate extraction of acidified reaction mixture (see Volume XVIIA [47]).
Phosphoribosyltransferase Principle. PRT activity is assayed fluorometrically by measuring the rate of disappearance of anthranilate. Reaction Mixture. The reaction mixture of 1.0 ml contains 10 /zM anthranilic acid, 0.3 mM PRPP, I0 mM MgCI2, 100 mM Tricine buffer, pH 7.6, and enzyme. Procedure. The procedure is identical to that described above for the assay of AS activity except that the rate of disappearance of anthranilate is measured. Definition of Unit and Specific Activity. One unit of PRT activity is defined as the amount of enzyme catalyzing the removal of 1 nmol of anthranilate per min at 22 °. Specific activity is expressed as units per milligram of protein. Comments on the Assay. In assays of purified PRT preparations and of crude extracts lacking PRA isomerase-indoleglycerolphosphate synthase (PRI-InGPS), the product of the trpC gene (see Volume XVIIA [46]), the reaction proceeds at the maximum linear rate for only a limited period because of product inhibition by PRA. An extended period of linearity can be achieved by including in the reaction mixture 5 units of purified PRI-InGPS. PRI-InGPS converts the PRA product to InGP, preventing inhibition. Glutaminase Principle. Glutaminase activity is assayed spectrophotometrically by measuring the formation of glutamate in a coupled reaction with glutamate dehydrogenaseJ 9 Reaction Mixture. The reaction mixture of 1.0 ml contains 5 mM Lglutamine, 0.1 mM chorismate, 5 mM EDTA, 1 mM acetylpyridine NAD ÷, 100 mM tricine buffer, pH 8.0, 0.37 mg bovine liver glutamate dehydrogenase, and enzyme. Procedure. The reaction mixture is placed in a cuvette and equilibrated to 22 °. The reaction is started by the addition of enzyme. The rate of reduction of acetylpyridine NAD ÷ is monitored continuously in a recording spectrophotometer by measuring the increase in absorbance of the solution at 365 nm. The amount of glutamate formed is calculated I9 H. Nagano, H. Zalkin, and E. J. Henderson, J. Biol. Chem. 245, 3810 (1970).
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B I O S Y N T H E S I S O F T H E A R O M A T I C A M I N O ACIDS
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using a molar extinction coefficient of 9100 for acetylpyridine NADH at 365 nm. Definition of Unit and Specific Activity. One unit of glutaminase activity is defined as the amount of enzyme catalyzing the formation of 1 nmol of glutamate per min at 22°. Specific activity is expressed as units per milligram of protein. Comments on the Assay. The assay can be used for the A S - P R T complex and the AS partial complex. Glutaminase activity (units/mg) is equivalent to the AS-GIn activity of the preparation. Chorismate is required in the reaction as an activator. The presence of EDTA prevents the synthesis of anthranilate by the enzyme by chelating divalent cations required for the amination of chorismate. A second assay method is available in which the formation of glutamyl hydroxamic acid is measured using hydroxylamine as acceptor instead of H20.~9 Purification of Enzymes Purification of the enzymes is facilitated by three factors: (1) the use of strains carrying the trp operons of appropriate mutants cloned on multicopy plasmids; (2) the growth of these auxotrophs with limiting supplements of tryptophan to effect derepression of operon expression in the terminal stages of growth; and (3) the application of dye-ligand chromatography as a fractionation method. In those cases where these approaches can be applied together, it is possible to isolate substantial amounts of homogeneous enzyme in high yield from small volumes of culture in a minimum of time.
Bacterial Strains Although specific strains are listed here, many others are available that are just as appropriate as sources of the various enzymes. The A S PRT complex was purified from S. typhimurium strain TB 1409/pSTP89. Plasmid pSTP89 contains the entire trp operon of LT2 auxotroph TB79, which has an amber nonsense mutation (trpA71) in the trpA gene. The AS partial complex was purified from strain TB1409/pSTP38. Plasmid pSTP38 contains the trp operon of LT2 auxotroph TB37, which has an ochre nonsense mutation (trpD479) at the beginning of the PRT-coding region of trpD.l° Component I subunit was purified from strain TB 1409/ pSTP66. Plasmid pSTP66 contains the trp operon of LT7 strain TB8, which has a deletion (AtrpDCBA43) that removes all structural genes but trpE. The component II subunit was purified from the single copy LT2 strain TB41, which has a UGA nonsense mutation (trpE703) in the trpE
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gene. All recombinant plasmids are derivatives of plasmid pBR322 that were constructed by an in viuo cloning procedure, z° Host strain TB 1409 has a deletion of the entire trp operon (AtrpEDCBAI67) and is RecA(recA1). Plasmid subclones with smaller inserts have been constructed from those described above, including several that may be used for the production of the component II subunit/6 These are recommended for future applications.
Growth of Bacteria Bacteria are grown in minimal salts medium (KzHPO4, 10.5 g/liter; KHzPO4, 4.5 g/liter; ( N H 4 ) z S O 4 , 1.0 g/liter; MgSO4, 0.1 g/liter) supplemented at the time of inoculation with glucose (2.5 g/liter), Difco casamino acids (2.0 g/liter), and a growth limiting amount of L-tryptophan (4 or 5 mg/liter). Small preparations (5-10 liters) are grown in 1 liter volumes in 2-liter baffled flasks. Larger cultures (150-300 liters) are grown in a batch fermentor. The medium is inoculated with 50 ~l/liter of an overnight broth culture of the strain containing, in the case of the plasmidbearing strains, ampicillin (50 mg/liter). The culture is incubated with vigorous aeration at 37°. The tryptophan supplement becomes exhausted after about 10 hr (OD550 nm ~ 1.5-1.7) at which time derepression of trp operon expression ensues. The culture is incubated for an additional 1-2 hr before the cells are harvested by centrifugation.
Binding Interactions of the Enzymes with Dye-Conjugated Agaroses The A S - P R T and AS partial complexes and the component I and component II subunits exhibit distinct patterns of binding to the five dyeconjugated M~ttrex Gel chromatographic media manufactured by Amicon Corp. zl (Table II). While salt elution uniformly displaces the bound enzymes from the gel media, low concentrations of tryptophan and, to a lesser extent, chorismate, are effective in selectively eluting the AS-PRT complex from Orange A gel, the AS partial complex from the Orange A and Red A gels, and the component I subunit from the Blue A and Red A gels. There is no similar elution of the bound component II subunit by its ligands, whether applied singly or in combinations. The specificities of adsorption of the enzymes to the various gel media suggest differences in the surface structures of the complexed and uncomplexed subunits. The effectiveness of L-tryptophan and chorismate in 2o T. Patterson and R. Bauerle, Plasmid 12, 149 (1984). 2~ In " D y e - L i g a n d C h r o m a t o g r a p h y . " Amicon Corporation, Lexington, M a s s a c h u s e t t s , 1980.
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BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
T A B L E II BINDING AND LIGAND ELUTION OF ANTHRANILATE SYNTHASE-PHOSPHORIBOSYLTRANSFERASE COMPLEX AND SUBUNITS TO AMICON M.~TREX AGAROSE GELS" Percentage of applied activity
Enzyme AS-PRT complex
AS partial c o m p l e x
Component I subunit
C o m p o n e n t II subunit
Bound/eluted Bound Eluted by L-Tryptophan Chorismate L-Glutamine Bound Eluted by L-Tryptophan Chorismate L-Glutamine Bound Eluted by L-Tryptophan Chorismate Bound Eluted by Anthranilate L-Glutamine PRPP L-Tryptophan
M~trex Blue A
M~trex Orange A
Mhtrex Red A
M~ttrex Green A
Mfitrex Blue B
97
92
99
97
8
0 0 0 38
80 8 2 78
0 0 0 100
0 0 0 17
---4
---97
67 16 7 6
91 34 0 89
---28
---13
92 29 97
--27
61 24 97
--93
--20
0 6
---
5 5 5 7
0 5 7 5
-----
6
--
6
--
a T h e results are from e x p e r i m e n t s using crude extracts o f derepressed cultures of appropriate m u t a n t strains o f S. typhimurium. Essentially identical results were obtained using purified e n z y m e s . C o l u m n s (2 ml) o f each M~ttrex gel were regenerated at r o o m temperature with 10 ml o f 8 M urea a n d w a s h e d with 30 ml of buffer B. After this, all steps were p e r f o r m e d at 4 °. C r u d e extract (0.5 ml) containing approximately 1.5 m g protein was applied to the gel and w a s h e d in with 0.3 ml of buffer B. T h e c o l u m n was closed off for 30 rain, w a s h e d with 10 ml o f buffer B, and then eluted with 10 ml of buffer B containing the indicated ligand. Ligand concentrations were as follows: L-tryptophan, 0.5 m M ; c h o r i s m a t e , 0.5 m M ; L-glutamine, l0 m M ; anthranilate, 0. l raM; PRPP, 0.5 m M . A S - P R T c o m p l e x a n d AS partial c o m p l e x were a s s a y e d by A S - G l n activity, compon e n t I s u b u n i t by A S - N H 3 activity, and c o m p o n e n t II subunit (a mixture of m o n o m e r a n d dimer) by P R T activity. T h e percentage of activity indicated as " b o u n d " is the difference b e t w e e n the activity applied to the column and that present in the wash.
SalmonellaANTHRANILATESYNTHASE-PRTRANSFERASE
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eluting the bound enzymes containing the component I subunit indicates that, in both the complexed and uncomplexed state, significant conformational changes accompany the binding of these two ligands to the subunit. Further analysis has established that when coupled with the specificity of tryptophan elution, M~ttrex Gel Orange A is a superior chromatographic medium for the purification of both the AS-PRT complex and the AS partial complex, while M~trex Gel Blue A is best for the preparation of the component I subunit. The critical concentration of tryptophan in the column buffer, as determined by gradient analysis, is about 5/zM in all cases. Mhtrex Gel Red A is not as useful as the Orange A gel for the preparation of the AS partial complex since there is a significant amount of uncomplexed component I subunit in the trpD mutant extract which is coeluted by tryptophan. The Blue A gel yields a more highly purified preparation of component I subunit than does the Red A gel. Although specific ligand elution of the component II subunit bound to the Blue A, Red A, and Green A gels does not occur, these gels are very effective chromatographic media for the purification of the subunit when used with salt gradient elution.
Buffers Buffer A: Potassium phosphate, 100 raM, pH 7.0, containing glycerol, 1 mM dithiothreitol, and 0.1 mM EDTA Buffer B: Potassium phosphate, 100 mM, pH 7.5, containing glycerol, 1 mM dithiothreitol, and 0.1 mM EDTA Buffer C: Potassium phosphate, 50 mM, pH 7.5, containing glycerol, 0. I mM dithiothreitol, and 0.1 mM EDTA Buffer D: Potassium phosphate, 10 mM, pH 7.5, containing glycerol, 0.1 mM dithiothreitol, and 0.1 mM EDTA
10% 10% 10% 10%
Preparation of Crude Extracts After harvest, the cells are washed once with cold saline (0.9% NaCI) and resuspended in either buffer A or B, as indicated, using 4 ml per gram packed cells. Small preparations are sonified for 45 sec in 10-12 aliquots in an ice-jacketed 50 ml stainless-steel beaker using a Branson model W185D sonifier operated at 75 W. Large preparations are sonified at 100 W in a 60 ml water-jacketed (0°) continuous-flow Rosette cell using an exchange rate of 60 ml of cell suspension per min. The sonicate is centrifuged at 48,000 g for 60 min (27,000 g for large preparations) and the supernatant collected. All subsequent steps are carried out at 0-4 °.
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B I O S Y N T H E S I S O F T H E A R O M A T I C A M I N O ACIDS
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Preparation of Mgttrex Gel Columns for Enzyme Purification Immediately before use, gel columns are cleared of unconjugated dye and other adsorbed material by washing with at least one column volume of 8.0 M urea and then equilibrated with 2-3 column volumes of buffer. Buffer A is used for M~ttrex Gel Orange A and buffer B for M~ttrex Gel Blue A. These preliminary steps are done at room temperature because of the solubility limits of urea. After use, the gel columns can be regenerated for reuse by first washing with buffer containing 1.5 M KC1 and then with urea and buffer as described above.
Purification of Anthranilate Synthase-Phosphoribosyltransferase Complex Step 1. Preparation of Crude Extract. Cells (22 g) obtained from 5 liters of culture of S. typhimurium strain TB1409/pSTP89 limited with 5 mg/liter of L-tryptophan are resuspended in buffer A and crude extract is prepared as described above. Step 2. Ammonium Sulfate Fractionation. Crude extract (96 ml) is brought to 40% saturation by the addition of 23.3 g solid ultrapure ammonium sulfate with constant stirring. The solution is stirred for an additional hour and then centrifuged at 12,000 g for 30 min. The precipitate is dissolved in 10 ml buffer A and dialyzed against 500 ml of the same buffer for 12 hr with 3 changes. Step 3. Mgttrex Gel Orange A Chromatography. The dialyzed preparation is diluted to 35 ml with buffer A and applied to a 2.0 × 22 cm column of Mhtrex Gel Orange A (70 ml bed volume). After the enzyme solution is loaded into the gel bed, the column is closed off and allowed to stand for 2 hr. The column is washed with 100 ml buffer A and then eluted with buffer A containing 50/xM L-tryptophan at a flow rate of 10 ml/hr. Fractions of 5 ml are collected. The bulk of the A S - P R T activity (75% of that applied) emerges in 6 fractions beginning approximately 70 ml after elution with tryptophan is begun. A minor portion of the activity (about 15%) appears in the wash. This is presumably the membrane-bound particulate form of the enzyme present in crude cell-free preparations, since its PRT activity is masked relative to its AS activity) The results of the purification are presented in Table III. The isolated A S - P R T complex is essentially homogeneous as judged by SDS-polyacrylamide gel electrophoresis (SDS PAGE) which resolves the component I and component II subunits (Fig. 1, lane B). The enzyme is stable when stored as a solution at - 2 0 ° in buffer A containing 50% glycerol, losing less than 10% of its activity per year.
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TABLE 111 PURIFICATION OF ANTHRANILATE SYNTHASE--PHOSPHORIBOSYLTRANSFERASECOMPLEX
Total activity
Fraction 1. Crude extract 2. Ammoniumsulfate (0-40% saturation) 3. M~ttrexGel Orange A
Recovery Specificactivity
Total Total AS-GIn PRT volume protein~ AS-GIn PRT AS-GIn PRT (units/ (units/ (ml) (mg) (units) (units) (%) (%) mg) rag) 96 35
1277 363
63,900 100,800 47,000 69,750
100 74
100 69
50 130
79 192
28
31
31,500 43,700
52
44
1015
1410
" Determinedby the methodofM. M. Bradford[Anal.Biochem.72, 248 (1976)] usingbovine serum albumin as standard.
Comments on the Purification Procedure. The affinity of the AS-PRT complex, and of proteins in general, for the M~trex Gel Orange A is weak. The use of buffer A (pH 7.0) instead of buffer B (pH 7.5), which was used in the preliminary binding experiments of Table II, greatly enhances the performance of the Orange A gel as an affinity adsorbent. It is also important to adjust the sample load to a volume equivalent to the void volume of the gel bed (about 50% of the total bed volume) and to interrupt the column flow for several hours immediately after the sample is applied. Under the conditions described here, about 15% of the protein applied to the gel is tightly bound. More than half of this is the AS-PRT complex. The bulk of the applied protein (about 65%) and the nucleic acid fraction appears in the first half of the column wash. A smaller protein fraction (about 20%) is retarded by the gel and emerges in the second half of the wash, immediately before the tryptophan-eluted protein peak. This method of purification of the AS-PRT complex effectively replaces procedures used previously. 2,~z Purification of Anthranilate Synthase Partial Complex Step 1. Preparation of Crude Extract. Cells (28 g) obtained from 5 liters of culture of S. typhimurium stain TB 1409/pSTP38 limited with 5 mg/liter of L-tryptophan are resuspended in buffer A and crude extract is prepared as described above. Step 2. Ammonium Sulfate Fractionation. Crude extract (117 ml) is brought to 38% saturation by the addition of 26.9 g solid ultrapure ammo-
378
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
A
B
C
D
[47]
E
kD
92.5 66.2
45
31
21.5 14.4 FIG. 1. SDS-polyacrylamide gel electrophoresis of AS-PRT complex, AS partial complex, and component I subunit purified by dye ligand chromatography. Lane A, molecular weight standards (Bio-Rad Labs.); lane B, AS-PRT complex (6 /zg); lane C, AS-PRT complex (6/xg) digested with trypsin; lane D, AS partial complex (4/~g) isolated from the trpD479 ochre mutant; lane E, component I subunit (3/zg). Electrophoresis was in a 10% polyacrylamide slab gel using the discontinuous buffer system of Laemmli (see this series, Vol. 104 [12]).
nium sulfate with constant stirring. The solution is stirred for an additional hour and then centrifuged at 12,000 g for 30 min. The precipitate is dissolved in 10 ml buffer A and dialyzed against 500 ml of the same buffer for 12 hr with 3 changes.
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TABLE IV PURIFICATION OF ANTHRANILATE SYNTHASE PARTIAL COMPLEX
Fraction 1. Crude extract 2. Ammonium sulfate (0-38% saturation) 3. Mhtrex Gel Orange A
Total Total Totalactivity Recovery Specificactivity volume proteina AS-GIn AS-GIn AS-GIn (ml) (mg) (units) (%) (units/rag) 117 35
1100 425
149,000 130,000
100 87
135 306
37
59
102,000
68
1730
Determined by the method of M. M. Bradford [Anal. Biochem. 72, 248 (1976)] using bovine serum albumin as standard.
Step 3. Mdtrex Gel Orange A Chromatography. The dialyzed preparation is diluted to 35 ml with buffer A and applied to a 2.0 × 22 cm column of M~trex Gel Orange A (70 ml bed volume). The column is developed as described in step 3 of the previous section. Nearly 80% of the applied activity is recovered in the tryptophan-eluted peak; only 4% is present in the unbound wash fraction. The results of the purification are presented in Table IV. The isolated AS partial complex is essentially homogeneous as judged by SDS-PAGE (Fig. 1, lane D). The component II* fragment of this mutant complex has an estimated molecular weight of 25,000, consistent with the location of the trpD479 ochre mutation. It is slightly larger than the component II* fragment generated by trypsin digestion of the purified AS-PRT complex (see below). The component I subunit is intact, as expected. Comments on the Purification Procedure. Strain TB1409/pSTP38 is ideal as a source of a mutationally generated AS partial complex since its nonsense mutation lies very close if not within the hinge region between the amino-terminal glutaminase domain and the carboxy-terminal PRT domain of the component II subunit. Unlike other trpD nonsense mutants of the PRT domain, its AS-GIn activity is near the wild-type level and no activity is found in the particulate form in crude extracts. However, as determined by Coil complementation, about 25% of the component I of TB1409/pSTP38 is uncomplexed. Uncomplexed component I molecules are completely resolved from the AS partial complex by Mhtrex Gel Orange A chromatography, as they emerge in the retarded fraction of column wash. All comments included in the previous section on the purification of the A S - P R T complex apply here as well.
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B I O S Y N T H E S OF I S THE AROMATIC AMINO ACIDS
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Preparation of AS Partial Complex by Tryptic Digestion of AS-PR T Complex An alternate method for the preparation of AS partial complex is by proteolytic removal of the PRT domain of the intact AS-PRT complex. 22 Trypsin digestion of homogeneous AS-PRT complex is followed by Sephadex G-200 gel filtration of the digest mixture to remove the proteolyzed fragments, any undigested complex molecules, and the trypsin. It is also possible to treat crude extract containing the AS-PRT complex with trypsin 3 and then to purify the AS partial complex by M~trex Gel Orange A chromatography as described in the previous section. In either case, 40/~g of trypsin is added per mg protein, the mixture incubated at 20 ° for 1-2 hr, and then an amount of soybean trypsin inhibitor equal to the amount of trypsin used is added. PRT activity is completely destroyed by the treatment while AS-GIn activity remains unchanged. The component II* fragment of the AS partial complex produced by trypsin digestion is slightly smaller (estimated molecular weight of 24,000) than that of the partial complex isolated from the TB1409/pSTP38 trpD ochre mutant strain (Fig. 1, lanes C and D). Other than this, the two enzymes are indistinguishable in properties. Considering the estimated molecular weights and the specific origins of the two component II* fragments, it can be speculated that the carboxy-terminus of the trypsingenerated fragment is Lys-193 and that of the ochre fragment is Ala-210. The component I subunit is not "nicked" or significantly degraded by trypsinolysis of the AS-PRT complex (Fig. 1, Lane C).
Purification of Component I Subunit Step 1. Preparation of Crude Extract. Cells (38 g) obtained from 10 liters of culture of S. typhimurium strain TB1409/pSTP66 limited with 4 mg/liter of L-tryptophan are resuspended in 76 ml of buffer B and crude extract is prepared as described above. Step 2. Ammonium Sulfate Fractionation. Crude extract (93 ml) is brought to 40% saturation by the addition of 22.6 g solid ultrapure ammonium sulfate with constant stirring. The solution is stirred for an additional hour and then centrifuged at 12,000 g for 30 min. The precipitate is dissolved in I0 ml buffer B and dialzed against 500 ml of the same buffer for 12 hr with 3 changes. ,_2The previous report in this series on the anthranilate synthase of S. typhimurium (Volume XVIIA [48a]) dealt unknowingly with the AS partial complex. Proteolytic enzyme contaminants present in the crude intestinal lipase used in an early step of the purification procedure to solubilize the particulate enzyme fraction digested the AS-PRT complex to the AS partial complex (see Ref. 3).
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TABLE V PURIFICATION OF COMPONENT 1 SUBUN1T OF ANTHRANILATE SYNTHASE--PHOSPHORIBOSYLTRANSFERASE COMPLEX
Fraction 1. Crude extract 2. Ammoniumsulfate (0-40% saturation) 3. M~trexGel Blue A
Total Total Totalactivity Recovery Specificactivity volume proteina AS-NH3 A S - N H 3 AS-NH3 (ml) (mg) (units) (%) (units/rag) 93 30
1620 460
28,300 23,500
100 83
17 51
24
63
19,000
67
300
" Determinedby the method of M. M. Bradford [Anal. Biochem. 72, 248 (1976)] using bovine serum albuminas standard.
Step 3. M~trex Gel Blue A Chromatography. The dialyzed preparation from step 2 is brought to 30 ml with buffer B and is applied to a 2.0 x 19 cm column of M~trex Gel Blue A (60 ml bed volume) equilibrated with buffer B. The column is developed as described in step 3 of the section on the purification of the AS-PRT complex, with the exception that buffer B is used throughout. Nearly 70% of the applied activity is recovered in 5 fractions beginning about 60 ml after the start of the tryptophan elution. The results of the purification are presented in Table V. The isolated component I subunit is essentially homogeneous when analyzed by SDSPAGE (Fig. 1, lane E) and by HPLC gel filtration. The enzyme is stable when stored at -20 ° in buffer B containing 50% glycerol. Comments on the Purification Procedure. M~trex Gel Blue A has an excellent binding capacity for proteins. Loading experiments indicate that, under the conditions described here, 35 to 40 mg of crude cell extract can be applied per ml of gel bed before the efficiency of binding of component I subunit begins to be compromised. In the purification procedure summarized in Table V, about 25% of the protein applied to the gel is tightly bound, as a major portion (75%) is present in the column wash along with the nucleic acid fraction of the preparation. About half of the bound protein is the component I subunit which is specifically eluted by the tryptophan-containing buffer. This method of purification of the component I subunit effectively replaces earlier procedures. 5 Purification o f Component H Subunit 6 Step 1. Preparation o f Crude Extract. Cells (700 g) obtained from 300 liters of culture of S. typhimurium strain TB41 grown in a batch fermentor
382
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[47]
with tryptophan limitation at 4 mg/liter are resuspended in buffer C and crude extract is prepared as described above. Step 2. Treatment with Streptomycin Sulfate. Nucleic acids are precipitated from the crude extract (1200 ml) by the addition, with constant stirring, of 120 ml of 20% streptomycin sulfate in buffer C. After 30 min the precipitate is removed by centrifugation at 27,000 g for 30 min. Step 3. Ammonium Sulfate Fractionation. The supernatant from step 2 (1340 ml) is brought to 40% saturation by the addition of 325 g solid ultrapure ammonium sulfate with constant stirring. After 30 min the precipitate is collected by centrifugation at 24,000 g for 30 min and redissolved in 90 ml buffer C containing 1 mM dithiothreitol. The preparation is dialyzed against 4 liters of the same buffer for 8 hr with two changes. Step 4. Ultracentrifugation. The dialyzed extract is centrifuged at 135,000 g for 3 hr and the pellet discarded. Step 5. DEAE-Sephadex Chromatography. The supernatant is applied to a column (7.5 x 80 cm) of DEAE-Sephadex A50 equilibrated with buffer D. The gel column is washed with 6 liters of buffer D and then eluted with a 4 liter KCI gradient (0-0.5 M) prepared in buffer D, at a flow rate of I00 ml/hr. Fractions of 100 ml are collected. Peak fractions containing PRT activity are pooled and brought to 60% saturation by the addition of solid ammonium sulfate (312 g/800 ml of pooled material). The precipitate is collected by centrifugation at 24,000 g for 30 min, dissolved in 20 ml of buffer C, and dialyzed against 4 liters of buffer C for 6 hr with one change. Step 6. Sephadex G-150 Gel Filtration. The dialyzed preparation is applied to the bottom of a column (10 x 115 cm) of Sephadex G-150 equilibrated with buffer C. The column is developed by upward flow with buffer C at a flow rate of 100 ml/hr. Fractions of 150 ml are collected. Those containing the PRT activity (between 5.8 and 7.0 liters of effluent) are pooled and concentrated to 30 ml by ultrafiltration. Step 7. Hydroxylapatite Fractionation. The enzyme concentrate is dialyzed against 1 liter of buffer D for 12 hr with 3 changes and applied to a column (1.5 x 27 cm) of hydroxylapatite (BioGel HT) equilibrated with buffer D. The column is washed with 80 ml of buffer D and then eluted with 100 ml of buffer C at a flow rate of 10 ml/hr. Fractions of 4 ml are collected throughout the wash and elution. Component II dimer does not adsorb to the hydroxylapatite and is recovered in the wash in 8 fractions; component II monomer elutes as a sharp peak beginning about 50 ml after the change to buffer C. Both component I monomer and dimer are stable when stored at - 2 0 ° in buffer C containing 50% glycerol, each losing about 5% of its PRT activity per year.
[47]
Salmonella ANTHRANILATESYNTHASE-PR TRANSFERASE
383
TABLE VI PURIFICATION OF COMPONENT I I SUBUNIT OF ANTHRANILATE SYNTHASE--PHOSPHORIBOSYLTRANSFERASE COMPLEX a
Fraction 1. Crude extract 2. Streptomycin sulfate 3. Ammonium sulfate (0-40% saturation) 4. 135,000 g supernatant 5. DEAE-Sephadex 6. Sephadex G-150 7. Hydroxylapatite Wash (ColI Dimer) Eluate (ColI Monomer)
Total volume (mi)
Total Total activity proteinh PRT (mg) (units)
1200 1340 120
17,700
Recovery PRT (%)
Specific activity PRT (units/rag)
100 93 78
28
7,000
498,000 466,000 398,000
117 40 1300
6,700 560 200
402,000 316,000 217,000
81 63 44
59 590 1085
30 15
17 23
65,000 7 i ,000
13 14
3800 3100
56
a Taken from M. Grieshaber and R. Bauerle, Biochemistry 13, 373 (1974). b Determined by the method of Lowry (see this series, Vol. 3 [73]) using bovine serum albumin as standard.
C o m m e n t s on the Procedure. C o m p o n e n t II m o n o m e r and dimer copurify in steps 2 - 5 and are only partially resolved by Sephadex G-150 fractionation, as evidenced b y the disproportionate level of CoI complementing activity in the trailing fractions of the PRT activity peak. 6 The c o m p o n e n t II d i m e r is believed to be an artifact of preparations having high concentrations of u n c o m p l e x e d c o m p o n e n t II subunit, arising by the f o r m a t i o n of a disulfide linkage b e t w e e n the active site cysteines of two subunit molecules. 9 The final hydroxylapatite treatment effectively resolves the physiologically important c o m p o n e n t II m o n o m e r from the artifactual c o m p o n e n t II dimer. It is possible to increase the yield of m o n o m e r b y dissociating dimer molecules present in the preparation by raising the concentration o f dithiothreitol in the e n z y m e concentrate of step 6 to 10 m M and then incubating the preparation at 41 ° for 60 min immediately before the hydroxylapatite fractionation. The purified c o m p o n e n t I I subunits (T~,ble VI) are estimated to be 9 0 95% h o m o g e n e o u s b y S D S - P A G E . 6 Strains with the trpD gene cloned on a multicopy plasmid p r o d u c e 5 - 1 0 times as m u c h e n z y m e as the single c o p y strain. The use of these should m a k e it possible to simplify the purification p r o c e d u r e and to obtain e n z y m e of increased purity.
384
[47]
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS 1.0-~ern~_
~
=
?
w
0.8
~
0.7
~
_~0.5~~ )" p-
0.6
.¢
0.4
&
Complex
7
Co II D
v
~
0.3 D u) "'
Complex 0.2
~ 0.1
~t~
Co II M
~Co I I
0
10
I
20
--
I
I
I
30
40
50
I
60
MINUTES
FIG. 2. Effect of trypsin treatment on the enzymatic activities of the AS-PRT complex and subunits. Digest mixtures were prepared in buffer C and contained the following in a final volume of I mh (1) 500/zg A S - P R T complex + 20/xg trypsin; (2) 210/zg component I subunit + 0.3/xg trypsin; (3) 500/zg component II monomer + 7/xg trypsin; (4) 400 p.g component II dimer + 7 /zg trypsin. Incubation was at 25 °. Samples were removed to enzyme reaction mixtures at the indicated times for the assay of residual AS (closed symbols) and PRT (open symbols) activity. AS-PRT complex was assayed in the AS-GIn and PRT reactions; component I subunit in the AS-NH3 reaction; component II monomer and dimer in the PRT reaction.
Domain Structure of Component I and Component H Subunits The existence of independent functional domains for glutaminase and PRT activity in the component II subnit has been demonstrated by mutational analysis of the trpD gene and by in vitro proteolytic digestion of the trpD polypeptide. Monofunctional mutant polypeptides of both types exist as stable enzyme forms in various nonsense and deletion mutant strains. 1°,14-16The carboxy-terminal PRT domain of both complexed 3 and uncomplexed 6 component II is destroyed by in vitro trypsinolysis (Fig. 1), while the amino-terminal glutaminase domain remains structurally and functionally intact. The proteolytic attack entails the sequential removal of peptides from the carboxy-terminus, 1° rather than the nicking of an interdomain hinge. The uncomplexed monomeric subunit is much more susceptible to proteolysis than the complexed subunit, as indicated by the relative rates of inactivation of PRT activity upon trypsin treatment (Fig. 2). The dimeric subunit, however, is highly resistant to proteolytic attack under the same conditions.
[47]
SalmonellaANTHRANILATESYNTHASE-PRTRANSFERASE
385
MINUTES in
kD 92.5 66.2 45 31
21.5 14.4. FIG. 3. SDS-polyacrylamide gel electrophoresis of tryptic digest of component I subunit. Component I (140 p~g) in 700/zl of TE buffer (50 mM Tris-HCl, 2 mM EDTA, pH 7.6) was digested with 0.2/zg trypsin (TPCK treated) at 25°. Samples of 25/zl were removed at the indicated times to 35 /~1 of sample denaturing buffer (see this series, Vol. 104 [12]) and immediately heated at 100° for 3 min. The entire sample was then analyzed by SDS-PAGE as described in Fig. 1.
Mutational analysis and physical mapping of the trpE gene indicate that all amino acid residues critical for catalytic activity of the component I subunit are found within its carboxy-terminal 200 residues. 16 Critical residues identified by mutation include Thr-329, Glu-358, His-398, Thr425, Gly-485, and Glu-495. Chemical modification studies indicate that Cys-377 is also an essential residue. 23 The results of limited proteolysis experiments suggest the possibility of domain substructures in the polypeptide. While the complexed component I subunit is remarkably resistant to trypsin digestion, the uncomplexed subunit is extremely susceptible (Figs. 1 and 2). Trypsin cleaves the component I subunit into several metastable fragments which appear in sequence (Fig. 3). Initially, a doublet of large fragments with molecular 23 H. Zalkin, J. Paluk, M. van Cleemput, S. Moye, and C. Yanofsky, J. Biol. Chem. 259, 3985 (1984).
386
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[48]
weights of 45,000 and 42,000 is formed from the 58-kDa polypeptide. The loss of catalytic activity is congruent with the initial cleavage as determined by enzymatic assay and gel densitometry. The initial doublet is then reduced to a doublet of fragments with molecular weights of 33,000 and 30,000, respectively. Several smaller fragments appear which may be the products of the initial cleavages. The presence of ligands of the subunit in the digest mixture (i.e., tryptophan, chorismate, Mg2+, and NH~) affects the rate of digestion in some cases, but does not alter the pattern of the proteolytic fragments.
[48] P h o s p h o r i b o s y l a n t h r a n i l a t e Isomerase-Indoleglycerol-Phosphate Synthase from Escherichia coli By KASPER KIRSCHNER, HALINA SZADKOWSKI, THEODORE S. JARDETZKY,
and V R E N I H A G E R
Introduction Phosphoribosylanthranilate (PRA) isomerase catalyzes the fourth step of tryptophan biosynthesis PRA --* I-(2-carboxyphenylamino)- I-deoxyribulose 5'-phosphate (CdRP)
( 1)
This reaction is a practically irreversible Amadori rearrangement. Indoleglycerol-phosphate (InGP) synthase catalyzes the fifth step CdRP ~ InGP + HCO~ + H20
(2)
in which ring closure to the indole nucleus occurs, again a practically irreversible reaction. These two reactions have been shown to occur in every organism capable of synthesizing tryptophan. In enteric bacteria (Escherichia coli, Salmonella typhimurium, Serratia marcescens) a monomeric protein with a single polypeptide chain of Mr 49,400 catalyzes both reactions. ~-4 In other gram-negative ( P s e u d o m o n a s putida, ~,2Acinei I. P. Crawford, CRC Crit. Rev. Biochem. 8, 175 (1980). 2 I. P. Crawford, in "MultifunctionalProteins" (H. Bisswanger and E. Schmincke-Ott, eds.), p. 151. Wiley, New York, 1980. 3j. F. McQuadeIII and T. E. Creighton,Eur. J. Biochem. 16, 199 (1970). 4j. M. Potts and G. Drapeau, J. Bacteriol. 111, 334 (1972).
METHODS IN ENZYMOLOGY,VOL. 142
Copyright © 1987by AcademicPress, Inc. All rights of reproductionin any form reserved.