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Regulation of threonine biosynthesis in Escherichia coli
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
78, 416-432 (1958)
Regulation of Threonine Biosynthesis in Escherichiu
coZi1~2
Eva H. Wormser3*4 and Arthur B. Pardee From the Department of Biochemistry and the Virus Laborator!/, University of California, Berkeley, California Received
May
28, 1958
INTRODUCTION
Escherichia coli can synthesize all of the metabolites necessary for growth in a medium containing only salts, an inorganic nitrogen source, and an organic energy source. When the cells are in the exponential phase of growth, this synthesis is so economical that, with the exception of some vitamins, only small amounts of the newly synthesized metabolites are released into the medium. If these same organisms are grown in a medium supplemented with one or more amino acids, purine or pyrimidine derivatives, or vitamins, the cells usually utilize the preformed substance to a large extent in preference to the internally synthesized compound. By adding glucose, randomly labeled with U4, as the principal carbon source and a nonradioactive amino acid to the minimal medium, it was shown that in some cases the cells continue their normal biosynthetic pattern, releasing the radioactive amino acid into the medium. In most cases, e.g., threonine, the exogenous amino acid suppresses the biosynthesis of the corresponding compound, and no radioactive metabolites are released into the medium (1). These experiments indicate that some enzyme systems are endowed with a feed-back control, i.e., an internal control which suppresses the synthesis of the end product when that end product is present in sufficiently 1 Aided by a grant from the University of California Cancer Research Funds and by U. S. Public Health Service Training Grant CRTY 5028. 2 Presented in part at the meeting of the Society of American Bacteriologists, Stanford University, August 27, 1957. 3 Some of this work was submitted in partial fulfillment of the requirement for the Ph.D. degree in Biochemistry at the University of California. 4 Present address: Department of Food Technology, University of California, Davis, Calif. 416
THREONINE
-117
BIOSYNTHESIS
high concentration. This hypothesis was suggested by Adelberg and Umbarger (2) when they showed that a valine-requiring mutant growing on limiting amounts of the missing metabolite releases large quantities of the substrate of the blocked reaction into the medium. However, if t,he required metabolite is added in excess, the accumulation of the intermediate is halted. The existence of feed-back controls has also been shown in brokencell preparations. Yates and Pardee (3), studying the control of pyrimidine biosynthesis in this laboratory, showed that even low levels of uracil in the medium inhibit the formation in v&o of the intermediate carbamyl aspartic acid in two pyrimidine-requiring mutants, and, in vitro, cytidine and cytidylic acid inhibit ureidosuccinate formation in competition with both substrates, aspartic acid and carbamyl phosphate. Umbarger (4), working with extracts of a different strain of E. co&, showed that the irreversible deamination of L-threonine to a-ketobutyric acid, postulated to be an essential step in the biosynthesis of L-isoleucine, is inhibited competitively by L-isoleucine. The study of the regulation of threonine biosynthesis was undertaken to determine whether the same type of control could be found in amino acid biosynthesis as had been found in pyrimidine biosynthesis. Threonine was chosen because its pathway of biosynthesis from aspartic acid (5) via homoserine and homoserine phosphate (HSP)3 (6) was known. Homoserine was the starting point of these st,udies. As the scheme below shows, the pathways of biosynthesis of threonine and methionine diverge at this point. Since threonine apparently does not interfere with methionine biosynthesis in this organism, it would hardly be expected that, if threonine exerted feed-back control on its own biosynthetic pathway, it would do so before homoserine. HSK aspartic acid ---f ----f homoserine _____f ATP, Mg++ L
homoserine phosphate
-
HSPM
L
methionine threonine
threonine ) a-ketobutyric deaminase
Since tjhrconine is it,self incorporated
acid ---f --+ isoleucine
int,o prot,ein and is also an int>er-
5 The following abbreviations will be used in this work: HSP, homoserine phosphate; HSK, homoserine kinase; HSPM, homoserine phosphate mutaphopphatase; ATP, adenosine triphosphate.
418
WORMSER AND l'ARDEE
mediate in isoleucine biosynthesis, the control of its concentration in the cell is probably influenced by several factors. Evidence is presented in this work that the synthesis [as well as the destruction (4)] of this amino acid is affected by a feed-back control. MATERIAL
AND METHODS
General Organisms. Yeast extracts were made from fresh Fleischmann’s Baker’s yeast. The bacterial strain was Escherichia coli B. Media. The minimal “C medium” of Roberts (1) was used throughout the work, supplemented when necessary with threonine. For most of the work, 10 ml. of sterile 10% glucose solution was added per liter of sterile medium.
Extracts and Enzymes Purification and Fractionation of Threonine Synthase from Yeast. Threonine synthase, the enzyme system which catalyzes the formation of threonine from homoserine, was purified by the method of Watanabe and Shimura (6). The enzyme system was separated into two fractions, HSK and HSPM by the method of Watanabe et al. (7). These catalyze the phosphorylation of homoserine, CH20H-CHZ-CH(NH+COOH
ATP ___3 Mg++
homoserine HeOaP-0-CHZ-CHZ-CH(NH2)-COOH homoserine phosphate and the subsequent formation of threonine from homoserine phosphate, respectively. Preparation of Bacterial Eztract. In the early part of this work, bacterial extract was prepared daily. The cells were grown to a concentration of approximately 108 cells/ml., centrifuged, suspended in a small volume of distilled water, and broken by a lo-min. treatment in a g-kc. Raytheon Magnetostriction Oscillator. This method was time-consuming and introduced numerous variations from day to day. In order to eliminate these disadvantages, the bacteria were harvested from a 20-l. culture in a Sharples centrifuge, the concentrated cells were treated in a IO-kc. oscillator, and the crude bacterial extract was lyophilized. The dry powder remained stable for months at -5°C. One milligram of the dry extract had approximately as much threonine synthase activity as 3.5 X lo9 cells.
Compounds Amino Acids. L-Threonine was obtained from the California Foundation for Biochemical Research, and nn-homoserine was purchased from Mann Research Laboratories. Most of the other amino acids, which had originally been purchased
THREONINE
BIOSYNTHESIS
419
from Mann Research Laboratories or Nutritional Biochemicals Corporation, were kindly supplied by Dr. F. H. Carpenter. Homoserine Phosphate. A solution of HSP, essentially free of threonine, for use as substrate in the HSPM reaction was prepared by a large-scale application of the HSK reaction with the use of the yeast enzyme as catalyst. Sixty milliliters of the incubation mixture (see Table I) were held at 37” for 3 hr., heated in a to remove the denatured protein. boiling water bath for 15 min., and centrifuged The supernatant solution was then forced through 5 ml. of Dowex 50 resin (length of column, approx. 25 cm.; i.d. 8 mm.) at an approximate rate of one drop per second. The first 10 ml. eluant contained no ninhydrin-positive material and was discarded. The remaining 50 ml. eluant contained essentially threonine-free HSP. The column was then rinsed free of HSP with 10 ml. of distilled water which was allowed to flow through the resin without, the use of air pressure. The volume of the solution was reduced by heating it to about 80°C. and directing a stream of air over the surface of the liquid. Excess salt was removed by centrifugation of the concentrated, refrigerated solution. The solution was stored at -5°C.
Assays for Compounds ‘I’hreonine Assay. The method was a combination and modification of the methods of Winnick (8) and Neidig and Hess (9). The basis of the method was the oxidation of threonine by periodic acid in plastic Conway units and the absorption of acetaldehyde by sodium bisulfite as described by Winnick. The time for the completion of the reaction was reduced from 5 hr. to 2 hr. by permitting the reaction to take place at 37°C. instead of at room temperature. The acetaldehydebisulfite complex was converted to a complex of p-hydroxydiphenyl, and t)he colored product was estimated in a Klett calorimeter with a green (540 mp) filter as described by Neidig and Hess. This method was useful from 0.03 to 0.5 pmole threonine; the error was within 0.01-0.03 pmole, depending on the concentration of the threonine. Attempts to estimate the acetaldehyde directly by absorbing it in a solution of p-hydroxydiphenyl in concentrated sulfuric acid ended in failure because t,he assay became extremely sensitive to minor variations in procedure. This sensitivity may have been caused by the absorption of water as well as acetaldehyde by the sulfuric acid, or by the impossibility of cooling the sulfuric acid-acetaldehyde mixture before adding the p-hydroxydiphenyl. Later experiments showed that either the addition of extra water or improper cooling caused a reduction of color. Homoserine Phosphate Assay. Homoserine phosphate was separated from the neutral amino acids, threonine and homoserine, with the aid of the sulfonate resin Dowex 50. A series of columns, 20 cm. long, was prepared from 5-mm. (i.d.) tubing by drawing out a tip at the bottom end and flanging the top. Each column was packed by pipetting a suspension of 1 ml. of 20&4O@mesh Dowex 50 in the hydrogen form in 2 ml. water onto a small pad of glass wool, and allowing the excess water to drain off. One milliliter of the solution to be assayed was pipetted into the column and forced through the bed of resin under air pressure in 20-30 sec. in order to prevent the resin from being clogged by denatured protein. This effluent was discarded. The HSP was rinsed from the resin with 3 ml. of distilled water,
420
WORMSER
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PARDEE
which was permitted to flow through the column without added pressure. After thoroughly mixing the effluent, a l-ml. aliquot was assayed by the ninhydrin method of Moore and Stein (10). The same resin bed could be used at least three times by rinsing with additional distilled water before adding the next sample. a-Keto Acids. A modification of the method of Friedemann and Haugen (11) was used. To 1.0 ml. of the sample, containing 0.01-0.50 pmole a-keto acid, was added 0.9 ml. of 0.05yo of 2,4-dinitrophenylhydrazine in 1.2 N HCl. The tubes were mixed and allowed to stand at room temperature for 20 min. before adding 1.7 ml. of 2.5 N NaOH. The optical density of the solution was read at 520 mp. a-Ketoglutarate was used as the standard.
Enzyme Assays Threonine Synthase Assay. The method of Watanabe and Shimura (6) was used almost without modification. It is based on the phosphorylation of homoserine by ATP in the presence of magnesium ion, the subsequent conversion of the phosphorylated intermediate to threonine, and, finally, the chemical estimation of threonine. The homoserine concentration was reduced by a factor of four to bring its concentration below the saturation level for the competitive inhibition studies. Hmoserine Kinase Assay. This assay was the same as the threonine synthase assay except that the incubation mixture was assayed for HSP instead of threonine. Since the first step in the HSP assay is the separation of HSP from other components of the incubation mixture on a column of Dowex 50, and since this procedure denatures and removes the protein, the enzyme reaction could be stopped either by heating as in the threonine synthase assay, or by forcing the incubation mixture through a Dowex 50 resin column with compressed air. Homoserine Phosphate Mutaphosphatase Assay. This assay was also based on the threonine synthase assay. However, the ATP, magnesium sulfate, and sodium fluoride were unnecessary, leaving only the phosphate buffer. The substrate was HSP rather than homoserine. In this assay the incubation mixture was assayed for both threonine formation and HSP disappearance. RESULTS
Threonine Synthase Crude extracts made from E. co& strain B, catalyzed the formation of threonine from homoserine when the substrate was incubated with ATP and magnesium sulfate in a phosphate buffer at pH 6.8. Sodium fluoride was also present, as a general inhibitor of phosphatases; it had no effect on the threonine synthase reaction. To saturate the system when an extract from 6.5 X lo9 cells/ml. of incubation mixture was used, 1.25 pmoles/ml. L-homoserine were required, and approximately 0.2 pmole threonine was formed. Attempts were made to determine whether this over-all reaction was inhibited by threonine. However, it
THREONINE
BIOSYKTHESTS
121
soon became evident that if threonine had an inhibitory effect it was only in concentrations which were very high compared to the amount of threonine formed. In the presence of these concentrations of threonine, it was impossible to determine with the necessary accuracy small changes in the amount, of threonine formed. It was, therefore, decided to work with the two known components of the threonine synthase system, HSK and HSPM. Homoserine Kinase In crude extracts of E. coli the formation of HSP was much more rapid than its transformation into threonine: In 1 ml. of incubation mixture, 0.2 mg. of lyophilized crude extract, approximately equivalent, to 7 X lOa cells, catalyzed the formation of 0.4 pmole HSP in 1 hr. accompanied by the formation of less than 0.02 pmole threonine from an initial concentration of 1.25 pmoles L-homoserine/ml. (Fig. 1). The conditions were optimal in regard to pH, ATP, and magnesium sulfate. This difference in rates between HSK and HSPM permitted the separation of the action of the two enzymes without purification of the enzymes. Homoserine kinase was inhibited competitively by threonine. Figure 2 shows the results of one experiment. Average values of the constants o Homoserinephosphote A Threonine
Mg. Extract
FIG. 1. The formation
of homoserine phosphate and threonine by bacterial threonine synthase. mg. of lyophiliaed E. coli B extract.
Abscissa: Conditions: The usual incubation mixture (see Table I), with 1.25 Mmoles L-homoserine/ml., was incubated for 1 hr. with varying amounts of bacterial extract. After denaturation and removal of the protein, the snpernatant solut,ion ws.s assayed for both homoserine phosphate and threonine.
422
FIG.
WORMSER
2. Competitive
Abscissa:
inhibition
AND
of bacterial
PARDEW
homoserine
kinaxe
by threonine.
Reciprocal
of the substrate concentration. ml./pmoles n-homoserine Ordinate: Reciprocal of the velocity of the reaction. ml.-hr./pmoles homoserine phosphate Conditions: 0.4 mg. of lyophilized bacterial extract was incubated for 1 hr. with the usual incubation mixture (see Table I), varying only the homoserine concentration. The inhibition constant was determined by the addition of 10 rmoles L-threonine/ml. of incubation mixture.
were calculated from the data by the method of least squares from experiments at three enzyme levels and varying incubation periods. The Michaelis constant of HSK was 6 X 10e4 M for homoserine and the inhibition constant was 4 X 1O-3 M for L-threonine. Thus, the affinity of the enzyme was greater for homoserine than for threonine. Since the purpose of these experiments was to detect a physiologically important feed-back mechanism, it was necessary to determine whether the inhibition by threonine was specific, or whether other amino acids had the same effect. A compound was considered inhibitory if in its presence the formation of HSP was inhibited by more than 20%. This
THREONINE
BIOSYNTHEL3IK
423
point of division between inhibitors and noninhibitors was chosen somewhat arbitrarily. The choice was based on the natural break in the series of values at this point, and the decision that an inhibition of less than 15 or 20% was insignificant. Each compound was tested at least four times to obtain a more accurate average. Table I shows a strong structural influence on inhibition. Threonine is not the only inhibitor of HSK, but it is the best inhibitor. The compounds which were tested fell into three groups: inhibitory compounds which are also structurally relat,ed to threonine, inhibitory amino acids which are basic, and noninhibitory amino acids or related compounds. The first group had two structural features in common: at least one of the y-carbons is in a methyl group, and a hydrogen atom is attached to the erythro position of the beta carbon. In addition, the two best inhibitors, threonine and a-aminobutyric acid were four-carbon a-amino acids. The Lu-amino group was not necessary, but in the case of the four-carbon compounds it distinctly enhanced the inhibitory power. The p-hydroxyl group in the L-threo position was unnecessary, but it could not be in the position of an L-erythro-hydroxyl group. Neither allothreonine nor I)-threonine hn.d any effect on the HSK reaction. The apparent exception to this patt’ern was cysteine. However, if the sulfhydryl group is considered to be equivalent to a methyl group, cysteine also falls into the structural pattern of the inhibitors. Nomoserine I’hosphate
Mutaphosphatasc
The mutaphosphatase reaction appeared to be much slower than the phosphorylat,ion of homoserine. The muximum rate of HSl’ formatioll wit,h extracts of E’. coli B was 1000 pmoles/hr.:‘g. wet weight of cells, i.e., about 1012cells, whereas the maximum rat’e of threonine formation was only 40 @moles/hr./g. cells. This rate in vitro is about one-half the rate in viva, since cells growing at an exponential rate in minimal medium with a generation time of 1 hr. must synthesize 75 Mmoles threonine/ hr./g. cells to supply the threonine and isoleucine of cellular protein. Figure 1 shows the relationship of the amounts of threonine and HSP formed from homoserine under the same conditions. When 1.25 pmoles L-homoserine was incubated for 1 hr. with lyophilized extract equivalent t)o 2.5-7.5 X log cells, the final incubation mixture contained approximately 0.8 pmole HSP and 0.07-0.2 pmole threonine. At these enzyme levels the rate of threonine synthesis was proportional to the concentration of enzyme, and the amount of HSP remained constant. At lo\vel
424
WORMSER
AND
TABLE
PARDEE
I
Kinase by Amino Acids Conditions: 0.2 or 0.4 mg. of lyophilized E. coli B extract was incubated for 1 hr. with the incubation mixture used throughout this work. It contained 200 pmoles NaF, 100 pmoles MgSOa , and 50 pmoles ATP in 1 ml. of 0.1 M phosphate buffer at pH 6.8; 1.25 pmoles L-homoserine and 10 rmoles L-amino acid (or 20 rmoles m-amino acid)/ml. of incubation mixture were added. I. Inhibitory compounds which are structurally related to threonine. II. Basic amino acids. III. Noninhibitory amino acids. Inhibition
Group
of Bacterial
Amino
Homoserine
Average inhibition
acid
Range
Number of experiments
% I
II
III
n-Threonine nn-or-Aminobutyric L-Cysteine Butyric acid n-Valine L-Isoleucine Isovaleric acid
55” 49 37 31 27 26 24
4961 44-54 3242 29-33 20-33 1931 1833
23 4 7 4 6 9 6
L-Arginine L-Lysine L-Histidine
24 24 21
20-31 15-31 7-30
8 7 4
nn-Alanine n-Methionine L-Tryptophan n-Aspartic acid L-Leucine L-Norleucine Iso-a-aminobutyric nn-Allothreonine nn-Norvaline L-Serine n-Asparagine Glycine n-Threonine
17 14 14 12 12 12 12 10 9 8 6 4 4
12-22 8-17 8-23 8-20 7-17 O-22 9-20 O-16 2-11 O-20 O-17 o-8 o-8
6 5 9 6 5 6 8 6 5 6 5 6 4
acid
acid
(1An indication of the accuracy of the data may be obtained from the standard deviation of the “To inhibition”, c = 3.3, in the presence of L-threonine, the only amino acid which was tested frequently enough for such a calculation.
THREONINE
425
BIOSYNTHESIS
c~lzyme levels the rate of HSP synthesis was proportional to the enzyme concentratBion. Attempts to increase the rate of t,he HSPM reaction I)y the addition of pyridoxal phosphate, suggested by Nisman ct al. (12) to he a cofactor, were unsuccessful. For the purpose of inhibition studies with amino acids, HSPM was assayed separately from HSK by using the product of the kinase reaction, HSP, as the substrate. It was difficult to obtain consistent results, and especially difficult to achieve a balance of HSP disappearance and threonine appearance (Table II) owing to t#he difficulty of determining a small increment in the threonine concentration. However, it,
Inhibition
of Bacterial
Homoserine
TABLE II Phosphate Mutaphosphatase
by Amino
Acids
Conditions: 1.4, 2.8, or 4.2 mg. of lyophilized E. coli B extract was incubated for 1 hr. with the usual incubation mixture (see Table I) including 0.5 pmole homoserine phosphate/ml.; 10 rmoles L-amino acid (or 20 rmoles m-amino acid)/ ml. of incubation mixture was added. After denaturation and removal of the protein, the supernatant solution was assayed for homoserine phosphate disappearance and for threonine formation, except in the samples to which threonine had been added. Average inhibition of HSP disappearance
Amino acid
Average inhibition of threonine formation
%
%
100 62 38 52
78 52 38 21
L-Serine r,-Arginine Valerie acid nI,-Alanine
32 24 27 68
44 25 0
Glycine r>-Histidine I,-Methionine L-Valine uL-cw-Aminobut,yric I>-Isoleucine
19 3 8 12 22 0
3 12 11 6 0 0
I,-Cysteine Butyric acid klysine I,-Aspartic acid
L-Threonine n-Threonine nL-Allothreonine
acid
0 0 0
No. of expts.
-
10 5 5
426
WORMSER
AND
PARDEE
is probably safe to conclude that the concentration of threonine which inhibited HSK approximately 50% did not inhibit HSPM. Two other amino acids which inhibited the kinase strongly, cu-aminobutyric acid and isoleucine, also had no inhibitory effect on the mutaphosphatase. On the other hand, butyric acid and cysteine, both of which inhibited the kinase less strongly than threonine, were strong inhibitors. Cysteine, in fact, inhibited this enzyme at a much lower concentration than any other amino acid tested. Yeast Enzymes The threonine synthase system was also found in crude extracts of baker’s yeast, prepared according to the directions of Watanabe and Shimura (6). Inhibition studies with threonine yielded the same ambiguous results as the analogous experiments with bacterial extracts. Therefore, HSK was separated from HSPM by the method of Watanabe et al. (7) by ammonium sulfate fractionation. The HSK fraction had a much greater specific activity (sixfold) than unfractionated threonine synthase and appeared to be almost free of HSPM activity. However, the mutaphosphatase fraction contained much residual kinase activity and showed no increase in specific activity with respect to threonine formation. No further work was done with the latter fraction. Yeast HSK, like the bacterial enzyme, was inhibited competitively by threonine. The Michaelis and inhibition constants of HSK from both sources were of the same order of magnitude, approximately 1O-3 M for t-homoserine and 3 X 10e3 M for L-threonine, respectively, in yeast, compared to 6 X lo-* M and 4 X 10e3 M for the corresponding constants in E. coli. All of the values are the averages of constants obtained from several different experiments, and each constant was calculated from the data by the method of least squares. Assays performed with threonine replacing the substrate showed neither a change in the concentration of threonine nor the appearance of a ninhydrin-positive substance which could be mistaken for HSP. It is evident, therefore, that the inhibition by threonine is not caused by any chemical changes in the threonine. Watanabe et al. (13) recently published their studies on the properties of yeast homoserine kinase. In agreement with the present work they found that threonine is not a substrate of yeast HSK. They did not, however, detect the inhibitory property of L-threonine since they used a saturating concentration of homoserine (6.25 mM) in their assays and did not compensate for this by an even higher threonine concentration.
THREONINE
TABLE Inhibition
427
BIOSYNTHESIS
of Yeast Homoserine
III
Kinase
by Several Amino
Acids
Conditions: 0.25 mg. of lyophilized homoserine kinase was incubated for 1 hr. with the usual incubation mixture (see Table I) including 0.5 pmole L-homoserine/ml.; 25 /Imoles L-amino acid (or 50 firmoles DL-amino acid)/ml. incubation mixture was added. Controls, incubated with all ingredients of the incubation mixture including the amino acid, but excluding ATP, formed no HSP. Average inhibition
Amino acid
Range
% r,-Threonine or,-ol-Aminobutyric or,-Alanine m-Isoleucine IA-Methionine L-Serine
62 77 54 37(?) 14 13
acid
51-68 73-80 46-62 17-61 4-24 3-20
Comparison of inhibition studies with amino acids other than threonine showed that the kinases from the two sources were similar, but by no means identical (Table III). Five of the six amino acids which were tested with both enzymes behaved in the same mamler qunlitatively, but there were significant quantitative differences. Alanine was the only amino acid which inhibited yeast HSK but did not inhibit bacterial HSK. In contrast to the bacterial enzyme, which was inhibited most st,rongly by threonine, yeast HSK was inhibited most strongly by cu-aminobutyric acid. However, in both cases t,he two best inhihit,ors were threonine and a-aminobutyric acid. Enzyme Concentration Another mode of metabolic control is by enzyme repression. Vogel (14) and Gorini and Maas (15) showed that the concentrations of some enzymes of arginine biosynthesis are reduced below the basal level when adequate amounts of arginine are provided in the medium. They also showed the reverse-the levels of these enzymes can be increased when arginine is limiting. The latter phenomenon was also shown in this laboratory by Yates and Pardee (17) in the biosynthesis of uracil. No attempt has been made as yet to determine whether diminishing the internal concentration of threonine raises the level of threonine synthase, but the observations of Cohen and Hirsch (16), that the level of threonine synthase is not affected by threonine in the medium, was confirmed by the present work. The level of homoserine kinase in E. coli B was not, affected by the presence of 1O-3 M threonine in the growth medium.
428
WORMSER
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PARDEE
Thus, control of threonine synthesis through the mechanism of enzyme repression by exogenous end product could not be detected. Rate of Removal of Threonine from the Growth Medium The rate of removal of threonine from minimal medium supplemented with both threonine and isoleucine was studied, in order to determine whether the apparent feed-back controls of the biosynthesis of threonine and of isoleucine (4) observed in extracts of E. coli are valid indications of the control mechanism in the growing cells. E. coli cells contain approximately 0.4 pmole threonine and the same amount of isoleucine per milligram of protein (1). If exogenous threonine were used only for protein synthesis in the form of threonine and isoleucine, the cells would remove 0.8 pmole threonine from the medium per milligram of protein formed in the absence of isoleucine, and 0.4 pmole in the presence of isoleucine. Cultures of E. coli in the exponential phase of growth, growing in a minimal medium with 0.1% glucose, were added to flasks containing TABLE
IV
by E. coli from Growth Medium
Supplemented with Zsoleucine Conditions: Cultures of E. coli (initially, 1.7 X 108 cells/ml.) in the exponential phase of growth were shaken at 37°C. in minimal medium supplemented with 40 mM n-threonine and varying concentrations of n-isoleucine. Aliquots were assayed hourly for turbidity, protein, threonine, and a-keto acids. Removal of Threonine
-
-
Incubation time hr.
1
2
-____ 3
Is&u&e concentration
Thm&ne 1
no./ml.
?nM
3.1 x 108
0.00
0.25 0.50 1.00 ___0.00 0.25 0.50 1.00 ---0.00 0.25 0.50 1.00
Protein formed
(
--
--
-
%/ml.
24 22 24 26
_--__ -_c_7.3 x 108 73 75 74 76 --__---15 x 108 213 198 240 206
_-
-
0.10 0.10 0.12 0.12 0.29 0.20 0.18 0.20
_--_
1.3 1.3 1.6 1.6 1.4 1.0 0.8 1.0
0.02 0.03 0.04 0.04 _---0.07 0.04 0.07 0.07
THREONINE
BIOSYNTHESIS
429
minimal medium, glucose, 40 mM L-threonine, and varying concentrations of L-isoleucine. Aliquots taken at hourly intervals were assayed for growth by turbidity measurements, and Folin protein determinations were made. After removal of the cells by centrifugation, the supernatant was assayed for threonine and cu-keto acids. Table IV shows that the cells removed from two to four times as much threonine from the medium as can be accounted for in terms of the assumption that in the absence of exogenous isoleucine threonine gives rise only to the threonine and isoleucine of cell protein and the metabolic pool, and, in the presence of exogenous isoleucine, threonine is not used for isoleucine synthesis. Even high concentrations of isoleucine had no effect on this removal. The formation of a-keto acids was slow and was not affected by the presence of isoleucine. DISCUSSION
The concentration of threonine in the cell appears to be a factor in t#hequantitative control of the biosynthesis of threonine, i.e., a feed-back mechanism operates. The present data show that homoserine kinase in crude extracts of both E. coli and yeast was inhibited competitively by threonine. The phosphorylation of homoserine to HSP is the first step in the biosynthesis of threonine which does not also lead to the synthesis of methionine; therefore, it is also the first step which can be inhibited by threonine without inhibiting the synthesis of methionine. This inhibition was not very strong, the affinity of the enzyme being somewhat greater for the substrate than for the inhibitor in both cases. The similarity in the affinity of the enzyme for the substrate and the inhibitor has also been found in the feed-back control of the biosynthesis of pyrimidines (3); on the other hand, in the third known case of feed-back control, the inhibition of threonine deaminase by isoleucine (4), the enzyme has a much greater affinity for t)he inhibitor than for its substrate. Threonine was a stronger inhibitor of HSK than were other amino acids, even those which are structurally related to threonine, indicating that the inhibition by threonine is specific. The second step in the transformation of homoserine to threonine, the step catalyzed by HSPM, was inhibited by several amino acids, but not by the same amino acids as the kinase, specifically not by threonine. This shows that the inhibitory effect of threonine on its own synthesis is not the mass action effect of a. product, affecting t#he equilibrium of n reaction.
430
WORMSER
AND
P.4RDEE
The question remains whether this feed-back inhibition plays a significant role in the control of threonine synthesis in viva. The concentration of threonine is governed by three factors: the rate of synthesis from homoserine, the rate of incorporation into protein, and the rate of catabolism. If threonine were synthesized at the rate necessary to provide the threonine and isoleucine needed for protein synthesis, but failed to be removed by deamination or incorporation into protein, the concentration of threonine in the cell would reach 5 X 1O-3 M in approximately 3.5 min. This level of threonine would be sufficient to inhibit homoserine kinase by 50%, if homoserine is present at low concentrations. It is evident that the concentration of threonine can build up sufficiently to inhibit HSK, provided that threonine fails to be removed, e.g., by deamination. The relative contributions of deamination and feed-back inhibition in preventing the accumulation of an excess of threonine in vivo are unknown. But isoleucine is a very potent inhibitor of threonine deaminase (4), and the feed-back inhibition could become important either when isoleucine is not removed by incorporation into protein, or when it is provided in the medium. It seems most reasonable to conclude that the concentration of threonine in the cell is governed by a number of coordinated mechanisms. In the normally growing cells HSPM probably limits threonine synthesis. In the absence of protein synthesis the accumulation of isoleucine and threonine probably stops threonine synthesis. The apparent coordinated functioning of two consecutive feed-back mechanisms-isoleucine permitting the accumulation of threonine by inhibiting the activity of threonine deaminase, and threonine inhibiting its own synthesis by inhibiting the activity of homoserine kinase-is a strong indication of the ubiquity of feed-back mechanisms in controlling the synthesis of small molecules by E. co& ------------l
homoserine
---+:------- homoserine
i
phosphate
--+
threonine
+--------7 1 a-ketobutyric acid I isoleucine
- - - - J
The metabolism of exogenous threonine is complex. When a culture of E. coli was grown in a minimal medium supplemented with threonine, more threonine was removed from the medium than was incorporated
THREONINE
BIOSYNTHESIS
431
into protein as threonine and isoleucine. Since threonine is considered to he a precursor of isoleucine and since isoleucine inhibits the deamination of threonine in vitro, one would expect the addition of isoleucine to reduce the amount of threonine removed by the cells. However, neither the removal of threonine, nor the slow format,ion of cY-keto acids, was affected by the presence of isoleucine. This apparent discrepancy between data obtained in experiments in vivo and in vitro is very probably explained in terms of another pathway of threonine catabolism, perhaps a threonine aldolase (18). Whereas only minute amounts of carbon from endogenous threonine are found in glycine, a high proportion of carbon from exogenous threonine can be detected in this amino acid (l), indicating that endogenous threonine is metabolized almost exclusively by a single pathway, whereas a major portion of exogenous threonine is metabolized by a second pathway. The pathway of threonine metabolism via glycine was confirmed by Miller and Simmonds (19) who showed that a mutant of E. coli requiring either glycine or serine for growth can be adapted to use either of these amino acids or L-threonine. SUMMARY
Threonine synthase, the enzyme system which catalyzes the formation of threonine from homoserine, and its two components, homoserine kinase and homoserine phosphate mutaphosphatase, were studied in broken cell preparations of Escherichia coli and of yeast. The purpose of these studies was to determine whether the concentration of threonine within the cell has an effect on the biosynthesis of this amino acid. A feed-back mechanism whereby threonine inhibits the formation of its precursor, homoserine phosphate, appears to be a factor in the control of the rate of threonine biosynt,hesis in E. coli. Although yeast was studied in less det’ail, the data indicate that, a similar mechanism exists in this organism. REFERENCES 1. ROBERTS, R. B., ABELSON, P. H., COWIE, D. B., BOLTON, E. T., AND BRITTEN, R. J., Carnegie Inst. Wash. Publ. No. 607 (1955). 2. ADELBERG, E. A., AND UMBARGER, H. E., J. Biol. Chem. 206, 475 (1953). 3. 4. 5. 6.
YATES, R. A., AND PARDEE, A. B., J. Biol. Chem. 221, 757 (1956). UMBARGER, H. E., Science 143, 848 (1956). BLACK, S., AND WRIGHT, N. G., J. Biol. Chem. 213, 27, 39, 51 (1955). WATANABE, Y., AND SHIMURA, K., J. Biochem. (Tokyo) 42, 181 (1955).
432
WORMSER
AND PARDEE
7. WATANABE, Y., KONISHI, S., AND SHIMURA, K., J. Biochem. (Tokyo) 42, 837 (1955). 8. WINNICK, T., J. Biol. Chem. 143, 461 (1942). 9. NEIDIG, B. A., AND HESS, W. C., Anal. Chem. 24, 1627 (1952). 10. MOORE, S., AND STEIN, W. H., J. Biol. Chem., 211, 907 (1954). 11. FRIEDEMANN, T. E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415 (1943). 12. NISMAN, B., COHEN, G. N., WIESENDANGER, S. B., AND HIRSCH, M-L., Compt. rend. 238, 1342 (1954). 13. WATANABE, Y., KONISHI, S., AND SHIMURA, K., J. Biochem. (Tokyo) 44, 299 (1957). 14. VOGEL, H. J., in “The Chemical Basis of Heredity” (McElroy, W. D., and Glass, B., eds.) p. 276. Johns Hopkins Press, Baltimore, (1957). 15. GORINI, L., AND MAAS, W. K., Biochim. et Biophys. Acta 26, 208 (1957). 16. COHEN, G. N., AND HIRSCH, M-L., J. Bacterial. 87, 182 (1954). 17. YATES, R. A., AND PARDEE, A. B., J. Biol. Chem. 227, 677 (1957). 18. LIN, S. C., AND GREENBERG, D. M., J. Gen. Physiol. 38, 181 (1954). 19. MILLER, D. A., AND SIMMONDS, S., Proc. Nat.!. Acad. Sci. U. S. 43, 195 (1957).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
78, 416-432 (1958)
Regulation of Threonine Biosynthesis in Escherichiu
coZi1~2
Eva H. Wormser3*4 and Arthur B. Pardee From the Department of Biochemistry and the Virus Laborator!/, University of California, Berkeley, California Received
May
28, 1958
INTRODUCTION
Escherichia coli can synthesize all of the metabolites necessary for growth in a medium containing only salts, an inorganic nitrogen source, and an organic energy source. When the cells are in the exponential phase of growth, this synthesis is so economical that, with the exception of some vitamins, only small amounts of the newly synthesized metabolites are released into the medium. If these same organisms are grown in a medium supplemented with one or more amino acids, purine or pyrimidine derivatives, or vitamins, the cells usually utilize the preformed substance to a large extent in preference to the internally synthesized compound. By adding glucose, randomly labeled with U4, as the principal carbon source and a nonradioactive amino acid to the minimal medium, it was shown that in some cases the cells continue their normal biosynthetic pattern, releasing the radioactive amino acid into the medium. In most cases, e.g., threonine, the exogenous amino acid suppresses the biosynthesis of the corresponding compound, and no radioactive metabolites are released into the medium (1). These experiments indicate that some enzyme systems are endowed with a feed-back control, i.e., an internal control which suppresses the synthesis of the end product when that end product is present in sufficiently 1 Aided by a grant from the University of California Cancer Research Funds and by U. S. Public Health Service Training Grant CRTY 5028. 2 Presented in part at the meeting of the Society of American Bacteriologists, Stanford University, August 27, 1957. 3 Some of this work was submitted in partial fulfillment of the requirement for the Ph.D. degree in Biochemistry at the University of California. 4 Present address: Department of Food Technology, University of California, Davis, Calif. 416
THREONINE
-117
BIOSYNTHESIS
high concentration. This hypothesis was suggested by Adelberg and Umbarger (2) when they showed that a valine-requiring mutant growing on limiting amounts of the missing metabolite releases large quantities of the substrate of the blocked reaction into the medium. However, if t,he required metabolite is added in excess, the accumulation of the intermediate is halted. The existence of feed-back controls has also been shown in brokencell preparations. Yates and Pardee (3), studying the control of pyrimidine biosynthesis in this laboratory, showed that even low levels of uracil in the medium inhibit the formation in v&o of the intermediate carbamyl aspartic acid in two pyrimidine-requiring mutants, and, in vitro, cytidine and cytidylic acid inhibit ureidosuccinate formation in competition with both substrates, aspartic acid and carbamyl phosphate. Umbarger (4), working with extracts of a different strain of E. co&, showed that the irreversible deamination of L-threonine to a-ketobutyric acid, postulated to be an essential step in the biosynthesis of L-isoleucine, is inhibited competitively by L-isoleucine. The study of the regulation of threonine biosynthesis was undertaken to determine whether the same type of control could be found in amino acid biosynthesis as had been found in pyrimidine biosynthesis. Threonine was chosen because its pathway of biosynthesis from aspartic acid (5) via homoserine and homoserine phosphate (HSP)3 (6) was known. Homoserine was the starting point of these st,udies. As the scheme below shows, the pathways of biosynthesis of threonine and methionine diverge at this point. Since threonine apparently does not interfere with methionine biosynthesis in this organism, it would hardly be expected that, if threonine exerted feed-back control on its own biosynthetic pathway, it would do so before homoserine. HSK aspartic acid ---f ----f homoserine _____f ATP, Mg++ L
homoserine phosphate
-
HSPM
L
methionine threonine
threonine ) a-ketobutyric deaminase
Since tjhrconine is it,self incorporated
acid ---f --+ isoleucine
int,o prot,ein and is also an int>er-
5 The following abbreviations will be used in this work: HSP, homoserine phosphate; HSK, homoserine kinase; HSPM, homoserine phosphate mutaphopphatase; ATP, adenosine triphosphate.
418
WORMSER AND l'ARDEE
mediate in isoleucine biosynthesis, the control of its concentration in the cell is probably influenced by several factors. Evidence is presented in this work that the synthesis [as well as the destruction (4)] of this amino acid is affected by a feed-back control. MATERIAL
AND METHODS
General Organisms. Yeast extracts were made from fresh Fleischmann’s Baker’s yeast. The bacterial strain was Escherichia coli B. Media. The minimal “C medium” of Roberts (1) was used throughout the work, supplemented when necessary with threonine. For most of the work, 10 ml. of sterile 10% glucose solution was added per liter of sterile medium.
Extracts and Enzymes Purification and Fractionation of Threonine Synthase from Yeast. Threonine synthase, the enzyme system which catalyzes the formation of threonine from homoserine, was purified by the method of Watanabe and Shimura (6). The enzyme system was separated into two fractions, HSK and HSPM by the method of Watanabe et al. (7). These catalyze the phosphorylation of homoserine, CH20H-CHZ-CH(NH+COOH
ATP ___3 Mg++
homoserine HeOaP-0-CHZ-CHZ-CH(NH2)-COOH homoserine phosphate and the subsequent formation of threonine from homoserine phosphate, respectively. Preparation of Bacterial Eztract. In the early part of this work, bacterial extract was prepared daily. The cells were grown to a concentration of approximately 108 cells/ml., centrifuged, suspended in a small volume of distilled water, and broken by a lo-min. treatment in a g-kc. Raytheon Magnetostriction Oscillator. This method was time-consuming and introduced numerous variations from day to day. In order to eliminate these disadvantages, the bacteria were harvested from a 20-l. culture in a Sharples centrifuge, the concentrated cells were treated in a IO-kc. oscillator, and the crude bacterial extract was lyophilized. The dry powder remained stable for months at -5°C. One milligram of the dry extract had approximately as much threonine synthase activity as 3.5 X lo9 cells.
Compounds Amino Acids. L-Threonine was obtained from the California Foundation for Biochemical Research, and nn-homoserine was purchased from Mann Research Laboratories. Most of the other amino acids, which had originally been purchased
THREONINE
BIOSYNTHESIS
419
from Mann Research Laboratories or Nutritional Biochemicals Corporation, were kindly supplied by Dr. F. H. Carpenter. Homoserine Phosphate. A solution of HSP, essentially free of threonine, for use as substrate in the HSPM reaction was prepared by a large-scale application of the HSK reaction with the use of the yeast enzyme as catalyst. Sixty milliliters of the incubation mixture (see Table I) were held at 37” for 3 hr., heated in a to remove the denatured protein. boiling water bath for 15 min., and centrifuged The supernatant solution was then forced through 5 ml. of Dowex 50 resin (length of column, approx. 25 cm.; i.d. 8 mm.) at an approximate rate of one drop per second. The first 10 ml. eluant contained no ninhydrin-positive material and was discarded. The remaining 50 ml. eluant contained essentially threonine-free HSP. The column was then rinsed free of HSP with 10 ml. of distilled water which was allowed to flow through the resin without, the use of air pressure. The volume of the solution was reduced by heating it to about 80°C. and directing a stream of air over the surface of the liquid. Excess salt was removed by centrifugation of the concentrated, refrigerated solution. The solution was stored at -5°C.
Assays for Compounds ‘I’hreonine Assay. The method was a combination and modification of the methods of Winnick (8) and Neidig and Hess (9). The basis of the method was the oxidation of threonine by periodic acid in plastic Conway units and the absorption of acetaldehyde by sodium bisulfite as described by Winnick. The time for the completion of the reaction was reduced from 5 hr. to 2 hr. by permitting the reaction to take place at 37°C. instead of at room temperature. The acetaldehydebisulfite complex was converted to a complex of p-hydroxydiphenyl, and t)he colored product was estimated in a Klett calorimeter with a green (540 mp) filter as described by Neidig and Hess. This method was useful from 0.03 to 0.5 pmole threonine; the error was within 0.01-0.03 pmole, depending on the concentration of the threonine. Attempts to estimate the acetaldehyde directly by absorbing it in a solution of p-hydroxydiphenyl in concentrated sulfuric acid ended in failure because t,he assay became extremely sensitive to minor variations in procedure. This sensitivity may have been caused by the absorption of water as well as acetaldehyde by the sulfuric acid, or by the impossibility of cooling the sulfuric acid-acetaldehyde mixture before adding the p-hydroxydiphenyl. Later experiments showed that either the addition of extra water or improper cooling caused a reduction of color. Homoserine Phosphate Assay. Homoserine phosphate was separated from the neutral amino acids, threonine and homoserine, with the aid of the sulfonate resin Dowex 50. A series of columns, 20 cm. long, was prepared from 5-mm. (i.d.) tubing by drawing out a tip at the bottom end and flanging the top. Each column was packed by pipetting a suspension of 1 ml. of 20&4O@mesh Dowex 50 in the hydrogen form in 2 ml. water onto a small pad of glass wool, and allowing the excess water to drain off. One milliliter of the solution to be assayed was pipetted into the column and forced through the bed of resin under air pressure in 20-30 sec. in order to prevent the resin from being clogged by denatured protein. This effluent was discarded. The HSP was rinsed from the resin with 3 ml. of distilled water,
420
WORMSER
AND
PARDEE
which was permitted to flow through the column without added pressure. After thoroughly mixing the effluent, a l-ml. aliquot was assayed by the ninhydrin method of Moore and Stein (10). The same resin bed could be used at least three times by rinsing with additional distilled water before adding the next sample. a-Keto Acids. A modification of the method of Friedemann and Haugen (11) was used. To 1.0 ml. of the sample, containing 0.01-0.50 pmole a-keto acid, was added 0.9 ml. of 0.05yo of 2,4-dinitrophenylhydrazine in 1.2 N HCl. The tubes were mixed and allowed to stand at room temperature for 20 min. before adding 1.7 ml. of 2.5 N NaOH. The optical density of the solution was read at 520 mp. a-Ketoglutarate was used as the standard.
Enzyme Assays Threonine Synthase Assay. The method of Watanabe and Shimura (6) was used almost without modification. It is based on the phosphorylation of homoserine by ATP in the presence of magnesium ion, the subsequent conversion of the phosphorylated intermediate to threonine, and, finally, the chemical estimation of threonine. The homoserine concentration was reduced by a factor of four to bring its concentration below the saturation level for the competitive inhibition studies. Hmoserine Kinase Assay. This assay was the same as the threonine synthase assay except that the incubation mixture was assayed for HSP instead of threonine. Since the first step in the HSP assay is the separation of HSP from other components of the incubation mixture on a column of Dowex 50, and since this procedure denatures and removes the protein, the enzyme reaction could be stopped either by heating as in the threonine synthase assay, or by forcing the incubation mixture through a Dowex 50 resin column with compressed air. Homoserine Phosphate Mutaphosphatase Assay. This assay was also based on the threonine synthase assay. However, the ATP, magnesium sulfate, and sodium fluoride were unnecessary, leaving only the phosphate buffer. The substrate was HSP rather than homoserine. In this assay the incubation mixture was assayed for both threonine formation and HSP disappearance. RESULTS
Threonine Synthase Crude extracts made from E. co& strain B, catalyzed the formation of threonine from homoserine when the substrate was incubated with ATP and magnesium sulfate in a phosphate buffer at pH 6.8. Sodium fluoride was also present, as a general inhibitor of phosphatases; it had no effect on the threonine synthase reaction. To saturate the system when an extract from 6.5 X lo9 cells/ml. of incubation mixture was used, 1.25 pmoles/ml. L-homoserine were required, and approximately 0.2 pmole threonine was formed. Attempts were made to determine whether this over-all reaction was inhibited by threonine. However, it
THREONINE
BIOSYKTHESTS
121
soon became evident that if threonine had an inhibitory effect it was only in concentrations which were very high compared to the amount of threonine formed. In the presence of these concentrations of threonine, it was impossible to determine with the necessary accuracy small changes in the amount, of threonine formed. It was, therefore, decided to work with the two known components of the threonine synthase system, HSK and HSPM. Homoserine Kinase In crude extracts of E. coli the formation of HSP was much more rapid than its transformation into threonine: In 1 ml. of incubation mixture, 0.2 mg. of lyophilized crude extract, approximately equivalent, to 7 X lOa cells, catalyzed the formation of 0.4 pmole HSP in 1 hr. accompanied by the formation of less than 0.02 pmole threonine from an initial concentration of 1.25 pmoles L-homoserine/ml. (Fig. 1). The conditions were optimal in regard to pH, ATP, and magnesium sulfate. This difference in rates between HSK and HSPM permitted the separation of the action of the two enzymes without purification of the enzymes. Homoserine kinase was inhibited competitively by threonine. Figure 2 shows the results of one experiment. Average values of the constants o Homoserinephosphote A Threonine
Mg. Extract
FIG. 1. The formation
of homoserine phosphate and threonine by bacterial threonine synthase. mg. of lyophiliaed E. coli B extract.
Abscissa: Conditions: The usual incubation mixture (see Table I), with 1.25 Mmoles L-homoserine/ml., was incubated for 1 hr. with varying amounts of bacterial extract. After denaturation and removal of the protein, the snpernatant solut,ion ws.s assayed for both homoserine phosphate and threonine.
422
FIG.
WORMSER
2. Competitive
Abscissa:
inhibition
AND
of bacterial
PARDEW
homoserine
kinaxe
by threonine.
Reciprocal
of the substrate concentration. ml./pmoles n-homoserine Ordinate: Reciprocal of the velocity of the reaction. ml.-hr./pmoles homoserine phosphate Conditions: 0.4 mg. of lyophilized bacterial extract was incubated for 1 hr. with the usual incubation mixture (see Table I), varying only the homoserine concentration. The inhibition constant was determined by the addition of 10 rmoles L-threonine/ml. of incubation mixture.
were calculated from the data by the method of least squares from experiments at three enzyme levels and varying incubation periods. The Michaelis constant of HSK was 6 X 10e4 M for homoserine and the inhibition constant was 4 X 1O-3 M for L-threonine. Thus, the affinity of the enzyme was greater for homoserine than for threonine. Since the purpose of these experiments was to detect a physiologically important feed-back mechanism, it was necessary to determine whether the inhibition by threonine was specific, or whether other amino acids had the same effect. A compound was considered inhibitory if in its presence the formation of HSP was inhibited by more than 20%. This
THREONINE
BIOSYNTHEL3IK
423
point of division between inhibitors and noninhibitors was chosen somewhat arbitrarily. The choice was based on the natural break in the series of values at this point, and the decision that an inhibition of less than 15 or 20% was insignificant. Each compound was tested at least four times to obtain a more accurate average. Table I shows a strong structural influence on inhibition. Threonine is not the only inhibitor of HSK, but it is the best inhibitor. The compounds which were tested fell into three groups: inhibitory compounds which are also structurally relat,ed to threonine, inhibitory amino acids which are basic, and noninhibitory amino acids or related compounds. The first group had two structural features in common: at least one of the y-carbons is in a methyl group, and a hydrogen atom is attached to the erythro position of the beta carbon. In addition, the two best inhibitors, threonine and a-aminobutyric acid were four-carbon a-amino acids. The Lu-amino group was not necessary, but in the case of the four-carbon compounds it distinctly enhanced the inhibitory power. The p-hydroxyl group in the L-threo position was unnecessary, but it could not be in the position of an L-erythro-hydroxyl group. Neither allothreonine nor I)-threonine hn.d any effect on the HSK reaction. The apparent exception to this patt’ern was cysteine. However, if the sulfhydryl group is considered to be equivalent to a methyl group, cysteine also falls into the structural pattern of the inhibitors. Nomoserine I’hosphate
Mutaphosphatasc
The mutaphosphatase reaction appeared to be much slower than the phosphorylat,ion of homoserine. The muximum rate of HSl’ formatioll wit,h extracts of E’. coli B was 1000 pmoles/hr.:‘g. wet weight of cells, i.e., about 1012cells, whereas the maximum rat’e of threonine formation was only 40 @moles/hr./g. cells. This rate in vitro is about one-half the rate in viva, since cells growing at an exponential rate in minimal medium with a generation time of 1 hr. must synthesize 75 Mmoles threonine/ hr./g. cells to supply the threonine and isoleucine of cellular protein. Figure 1 shows the relationship of the amounts of threonine and HSP formed from homoserine under the same conditions. When 1.25 pmoles L-homoserine was incubated for 1 hr. with lyophilized extract equivalent t)o 2.5-7.5 X log cells, the final incubation mixture contained approximately 0.8 pmole HSP and 0.07-0.2 pmole threonine. At these enzyme levels the rate of threonine synthesis was proportional to the concentration of enzyme, and the amount of HSP remained constant. At lo\vel
424
WORMSER
AND
TABLE
PARDEE
I
Kinase by Amino Acids Conditions: 0.2 or 0.4 mg. of lyophilized E. coli B extract was incubated for 1 hr. with the incubation mixture used throughout this work. It contained 200 pmoles NaF, 100 pmoles MgSOa , and 50 pmoles ATP in 1 ml. of 0.1 M phosphate buffer at pH 6.8; 1.25 pmoles L-homoserine and 10 rmoles L-amino acid (or 20 rmoles m-amino acid)/ml. of incubation mixture were added. I. Inhibitory compounds which are structurally related to threonine. II. Basic amino acids. III. Noninhibitory amino acids. Inhibition
Group
of Bacterial
Amino
Homoserine
Average inhibition
acid
Range
Number of experiments
% I
II
III
n-Threonine nn-or-Aminobutyric L-Cysteine Butyric acid n-Valine L-Isoleucine Isovaleric acid
55” 49 37 31 27 26 24
4961 44-54 3242 29-33 20-33 1931 1833
23 4 7 4 6 9 6
L-Arginine L-Lysine L-Histidine
24 24 21
20-31 15-31 7-30
8 7 4
nn-Alanine n-Methionine L-Tryptophan n-Aspartic acid L-Leucine L-Norleucine Iso-a-aminobutyric nn-Allothreonine nn-Norvaline L-Serine n-Asparagine Glycine n-Threonine
17 14 14 12 12 12 12 10 9 8 6 4 4
12-22 8-17 8-23 8-20 7-17 O-22 9-20 O-16 2-11 O-20 O-17 o-8 o-8
6 5 9 6 5 6 8 6 5 6 5 6 4
acid
acid
(1An indication of the accuracy of the data may be obtained from the standard deviation of the “To inhibition”, c = 3.3, in the presence of L-threonine, the only amino acid which was tested frequently enough for such a calculation.
THREONINE
425
BIOSYNTHESIS
c~lzyme levels the rate of HSP synthesis was proportional to the enzyme concentratBion. Attempts to increase the rate of t,he HSPM reaction I)y the addition of pyridoxal phosphate, suggested by Nisman ct al. (12) to he a cofactor, were unsuccessful. For the purpose of inhibition studies with amino acids, HSPM was assayed separately from HSK by using the product of the kinase reaction, HSP, as the substrate. It was difficult to obtain consistent results, and especially difficult to achieve a balance of HSP disappearance and threonine appearance (Table II) owing to t#he difficulty of determining a small increment in the threonine concentration. However, it,
Inhibition
of Bacterial
Homoserine
TABLE II Phosphate Mutaphosphatase
by Amino
Acids
Conditions: 1.4, 2.8, or 4.2 mg. of lyophilized E. coli B extract was incubated for 1 hr. with the usual incubation mixture (see Table I) including 0.5 pmole homoserine phosphate/ml.; 10 rmoles L-amino acid (or 20 rmoles m-amino acid)/ ml. of incubation mixture was added. After denaturation and removal of the protein, the supernatant solution was assayed for homoserine phosphate disappearance and for threonine formation, except in the samples to which threonine had been added. Average inhibition of HSP disappearance
Amino acid
Average inhibition of threonine formation
%
%
100 62 38 52
78 52 38 21
L-Serine r,-Arginine Valerie acid nI,-Alanine
32 24 27 68
44 25 0
Glycine r>-Histidine I,-Methionine L-Valine uL-cw-Aminobut,yric I>-Isoleucine
19 3 8 12 22 0
3 12 11 6 0 0
I,-Cysteine Butyric acid klysine I,-Aspartic acid
L-Threonine n-Threonine nL-Allothreonine
acid
0 0 0
No. of expts.
-
10 5 5
426
WORMSER
AND
PARDEE
is probably safe to conclude that the concentration of threonine which inhibited HSK approximately 50% did not inhibit HSPM. Two other amino acids which inhibited the kinase strongly, cu-aminobutyric acid and isoleucine, also had no inhibitory effect on the mutaphosphatase. On the other hand, butyric acid and cysteine, both of which inhibited the kinase less strongly than threonine, were strong inhibitors. Cysteine, in fact, inhibited this enzyme at a much lower concentration than any other amino acid tested. Yeast Enzymes The threonine synthase system was also found in crude extracts of baker’s yeast, prepared according to the directions of Watanabe and Shimura (6). Inhibition studies with threonine yielded the same ambiguous results as the analogous experiments with bacterial extracts. Therefore, HSK was separated from HSPM by the method of Watanabe et al. (7) by ammonium sulfate fractionation. The HSK fraction had a much greater specific activity (sixfold) than unfractionated threonine synthase and appeared to be almost free of HSPM activity. However, the mutaphosphatase fraction contained much residual kinase activity and showed no increase in specific activity with respect to threonine formation. No further work was done with the latter fraction. Yeast HSK, like the bacterial enzyme, was inhibited competitively by threonine. The Michaelis and inhibition constants of HSK from both sources were of the same order of magnitude, approximately 1O-3 M for t-homoserine and 3 X 10e3 M for L-threonine, respectively, in yeast, compared to 6 X lo-* M and 4 X 10e3 M for the corresponding constants in E. coli. All of the values are the averages of constants obtained from several different experiments, and each constant was calculated from the data by the method of least squares. Assays performed with threonine replacing the substrate showed neither a change in the concentration of threonine nor the appearance of a ninhydrin-positive substance which could be mistaken for HSP. It is evident, therefore, that the inhibition by threonine is not caused by any chemical changes in the threonine. Watanabe et al. (13) recently published their studies on the properties of yeast homoserine kinase. In agreement with the present work they found that threonine is not a substrate of yeast HSK. They did not, however, detect the inhibitory property of L-threonine since they used a saturating concentration of homoserine (6.25 mM) in their assays and did not compensate for this by an even higher threonine concentration.
THREONINE
TABLE Inhibition
427
BIOSYNTHESIS
of Yeast Homoserine
III
Kinase
by Several Amino
Acids
Conditions: 0.25 mg. of lyophilized homoserine kinase was incubated for 1 hr. with the usual incubation mixture (see Table I) including 0.5 pmole L-homoserine/ml.; 25 /Imoles L-amino acid (or 50 firmoles DL-amino acid)/ml. incubation mixture was added. Controls, incubated with all ingredients of the incubation mixture including the amino acid, but excluding ATP, formed no HSP. Average inhibition
Amino acid
Range
% r,-Threonine or,-ol-Aminobutyric or,-Alanine m-Isoleucine IA-Methionine L-Serine
62 77 54 37(?) 14 13
acid
51-68 73-80 46-62 17-61 4-24 3-20
Comparison of inhibition studies with amino acids other than threonine showed that the kinases from the two sources were similar, but by no means identical (Table III). Five of the six amino acids which were tested with both enzymes behaved in the same mamler qunlitatively, but there were significant quantitative differences. Alanine was the only amino acid which inhibited yeast HSK but did not inhibit bacterial HSK. In contrast to the bacterial enzyme, which was inhibited most st,rongly by threonine, yeast HSK was inhibited most strongly by cu-aminobutyric acid. However, in both cases t,he two best inhihit,ors were threonine and a-aminobutyric acid. Enzyme Concentration Another mode of metabolic control is by enzyme repression. Vogel (14) and Gorini and Maas (15) showed that the concentrations of some enzymes of arginine biosynthesis are reduced below the basal level when adequate amounts of arginine are provided in the medium. They also showed the reverse-the levels of these enzymes can be increased when arginine is limiting. The latter phenomenon was also shown in this laboratory by Yates and Pardee (17) in the biosynthesis of uracil. No attempt has been made as yet to determine whether diminishing the internal concentration of threonine raises the level of threonine synthase, but the observations of Cohen and Hirsch (16), that the level of threonine synthase is not affected by threonine in the medium, was confirmed by the present work. The level of homoserine kinase in E. coli B was not, affected by the presence of 1O-3 M threonine in the growth medium.
428
WORMSER
AND
PARDEE
Thus, control of threonine synthesis through the mechanism of enzyme repression by exogenous end product could not be detected. Rate of Removal of Threonine from the Growth Medium The rate of removal of threonine from minimal medium supplemented with both threonine and isoleucine was studied, in order to determine whether the apparent feed-back controls of the biosynthesis of threonine and of isoleucine (4) observed in extracts of E. coli are valid indications of the control mechanism in the growing cells. E. coli cells contain approximately 0.4 pmole threonine and the same amount of isoleucine per milligram of protein (1). If exogenous threonine were used only for protein synthesis in the form of threonine and isoleucine, the cells would remove 0.8 pmole threonine from the medium per milligram of protein formed in the absence of isoleucine, and 0.4 pmole in the presence of isoleucine. Cultures of E. coli in the exponential phase of growth, growing in a minimal medium with 0.1% glucose, were added to flasks containing TABLE
IV
by E. coli from Growth Medium
Supplemented with Zsoleucine Conditions: Cultures of E. coli (initially, 1.7 X 108 cells/ml.) in the exponential phase of growth were shaken at 37°C. in minimal medium supplemented with 40 mM n-threonine and varying concentrations of n-isoleucine. Aliquots were assayed hourly for turbidity, protein, threonine, and a-keto acids. Removal of Threonine
-
-
Incubation time hr.
1
2
-____ 3
Is&u&e concentration
Thm&ne 1
no./ml.
?nM
3.1 x 108
0.00
0.25 0.50 1.00 ___0.00 0.25 0.50 1.00 ---0.00 0.25 0.50 1.00
Protein formed
(
--
--
-
%/ml.
24 22 24 26
_--__ -_c_7.3 x 108 73 75 74 76 --__---15 x 108 213 198 240 206
_-
-
0.10 0.10 0.12 0.12 0.29 0.20 0.18 0.20
_--_
1.3 1.3 1.6 1.6 1.4 1.0 0.8 1.0
0.02 0.03 0.04 0.04 _---0.07 0.04 0.07 0.07
THREONINE
BIOSYNTHESIS
429
minimal medium, glucose, 40 mM L-threonine, and varying concentrations of L-isoleucine. Aliquots taken at hourly intervals were assayed for growth by turbidity measurements, and Folin protein determinations were made. After removal of the cells by centrifugation, the supernatant was assayed for threonine and cu-keto acids. Table IV shows that the cells removed from two to four times as much threonine from the medium as can be accounted for in terms of the assumption that in the absence of exogenous isoleucine threonine gives rise only to the threonine and isoleucine of cell protein and the metabolic pool, and, in the presence of exogenous isoleucine, threonine is not used for isoleucine synthesis. Even high concentrations of isoleucine had no effect on this removal. The formation of a-keto acids was slow and was not affected by the presence of isoleucine. DISCUSSION
The concentration of threonine in the cell appears to be a factor in t#hequantitative control of the biosynthesis of threonine, i.e., a feed-back mechanism operates. The present data show that homoserine kinase in crude extracts of both E. coli and yeast was inhibited competitively by threonine. The phosphorylation of homoserine to HSP is the first step in the biosynthesis of threonine which does not also lead to the synthesis of methionine; therefore, it is also the first step which can be inhibited by threonine without inhibiting the synthesis of methionine. This inhibition was not very strong, the affinity of the enzyme being somewhat greater for the substrate than for the inhibitor in both cases. The similarity in the affinity of the enzyme for the substrate and the inhibitor has also been found in the feed-back control of the biosynthesis of pyrimidines (3); on the other hand, in the third known case of feed-back control, the inhibition of threonine deaminase by isoleucine (4), the enzyme has a much greater affinity for t)he inhibitor than for its substrate. Threonine was a stronger inhibitor of HSK than were other amino acids, even those which are structurally related to threonine, indicating that the inhibition by threonine is specific. The second step in the transformation of homoserine to threonine, the step catalyzed by HSPM, was inhibited by several amino acids, but not by the same amino acids as the kinase, specifically not by threonine. This shows that the inhibitory effect of threonine on its own synthesis is not the mass action effect of a. product, affecting t#he equilibrium of n reaction.
430
WORMSER
AND
P.4RDEE
The question remains whether this feed-back inhibition plays a significant role in the control of threonine synthesis in viva. The concentration of threonine is governed by three factors: the rate of synthesis from homoserine, the rate of incorporation into protein, and the rate of catabolism. If threonine were synthesized at the rate necessary to provide the threonine and isoleucine needed for protein synthesis, but failed to be removed by deamination or incorporation into protein, the concentration of threonine in the cell would reach 5 X 1O-3 M in approximately 3.5 min. This level of threonine would be sufficient to inhibit homoserine kinase by 50%, if homoserine is present at low concentrations. It is evident that the concentration of threonine can build up sufficiently to inhibit HSK, provided that threonine fails to be removed, e.g., by deamination. The relative contributions of deamination and feed-back inhibition in preventing the accumulation of an excess of threonine in vivo are unknown. But isoleucine is a very potent inhibitor of threonine deaminase (4), and the feed-back inhibition could become important either when isoleucine is not removed by incorporation into protein, or when it is provided in the medium. It seems most reasonable to conclude that the concentration of threonine in the cell is governed by a number of coordinated mechanisms. In the normally growing cells HSPM probably limits threonine synthesis. In the absence of protein synthesis the accumulation of isoleucine and threonine probably stops threonine synthesis. The apparent coordinated functioning of two consecutive feed-back mechanisms-isoleucine permitting the accumulation of threonine by inhibiting the activity of threonine deaminase, and threonine inhibiting its own synthesis by inhibiting the activity of homoserine kinase-is a strong indication of the ubiquity of feed-back mechanisms in controlling the synthesis of small molecules by E. co& ------------l
homoserine
---+:------- homoserine
i
phosphate
--+
threonine
+--------7 1 a-ketobutyric acid I isoleucine
- - - - J
The metabolism of exogenous threonine is complex. When a culture of E. coli was grown in a minimal medium supplemented with threonine, more threonine was removed from the medium than was incorporated
THREONINE
BIOSYNTHESIS
431
into protein as threonine and isoleucine. Since threonine is considered to he a precursor of isoleucine and since isoleucine inhibits the deamination of threonine in vitro, one would expect the addition of isoleucine to reduce the amount of threonine removed by the cells. However, neither the removal of threonine, nor the slow format,ion of cY-keto acids, was affected by the presence of isoleucine. This apparent discrepancy between data obtained in experiments in vivo and in vitro is very probably explained in terms of another pathway of threonine catabolism, perhaps a threonine aldolase (18). Whereas only minute amounts of carbon from endogenous threonine are found in glycine, a high proportion of carbon from exogenous threonine can be detected in this amino acid (l), indicating that endogenous threonine is metabolized almost exclusively by a single pathway, whereas a major portion of exogenous threonine is metabolized by a second pathway. The pathway of threonine metabolism via glycine was confirmed by Miller and Simmonds (19) who showed that a mutant of E. coli requiring either glycine or serine for growth can be adapted to use either of these amino acids or L-threonine. SUMMARY
Threonine synthase, the enzyme system which catalyzes the formation of threonine from homoserine, and its two components, homoserine kinase and homoserine phosphate mutaphosphatase, were studied in broken cell preparations of Escherichia coli and of yeast. The purpose of these studies was to determine whether the concentration of threonine within the cell has an effect on the biosynthesis of this amino acid. A feed-back mechanism whereby threonine inhibits the formation of its precursor, homoserine phosphate, appears to be a factor in the control of the rate of threonine biosynt,hesis in E. coli. Although yeast was studied in less det’ail, the data indicate that, a similar mechanism exists in this organism. REFERENCES 1. ROBERTS, R. B., ABELSON, P. H., COWIE, D. B., BOLTON, E. T., AND BRITTEN, R. J., Carnegie Inst. Wash. Publ. No. 607 (1955). 2. ADELBERG, E. A., AND UMBARGER, H. E., J. Biol. Chem. 206, 475 (1953). 3. 4. 5. 6.
YATES, R. A., AND PARDEE, A. B., J. Biol. Chem. 221, 757 (1956). UMBARGER, H. E., Science 143, 848 (1956). BLACK, S., AND WRIGHT, N. G., J. Biol. Chem. 213, 27, 39, 51 (1955). WATANABE, Y., AND SHIMURA, K., J. Biochem. (Tokyo) 42, 181 (1955).
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AND PARDEE
7. WATANABE, Y., KONISHI, S., AND SHIMURA, K., J. Biochem. (Tokyo) 42, 837 (1955). 8. WINNICK, T., J. Biol. Chem. 143, 461 (1942). 9. NEIDIG, B. A., AND HESS, W. C., Anal. Chem. 24, 1627 (1952). 10. MOORE, S., AND STEIN, W. H., J. Biol. Chem., 211, 907 (1954). 11. FRIEDEMANN, T. E., AND HAUGEN, G. E., J. Biol. Chem. 147, 415 (1943). 12. NISMAN, B., COHEN, G. N., WIESENDANGER, S. B., AND HIRSCH, M-L., Compt. rend. 238, 1342 (1954). 13. WATANABE, Y., KONISHI, S., AND SHIMURA, K., J. Biochem. (Tokyo) 44, 299 (1957). 14. VOGEL, H. J., in “The Chemical Basis of Heredity” (McElroy, W. D., and Glass, B., eds.) p. 276. Johns Hopkins Press, Baltimore, (1957). 15. GORINI, L., AND MAAS, W. K., Biochim. et Biophys. Acta 26, 208 (1957). 16. COHEN, G. N., AND HIRSCH, M-L., J. Bacterial. 87, 182 (1954). 17. YATES, R. A., AND PARDEE, A. B., J. Biol. Chem. 227, 677 (1957). 18. LIN, S. C., AND GREENBERG, D. M., J. Gen. Physiol. 38, 181 (1954). 19. MILLER, D. A., AND SIMMONDS, S., Proc. Nat.!. Acad. Sci. U. S. 43, 195 (1957).