- Email: [email protected]
Control of feedback repression of the enzymes of purine biosynthesis in Aerobacter aerogenes
349
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
26525
CONTROL BY F E E D B A C K R E P R E S S I O N OF T H E ENZYMES OF P U R I N E B I O S Y N T H E S I S IN AEROBACTER AEROGENES D O N A L D P. N I E R L I C H AND B O R I S M A G A S A N I K
Department of Bacteriology, University of California, Los Angeles, Calif. 90024 (U.S.A .) and Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. o2z39 (U.S.A.) (Received N o v e m b e r 3rd, 197 o)
SUMMARY
Studies have been made of the regulation of the synthesis of six purine biosynthetic enzymes : P-ribosyl-PP amidotransferase (I), P-ribosyl glycinamide synthetase (u), P-ribosyl formyl glycinamide amidotransferase (IV), adenylosuccinate lyase ( V I I I - I I A ) , adenylosuccinate synthetase (IA), and IMP dehydrogenase (IG). Wild type Aerobacter aerogenes and two purine requiring mutants derived from it, were grown with limiting or excess adenine or guanine, cell extracts prepared, and enzyme activities measured. Using as a reference the levels of enzymes found in the wild type bacteria grown in unsupplemented medium, the results indicate that all of the enzymes are repressed by excess purine, and derepressed by purine starvation. However, these controls are not coordinate throughout the pathway, since each segment of the pathway shows a unique response, apparently to accommodate the special functions of the enzymes within it. The levels of three early enzymes of the pathway are primarily responsive to adenine supplementation and therefore it is concluded that these are controlled by the intracellular levels of adenine nucleotides, or perhaps those of inosine (produced in cultures by deamination). Two of these (I and IV) are coordinately controlled, while the levels of the third enzyme (III) vary in parallel with the others, but with changes of a smaller magnitude. One enzyme ( V I I I - I I A ) was shown to be subject to a dual or multivalent control, apparently being regulated both by the supply of adenine and guanine nucleotides. This enzyme has a dual function, catalyzing one step in IMP synthesis, and another in the conversion of IMP to AMP. Strikingly a subsequent enzyme in IMP formation, IMP cyclohydrolase, has been shown by others to be primarily controlled b y guanine, In addition to its above mentioned function, this latter enzyme can be envisioned as necessary for an interconversion reaction in the pathway from adenine to guanine nucleotides. The synthesis of the enzymes studied subsequent to IMP formation, in the branches leading to AMP (IA) and GMP (IG), appear primarily controlled b y their respective endproducts alone. However, the possibility that the levels of the first enzyme of Abbreviations: succino-ACR, 5'-phosphoribosyl 5-amino-4-imidazole-N-succinocarboxa m i d e ; ACR, 5 ' - p h o s p h o r i b o s y l - 5 - a m i n o - 4 - i m i d a z o l e c a r b o x a m i d e .
Biochim. Biophys. Acta, 23 ° (1971) 3 4 9 - 3 6 I
350
D.P. NIERLICH, B. MAGASANIt~
GMP formation (IG) might be increased by the presence of substrate, as well as repressed by endproduct, was discussed.
INTRODUCTION Biosynthetic pathways are in general regulated by two specific control mechanisms: endproduct inhibition, controlling the activity of a pathway's first enzyme 2, and endproduct repression, serving to adjust the levels of the pathway's enzymes by controlling the rate of de novo enzyme synthesis a. Extensive study of the regulation of purine biosynthesis in bacteria has provided a view of an elegant system of controls by which the first of these, endproduct inhibition, regulates not only the initial reaction leading to IMP synthesis ~, but also the reactions involved in the synthesis and interconversion of the final products, AMP and GMP 5 7. On the other hand little is known about the control by repression of the enzymes of the pathway v, and detailed studies in bacteria have been restricted to only a few enzymes 7-12. In this work we present studies with six purine biosynthetic enzymes, chosen from different parts of the pathway*. The formation of these enzymes was measured under a variety of conditions of purine limitation and excess. It was hoped that in this way this work would not only provide more information on the regulation of purine biosynthesis, but also more generally indicate the types of controls involved in the regulation of a branched pathway. MATERIALS AND METHODS Chemicals
Beyond those previously listed 4, the chemicals used and their supplies are as follows : adenine, b-histidine-HC1 : California Corp. for Biochemical Research ; guanine (synthetic): Mann Research Laboratories; thiamine-HCl: Nutritional Biochemicals Corp. ; lithium acetyl phosphate: Cambridge Biochemical Co. Tile authors are indebted to Dr. John Buchanan for a gift of 5'-phosphoribosyl 5-amino-4-imidazole-N-succinocarboxamide, to Dr. Thayer French for 4'-phosphoribosyl formylglycinamide, and to Dr. Standish H a r t m a n for 5'-phosphoribosyl glycinamide. Cells and cell extracts Aerobacter aerogenes wild type strain lO33 and two purine mutants derived from
it, P D - I and P-I4, were used in this study 13. Cultures were grown in i-1 quantities in 2-1 flasks, shaken vigorously on a New Brunswick rotary shaker at 37 °. The basic mineral medium used TM was supplemented with 4 ° #g/ml of L-histidine-HC1 (to assure that the histidine supply would not limit protein synthesis during purine starvation of tile auxotrophs), 0.025 /,g/ml thiamine HC1 (required for strain PD-I), 2 mg/ml glucose, and adenine and guanine as indicated in the text. Where a high pH-high ammonia medium is indicated, the standard medium has been adjusted to pH 7.8 by increasing the proportion of dibasic potassium phosphate, and NH4C1 added to a final concentration of 8 mg/ml. * A preliminary report of this work has been given 1. Biochim. Biophys. Acta, 230 (1971) 349-36I
FEEDBACK REPRESSION IN PURINE BIOSYNTHESIS
351
Cultures were inoculated with 5-ml quantities of the appropriate freshly prepared overnight culture, grown with 20 #g/ml guanine. After 14 h the cultures were chilled and the cells were harvested b y centrifugation. They were then washed twice by centrifugation and resuspension in 15o ml of 0.03 M potassium phosphate buffer, p H 7-5; finally, they were resuspended in the same buffer, using io ml for each gram wet weight of cells. Cell extracts were prepared by treatment for 6 rain in a Raytheon Io-kcycle sonic oscillator. These extracts were then centrifuged at 29000 × g for I h. Each preparation was dialyzed for 6 h against two 2o-vol quantities of the extraction buffer. Such preparations contained 13-18 mg/ml protein. The extracts were stored in ice, under which conditions the enzymes were shown to be stable until tested in the course of several days.
Assay of 5'-P-ribosyl-z-P~ amidotransferase (ribosylamine 5'-phosphate:pyrophosphate phosphoribosyltransferase (glutamate-amidating), EC 2.4.2.I4) The assay used was a modification of the procedure of HARTMAN AND BUCHANAN 14, which we have described in detail 4. In these experiments to each reaction mixture (volume 0.35 ml) 4/,moles of lithium acetyl phosphate were added. This serves, with the acyl phosphokinase present in the crude bacterial extracts, as an ATP regenerating system.
Assay of 5'-P-ribosyl glycinamide synthetase (5'-phosphoribosylamine :glycine ligase (ADP), EC 6.3.I.3) The assay for 5'-P-ribosyl glycinamide synthetase has been previously described 1~. 5'-P-ribosyl glycinamide formed in a first incubation is in turn measured enzymatically by a modification of a technique described by WARREN AND BUCHANANTM. As above, 4/,moles of lithium acetyl phosphate were added to each incubation vessel.
Assay of 5'-P-ribosyl formylglycinamide amidotransferase (5'-phosphoribosyl-formylglycinamide :L-glutamine amido-ligase (ADP), EC 6.3.5.3) This enzyme was measured using the modification described b y FRENCH et al. 1~ of a procedure originally described by LEVENBERG AND BUCHANANTM. To each vessel 2.4 #moles of lithium acetyl phosphate were added. The pigeon liver enzyme fraction required for this procedure was the kind gift of Dr. T. French. An increase of I.O unit of absorbance in the assay was taken to indicate the formation of 22,35 nmoles of 5'-P-ribosyl formylglycinamide.
Assay of adenylosuccinate lyase (adenylosuccinate AMP-lyase, EC 4,3.2.2) This enzyme catalyses two reactions in purine biosynthesis, the removal of furnarate from 5-amido-4-imidazole-N-succino carboxamide ribonucleotide as well as from adenylosuceinate 19. The former activity was measured, i.e. the conversion of 5'-phosphoribosyl 5-amino-4-imidazole-N-succinocarboxamide (succino-ACR) to 5'phosphoribosyl-5-amino-4-imidazolecarboxamide (ACR). The ACR produced was measured by a modification of the Bratton and Marshall technique described by GOTS AND GOLLUB2°. This procedure distinguishes between succino-ACR and ACR b y virtue of the relative instability of the diazonium complex of the former at room temperature. The reaction mixture contained in o.35 ml the following: 20 nmoles succino-ACR; 30 #moles potassium phosphate buffer, p H 7.0; and enzyme. After IO rain incubation
Biochim. Biophys. Acta, 23o (1971) 349-361
352
D . P . NIERLICH, B. MAGASANIK
at 37 °, o.o5 ml of 15 % trichloroacetic acid in I M HC1 was added. The vessels were then briefly cooled and the following operations performed at room temperature. The mixtures were centrifuged for 15 min at 80o x g and then the following were added, mixing so as not to disturb the pellet: 0.05 ml NaNO2, o.I %; 0.05 ml ammonium sulfamate, 0.5 %; 0.05 ml N-I-napthylethylenediamine dihydrochloride, o.I %. The latter was added exactly 8 rain after addition of the ammonium sulfamate. After a centrifugation as above, the absorbance of the supernatant was determined. An increase of i.o absorbance unit at 54 ° nm was taken to indicate the formation of 20.8 nmoles ACR 21.
Assay of ade~Lvlosuccinate synthetase (AMP:L-aspartate lyase (GDP), EC 6.3.4.4) This enzyme was assayed by a procedure patterned after one (Assay I) described by LIEBERMAN22. The reaction mixtures contained: IOO/,moles glycine buffer, pH 8.0, 0.3/,mole IMP, 1/,mole aspartic acid (neutralized), o.oi/,mole GTP, o.o 4 #mole ATP, 4/,moles lithium acetyl phosphate, 4/,moles MgC12, and water to give o.69 ml. The temperature of the mixture was brought to 24 ° and o.oi ml of enzyme was added. The initial rate of absorbance change at 28o nm was followed and found proportional to the amount of extract added, although the reactions did not continue linearly after the first 5 min. Tile activities observed in the linear range were very low, from 0.0o2 to o.oi absorbance unit/min. An increase of I.O unit at 280 nm was taken to be equivalent to the formation of o.o51/,mole of adenylosuccinate in this system (based on the data given by CARTER AND COHEN23).
Assay of I M P dehydrogenase ( I M P : N A D ÷ oxidoreductase, EC 1.2.I.I4) The assay for this enzyme followed that described by MAGASANIKet al34 in which the formation of xanthosine-5'-P is followed by measuring the increase in absorbance at 290 nm. The reaction mixture contained: ioo #moles Tris-HC1, pH 8.4; I /~mole IMP; 0.66 #mole NAD+; 3.3 /~moles glutathione; 20/,moles KC1; water and enzyme to a final volume of I.O ml. The reactions were started by adding enzyme to the reaction mixture, prewarmed to 24 °, and following the initial rate of increase of absorbance at that same temperature. An increase of I.O absorbance unit at 290 nm was taken to indicate the formation of 0.208/zmole of xanthosine-5'-P in this system 25.
Protein determinations Protein was estimated by the LOWRY el al. 26 modification of the Folin phenol method. Bovine serum albumin Fraction V (Armour) was used as standard. All of the enzyme assay procedures gave results in proportion to the length of the incubation period, over the times indicated. However, in three of the assay procedures the results were not strictly proportional to the amount of extract added. For this reason, in the cases of 5'-P-ribosyl-I-P 2 amidotransferase, 5'-P-ribosyl glycinamide synthetase, and adenylosuccinate lyase, assays were performed with varying amounts of enzyme, and the data obtained after plotting the results and extrapolating the curve to infinite enzyme dilution. The corrections were not greater than IO %. For the data presented in Tables I and II, all the assays were performed on one set of extracts. In all cases multiple assays were performed on each extract, varying either the time of incubation and amount of extract, or both. An indication of the
Biochim. Biophys. Acta, 23o (1971) 349 361
353
FEEDBACK REPRESSION IN PURINE BIOSYNTHESIS
overall reproducibility of the measurements is gained b y comparing the values shown in Tables I and I I for Extracts I I a and I I b , two cultures grown with identical purine supplement, but varying two supposedly unrelated parameters : the ammonia concentration and medium p H ( i i a was obtained from the standard medium, while I I b was grown in the high p H - h i g h ammonia medium). The values are in good agreement. RESULTS
The activities of six enzymes of purine biosynthesis were measured in extracts of cells grown in media with different purine supplements. The enzymes studied are indicated in Fig. i with their roles in purine biosynthesis. Three of the enzymes studied, 5'-P- ribosyl-l-P2 amidotransferase 5'-P-ribosyl-l-P2 I
5'-P-ribosy[
5'-P-ribosyl glycinarnlde synthetase ornino
,~
5'-P-rJbosyl glycinarnide
1r
e. ~T
5'-P-r~ bosyl forrnylglycinarnlde arnidot ransferose 5'-P-ribosyl 5'-P-ribosyl forrny]glycinarnide ~ formylglycinornidine ]2
~
D
7 (~- ~ "-*- --~ --¢" histidirte
/ ~
---ll- iluccino ACR
"---- I ACR
VIII
/ ---i-
IX
/
/
~,suc;inoie ,, A
' .t~
I,,/_,__
l/'""'"'" -----)-IMP
X ~ I M P
\
mgilt~le "xMP
,
IIG
>GMP
Fig. i. P a t h w a y of purine biosynthesis in bacteria, in outline, s h o w i n g e n z y m e s studied. The p a t h s of exogenous adenine and guanine utilization a n d the p a t h w a y of histidine biosynthesis are indicated s,~, i0.
5 ' - P - r i b o s y l - I - P 2 amidotransferase (I), 5'-P-ribosyl glycinamide synthetase (II), and
5'-P-ribosyl formylglycinamide amidotransferase (IV), catalyse reactions early in the common pathway to IMP. The enzyme adenylosuccinate lyase (VIII-IIA) catalyses two reactions. One, the conversion of succino-ACR to ACR, is a step in IMP formation and the other, the formation of AMP from adenylosuccinate, is the last step in AMP synthesis. The remaining two enzymes studied, adenylosuccinate synthetase (IA) and IMP dehydrogenase (IG), catalyse the first steps in the branches of the pathway leading from IMP to AMP and to GMP, respectively. In these experiments, enzyme levels were measured on extracts of cells grown in .Biochim. Biophys. Acta, 23 ° (1971) 349 361
354
D.P.
N I E R L I C H , B. MAGASANIK
flask culture into the stationary phase. This procedure provides a sensitive means for observing control by repression of these enzymes. It has been shown for example that when a guanine-requiring mutant is starved of guanine, the growth of the cells is restricted, and protein synthesis is reduced to a relatively low, linear rate 9. Under these same conditions an enzyme of the blocked pathway (IMP dehydrogenase) is also formed linearly, but now with a greatly increased differential rate of synthesis. In this way the levels of these enzymes may mount over several hours, resulting in greatly increased specific activities. In contrast, growth of cells in the presence of an excess of the product of the pathway may repress the synthesis of the enzymes. Thus when growth finally ceases due to a limitation of other growth factors, the enzymes' specific activities are relatively low. The specific activities of the six enzymes studied are presented in Tables I and II. The data show the effect of purine limitation or supplementation on the final specific activities of the enzymes. To allow interpretation of the data in a systematic way, we have arranged the two tables from top to bottom according to decreasing supplementation of the growth media. That is, in the center of Tables I and II (Extract 7) are given the specific activities of the enzymes of the wild type strain Io33 in which the intracellular levels of the postulated repressors, the purine nucleotides, are maintained by the endogenous paths of synthesis. Above this in Tables I and II are data obtained for cultures supplemented with an excess of adenine and/or guanine, and below this are data for cultures of the two purine-requiring mutants used in this study, grown with limiting quantities of the purines. The two mutants used, strains PD-I and P-I 4 are both derived from the wild type strain lO33. PD-I is blocked at an early step* in the pathway leading to IMP, and will grow on any one of the products of the pathway, since it possesses the enzymes to interconvert these compounds 5 (Fig. I). Strain P-I 4 is a specific guanine requirer, lacking the enzyme XMP aminase. This guanine requirement cannot be met by adenine, in that the conversion of adenine to guanine involves first the formation of IMP by one of several routes, followed by the formation of GMP along the normal biosynthetic pathway 6. Thus with this mutant one can test specifically for the influence of a guanine limitation, independent of the degree of adenine supplementation. We assume in these studies that the intracellular purine ribonucleotides are the effective repressors. The free bases themselves are not normally found in these cells nor do they occur on the known biosynthetic pathways as intermediates. The ribonucleotides, on the other hand, which are the endproducts of the purine pathway, do not enter the cells from the medium when provided, whereas the free bases are readily utilized and converted to the ribonucleotide level. It must be added that since it is known that a portion of the adenine provided to cells is deaminated directly and enters the cells as hypoxantine 28,29, in those cases where adenine is implicated in the control of an enzyme, the role of the hypoxanthine derivatives cannot be excluded.
Steps prior to the formation of I M P Inspection of Tables I and I I shows readily that the levels of the six enzymes are highly dependent on the level of purine in the growth medium. The levels are low in * T h e e x a c t l o c a t i o n of t h e m u t a t i o n is n o t k n o w n b u t i t is p r o b a b l e t h a t i t l a c k s e i t h e r t h e e n z y m e c a t a l y z i n g r e a c t i o n I I [ or V (Fig. I). I t is p r o b a b l e t h a t i t is b l o c k e d b e f o r e e n z y m e V1 (ref. 27), a n d i t d o e s p o s s e s s I, [ I a n d IV.
Biochim. Biophys Acta, 23 ° (1971) 349-361
FEEDBACK
REPRESSION
IN PURINE
BIOSYNTHESIS
355
the supplemented media (Extracts 1-6), intermediate where the medium is unsupplemented (Extract 7), and, in most instances, elevated where a purine limitation is imposed (Extracts 8-ii), Table I presents the data for 5'-P-ribosyl-I-P 2 amidotransferTABLE
I
EFFECT OF P U R I N E S U P P L E M E N T A T I O N
Extract
Additions* Guanine
ON L E V E L S OF P U R I N E BIOSYNTHETIC E N Z Y M E S
Strain
Enzyme specific activity (nmoles/min per mg protein)
Adenine
5'-P-ribosylz.-P2 amidotransferase
5'-P-ribosylglycinamide synthetase
5'-P-ribosyl forrnylglycinamide amidotransferase
1 2 3 4 5 6
X X X X o o
X o o o X X
PD-I lO33 P-I 4 PD-I lO33 PD-I
0.8 1.5 0.6 2. 5 1.5 2. 4
I.I 1.9 i.o 4-3 2.3 3.2
0. 5 I.i 0, 7 1.4 I .I 1. 4
7
o
o
lO33
7.5
7-5
2.9
8 9 IO I Ia I tb
L L o L L
X o L o o
P-I 4 P-I 4 PD-I PD-I PD-I
-5.2 24.0 29.9 28.2
I.I 8.0 rS.I 15. 3 16. 7
-4.7 16. 3 19. 4 20. I
L, limiting o, n o t a d d e d . TABLE
(6 / , g / m l
adenine
X,
excess
(4 ° / ~ g / m l a d e n i n e
and/or
guanine)
II
EFFECT OF P U R I N E S U P P L E M E N T A T I O N
Extract
or guanine);
Additions* Guanine
ON L E V E L S OF P U R I N E BIOSYNTHETIC E N Z Y M E S
Strain Adenine
Enzyme specific activity (nmoles/min per mg protein) Succino-AMP Succino-AMP 11~lP dehysynthetase lyase drogenase
I 2 3 4 5 6
X X X X o o
X o o o X X
PD-I lO33 P-I 4 PD-I lO33 PD-I
0. 3 I.I 1.2 i.o I.I 0.8
I.I 1. 9 2. 4 2.1 2. 3 2.2
1.6 0. 4 0. 4 0. 7 1. 5 2. 4
7
o
o
lO33
1.7
3 .8
1.6
S 9 IO I ta lib
L L o L L
X o L o o
P-I 4 P-I 4 PD-I PD-I PD-I
1.5 2.5 3.o 4.4 3.4
5.7 8,5 9.o 8.5 11.6
--79.2 34.o 6.0 6.2
* L, limiting o, n o t a d d e d .
(6 / * g / i n l a d e n i n e
or guanine);
X,
excess
(4 ° / , g / m l
adenine
Biochim. Biophys. Mcta,
and/or
guanine)
23o (1971) 349-361
356
D.P. NIERLICH, B. MAGASANIK
ase (I), 5'-P-ribosylglycinamide synthetase (II) and 5'-P-ribosyl formylglycinamide amidotransferase (IV), three enzymes which appear to be very similarly controlled. The results of the assays of 5'-P-ribosyl glycinamide synthetase are representative. The activity of this enzyme was found to be 7.5 units in the wild type strain Io33 where the nucleotide pools are nlaintained by endogenous synthesis (Extract 7). Addition to the medium of 4 ° keg/ml adenine and 4 ° ktg/ml guanine (i5-2o/~g/ml of either purine was sufficient in the basic medium to satisfy the purine requirement and repress enzyme synthesis) leads to a sevenfold repression of the enzyme (Extract I). On the other hand, addition of an excess of only one of the two products, adenine or guanine, leads to a lesser repression of the enzyme level (Extracts 2-6.) The fact t h a t supplementation by adenine and guanine leads to greater enzyme repression than by either one alone m a y be explained in two ways. First this m a y reflect a dual control over the synthesis of this enzyme. Second, it is possible that through the interconversion reactions, the dual supplement leads to a higher endogenous level of that single purine directly responsible for the control. Inspection of the data obtained by purine limitation (Extracts 8--11) indicates that an adenine derivative, and not one of guanine is most likely responsible for the regulation of this enzyme. Although a general purine limitation leads to a derepression of the enzyme (Extracts Io and I I), a specific guanine limitation alone has no effect on the enzyme level (compare extract 9 with Extract 7), while tile importance of the adenine supplement is revealed in the fact that excess adenine-limiting guanine results in full repression (compare Extracts 8 and I). The conclusion that a derivative of adenine is responsible for the control of 5'-P-ribosyl glycinamide synthetase is also applicable to the other two enzymes concerned with early steps in the pathway, 5'-P-ribosyl-I-P 2 amidotransferase and 5'-P-ribosyl formylglycinamide amidotransferase. This m a y be seen by inspection of the date in Table I, but even more easily in Fig. 2 where these data are shown graphically. Fig. 2a shows the relation of the activity of 5'-P-ribosyl-I-P 2 amidotransferase to that of 5'-P-ribosyl formylglycinamide amidotransferase; the points on the graph each represent the activities of the two enzymes in a different cell extract. It can be seen from the fact that a straight line can be drawn through the points in the figure that these two enzymes maintain the same ratio of activities under all growth conditions; that is, they are coordinate 3°. Fig. 2b shows a graph of 5'-P-ribosyl-I-P 2 amidotransferase against 5'-P-ribosyl glyeinamide synthetase, whose control was described in detail above. In this case a smooth curve fits the points very well. This behavior indicates that the two enzymes whose control appear coordinate, are regulated in qualitatively the same manner as 5'-P-ribosyl glycinamide synthetase but are more responsive to regulation than this latter enzyme. A similar situation has been observed in studies of the regulation of two pyrimidine biosynthetic enzymes ~1.
Adenylosuccinate synthetase (IA) and lyase ( V I I I and IIA) Table I I shows the activities of these two enzymes in the cell extracts. Here the response to purine excess and purine limitation is generally similar to that of 5'-Pribosyl glycinamide synthetase, but of a distinctly smaller amplitude. Here again combined adenine and guanine supplementation leads to the greatest enzyme repression (Extract I); supplementation with an excess of only one of the purines gives a
Biochim. Biophys. Acta, 23o (1971) 349-36~
FEEDBACK REPRESSION IN PURINE BIOSYNTHESIS
357
slight repression (Extracts 2-6); the wild type strain in unsupplemented medium possesses intermediate enzyme levels (Extract 7); and a general purine limitation, obtained by starving strain PD-I of either adenine or guanine, produces derepression (Extracts io,ii). To test further if guanine plays an independent role in the regulation I
I
I
20'
0 _go
c ~o
0
0
0
I0 20 5 L P - P i b o s y I - 1 - P 2 AMIDOTRANSFERASE
I
3
I
3O
t
20 0
c
"E
a.
P I I lO 20 5 L p - r [ b o sy1-1-P2 AMIDOTRANSFERASE
I 30
Fig. 2. R e l a t i v e specific activities of t h r e e p u r i n e b i o s y n t h e t i c e n z y m e s in cell-flee e x t r a c t s . D a t a , in n m o l e s / m i n per m g protein, are from T a b l e I. a. 5 ' - P - r i b o s y l f o r m y l g l y c i n a m i d e a m i d o t r a n s f e r a s e a n d 5 - ' P - r i b o s y l - I - P 2 a m i d o t r a n s f e r a s e , b. 5 -P-ribosyl g l y c i n a m i d e s y n t h e t a s e a n d 5 -P-ribosylI-P2 a m i d o t r a n s f e r a s e .
of these two enzymes, measurements were carried out on the guanine-requiring mutant P-I4, in which the specific effects of guanine limitation and adenine supplementation can be tested. For the second enzyme of adenine synthesis, adenylosuccinate lyase, a distinctly different result is obtained than that already described for the early enzymes. Here the condition of excess adenine-limiting guanine (Extract 8) leads to an intermediate enzyme level, near that of the wild type grown in unsupplemented medium (Extract 7). In the case where adenine is provided by endogenous synthesis, and guanine is limiting (Extract 9), a derepression is seen relative to that in which both adenine and guanine are provided by endogenous synthesis. It therefore seems that for enzyme repression both adenine and guanine must be present ; that is, a dual control appears to exist for adenylosuceinate lyase. It should be evident from the data inTable II that the adenylosuecinate syntheBiochirn. Biophys. Acta, 230 (1971) 349-361
35 ~
D . P . NIERLICH, B. MAGASAN1K
tase and the lyase, if not coordinate in their control, are at least quite similarly regulated. The question, however, is complicated because of the general difficulty experienced in measuring the first of these enzymes in crude extracts. The measurements are performed by following the production of adenylosuccinate, and the accumulation of this compound is influenced by the presence of the second enzyme. However, in spite of the problem of measurement, there appears to be at least one significant difference in the control of these enzymes. While the lyase appears to be subject to dual control, and both adenine and guanine limitations lead to a partial derepression, in the case of adenylosuccinate synthetase, the condition excess adenine-limiting guanine leads to repression (Extract 8). Therefore it appears that this enzyme is controlled by adenine.
I M P dehydrogenase The data for this enzyme, the first in the conversion of IMP to GMP, are also given in Table II. This enzyme is repressed in cultures of the wild type and the mutants by excess guanine but not by excess adenine (Extracts 2-6). Therefore, it seems reasonable to conclude that a guanine derivative is responsible for its control. This departure from the pattern of control observed with the other enzymes can be further seen in the very great derepression obtained, 2oo-fold, when a specific guanine limitation is imposed (Extract 9). DISCUSSION
We have shown here that while tile synthesis of tile enzymes of tile purine pathway is subject to regulation by the supply of endproducts, adenine and guanine, there is no single unitary control. In fact each segment of the pathway seems uniquely regulated to accommodate the special function of the enzymes within it. This conclusion becomes evident on listing the various responses we have observed here, with additional ca.ses others have reported: (a) The synthesis of the three early enzymes of the pathway studied (5'-P-ribosyl-I-P2 amidotransferase, 5'-P-ribosyl glycinamide synthetase and 5'-P-ribosyl formylglycinamide amidotransferase), are similar and possibly coordinate in control. These respond primarily to the supplementation of tile medium with adenine. (b) The synthesis of the enzyme adenylosuccinate lyase, serving both a step prior to the formation of IMP and a step in the conversion of IMP to AMP, appears equally sensitive to supplementation by both adenine and guanine. (c) The enzyme IMP cyclohydrolase (studied in the same series of mutants by LEVIN AN[) MAGASANIKS), forming part of an enzyme complex which is responsible for the two steps (IX and X) following that catalyzed by adenylosuccinate lyasO °, is responsive to guanine supplementation, and is not repressed by adenine during guanine starvation. (d) The synthesis of the first enzyme of GMP biosynthesis, IMP dehydrogenase, responds primarily to guanine supplementation. This same control is probable as well for the second enzyme of GMP biosynthesis, XMP aminase, since it and the first enzyme have been shown to be under coordinate control in Escherichia coli al,az (e) The first enzyme of AMP synthesis, adenylosuccinate synthetase, m a y be controlled by adenine alone, although our experiments do not exclude the possibility that its control is the same dual control as that of adenylosuccinate lyase. These controls are summarized in Fig. 3The dual control over the synthesis of adenylosuccinate lyase is similar to that tJiochim, t3iophys, dcla, 230 (~97 I) 349 36~
F E E D B A C K R E P R E S S I O N IN P U R I N E B I O S Y N T H E S I S
359
of several of the enzymes responsible for the synthesis of the branched chain amino acids in Salmonella 32. These enzymes contribute to the formation of valine, leucine, and isoleucine, and are only completely repressed when the three amino acids are present in excess. This so-called "multivalent" control is seen here in the fact that adenyloA
A and G ~ H l s h d i n e
G
Fig. 3. Control over the synthesis of the enzymes of the purine pathway. A and G are meant to indicate adenine and guanine and their ribonucleotide derivatives. See text. succinate lyase is only maximally repressed when both adenine and guanine are present in excess, and only fully derepressed when both are limiting. Since this enzyme carries out a reaction in the pathway common to both the formation of AMP and GMP, and a second reaction in the branch leading specifically to AMP, such a control would be clearly advantageous to the cell. The specificity and apparent utility of these controls is further seen in the control of IMP cyclohydrolase, which, as mentioned above, carries out a reaction in IMP formation following that of adenylosuccinate ]yase, but unlike tile latter enzyme is apparently controlled by guanine. This enzyme, in addition to its role in de novo IMP formation, can be seen as catalyzing a step in tile conversion of AMP to GMP by way of the histidine pathway (Fig. I). A control somewhat similar to that of adenylosuccinate lyase was observed with the three early enzymes studied. Here an excess of the two purines led to maximal repression, which might be taken to indicate a dual control. However, it was not possible to demonstrate independently an influence of guanine on the synthesis; i.e. a guanine limitation, when adenine was present in excess, led to repression. Although this does not rule out the possibility of a dual control of some direct type, it does suggest a simpler explanation. That is that a guanine supplement increases the repression given by adenine, by sparing adenine utilization. For this reason we envision adenine derivatives as playing the principal role in the regulation of these enzymes. The enzyme IMP dehydrogenase seems regulated by the pool of guanine derivatives. The enzyme is repressed by excess guanine, but not by adenine. However, there are some anomalous features of its regulation that need explanation. First, whereas generally speaking, the greatest repression of the other purine enzymes is observed with combined adenine and guanine supplementation, this was not the case with IMP dehydrogenase. Here, on the contrary, the addition of adenine to guaninecontaining media actually raised the enzyme levels (Table II). Secondly, when the general purine auxotroph (strain PD-I) was grown on limiting amounts of purine, the extent of derepression observed was highly dependent on whether that purine was adenine or guanine; a much higher enzyme level was in fact observed when adenine was the limiting substance. There are a number of possible explanations. One would be that adenine interferes with the utilization of guanine, such that the intracellular Biochirn. Biophys. Acta, 230 (1971) 34q-361
360
D.P.
N I E R L I C H , B. MAGASANIK
pools of GMP are lower with adenine present than without it. This possibility derives from the observation that adenine is readily deaminated to hypoxanthine by bacterial cultures and that hypoxanthine and guanine compete for a common pyrophosphorylase in their conversion to nucleoside monopbosphates~8, 29. Such a competition has been used to explain an increase in the levels of IMP dehydrogenase in cultures of Salmonella, brought about by hypoxanthine, when the enzymes had been repressed by 8-azaguanine, a guanine analog aa. A more interesting possibility, however, from the standpoint of our knowledge of the operation of these controls is that tile increase in IMP dehydrogenase may be attributed to induction by IMP. This compound is the precursor to the GMP branch of tile purine pathway, and would be formed in the conversion of adenine to guanine ribonucleotides, known to take place. Although enzyme induction by a precursor in biosynthetic pathways has not been generally observed, Gorini has reported some effect on enzyme levels by glutamic acid, tile precursor to the arginine pathway, in studies of the regulation of ornithine transcarbamylase, an arginine biosynthetic enzyme a4. It must be added also that the induction of the guanine biosynthetic enzymes has been observed in cultures of Escherichia coli treated with 5-amino-4-imidazolecarboxamide ribonucleoside n. The effect of the latter compound was, however, considered to be indirect, since this compound seemed to interfere with guanine utilization. ACKNOWLEDGEMENT
This work was supported in part by a grant, GM-o7446, from the National Institutes of Health, U.S. Public Health Service. REFERENCES I D. P. NIERLICH AND B. MAGASANIK, Federation Proc., 22 (1963) 476. 2 H. E. UMBARGER, in D. M. BONNER, Control Mechanisms in Cellular Processes, The Ronald Press, N e w York, 1961, p. 67. 3 H. J. VOGEL, in D. M. BONNER, Control Mechanisms in Cellular Processes, The Ronald Press, N e w Y o r k , 196I, p. 23. 4 D. P. NIERLICH AND B. MAGANANIK, J. Biol. Chem., 240 (1965) 358. 5 B. ~IAGASANIK AND D. [~ARIBIAN, J. Biol. Chem., 235 (196o) 2672. 6 B. ~'~IAGASANIK, in I. E. GUNSALUS AND R. Y. STANIER, The Bacteria, Vol. 3, Academic Press, N e w Y o r k , I962, p. 295. 7 R. L. BLAKLE¥ AND E . VITOLS, Ann. Rev. Biochem., 37 (1968) 2Ol. 8 A. P. LEVIN AND B. MAGASANIK, J. Biol. Chem., 236 (1961) 184. 9 A. P. LEVlN AND B. MAGASANIK, ./r. Biol. Chem., 236 (1961) 181o. 1o J. S. GUTS, F. R. DALAL AND S. R. SHUMAS, J. Bacteriol., 99 (1969) 441I I H . K. KURAMITSU, S. UDAKA AND H. S. -'X~OYED, J. Biol. Chem., 239 (1964) 3425 . 12 H. J. J. NIJCAMP AND P. G. D E HAAN, Biochim. Biophys. Acta, 145 (1967) 31. 13 D. USHIBA AND B. MAGASANIK, Proc. Soc. E:@tl. Biol. 3led., 80 (1952) 626. 14 S. C. HARTMAN AND J. M. BUCHANAN, J. Biol. Chem., 233 (1958) 451. 15 D. P. NIERLICH AND B. MAGASANIK, J . Biol. Chem., 240 (1965) 366. 16 L. WARREN AND J. M. BUCHANAN, J. Biol. Chem., 229 (1957) 613. 17 T. C. FRENCH, I. B. DAWID, R. A. DAY AND J. M. BUCHANAN, J. Biol. Chem., 238 (1963) 2171. 18 B. LEVENBERG AND J. M. BUCHANAN, J. Biol. Chem., 224 (1957) lOl 9. 19 1{. \xv'. MILLER, L. N. LUKENS AND J. M. BUCHANAN, J. Am. Chem. Sue., 79 (1957) 15132o J. S. GuTs AND E. G. GOLLUB, Proc. Natl. Acad. Sei. U.S., 43 (1957) 826. 21 J. G. FLAKS, L. WARREN AND J. ~I. BUCHANAN, J . Biol. Chem., 228 (I957) 215. 22 1. LIEBERMAN, J. Biol. Chem., 223 (1956 ) 327 . 23 C. E. CARTER AYD L. H . COHEN, J. Am. Chem. Soc., 77 (1955) 49924 B. MAGASANIK, H. S. MOYED AND L. B. GEHRING, J. Biol. Chem., 226 (1957) 339. 25 H. S. MOYED AND B. MAGASANIK, J . Biol. Chem., 226 (1957) 351 .
Biochim. Biophys. Acta, 230 (1971) 3 4 9 - 3 6 i
FEEDBACK REPRESSION IN PURINE BIOSYNTHESIS
361
26 O. H LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. S. RANDALL, J . Biol. Chem., 193 (1951) 265 . 27 M. S. BROOKE AND ]3. MAGASANIK, J. Bacteriol., 68 (1954) 727 . 28 S. FRIEDMAN AND J. S. GOTS, J. Biol. Chem., 2Ol (1953) 125. 29 g F. ZIMMERMAN AND ]3. MAGASANIK, J. Biol. Chem., 239 (1964) 2933 ° F. JACOB, D. PERRIN, C. SANCHEZ AND J. MONOD, Compl. Rend., 25 ° (196o) 1727. 31 J. R. BECKWlTH, A. B. PARDEE, R. AUSTRIAN AND F. JACOB, J. ,~lol. Biol., 5 (1962) 618. 32 M. FREUNDLICH, R. O. BURNS AND H. E. UMBARGER, Proc. Natl. Acad. Sci. U.S., 48 (1962) 18o4. 33 A. P. LEVlN, D o c t o r a l D i s s e r t a t i o n , H a r v a r d U n i v e r s i t y , 1959. 34 L. GORINI, Bacteriol. Rev., 27 (1963) 182.
Biochim. Biophys. Acta, 23 ° (1971) 349-361
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
26525
CONTROL BY F E E D B A C K R E P R E S S I O N OF T H E ENZYMES OF P U R I N E B I O S Y N T H E S I S IN AEROBACTER AEROGENES D O N A L D P. N I E R L I C H AND B O R I S M A G A S A N I K
Department of Bacteriology, University of California, Los Angeles, Calif. 90024 (U.S.A .) and Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. o2z39 (U.S.A.) (Received N o v e m b e r 3rd, 197 o)
SUMMARY
Studies have been made of the regulation of the synthesis of six purine biosynthetic enzymes : P-ribosyl-PP amidotransferase (I), P-ribosyl glycinamide synthetase (u), P-ribosyl formyl glycinamide amidotransferase (IV), adenylosuccinate lyase ( V I I I - I I A ) , adenylosuccinate synthetase (IA), and IMP dehydrogenase (IG). Wild type Aerobacter aerogenes and two purine requiring mutants derived from it, were grown with limiting or excess adenine or guanine, cell extracts prepared, and enzyme activities measured. Using as a reference the levels of enzymes found in the wild type bacteria grown in unsupplemented medium, the results indicate that all of the enzymes are repressed by excess purine, and derepressed by purine starvation. However, these controls are not coordinate throughout the pathway, since each segment of the pathway shows a unique response, apparently to accommodate the special functions of the enzymes within it. The levels of three early enzymes of the pathway are primarily responsive to adenine supplementation and therefore it is concluded that these are controlled by the intracellular levels of adenine nucleotides, or perhaps those of inosine (produced in cultures by deamination). Two of these (I and IV) are coordinately controlled, while the levels of the third enzyme (III) vary in parallel with the others, but with changes of a smaller magnitude. One enzyme ( V I I I - I I A ) was shown to be subject to a dual or multivalent control, apparently being regulated both by the supply of adenine and guanine nucleotides. This enzyme has a dual function, catalyzing one step in IMP synthesis, and another in the conversion of IMP to AMP. Strikingly a subsequent enzyme in IMP formation, IMP cyclohydrolase, has been shown by others to be primarily controlled b y guanine, In addition to its above mentioned function, this latter enzyme can be envisioned as necessary for an interconversion reaction in the pathway from adenine to guanine nucleotides. The synthesis of the enzymes studied subsequent to IMP formation, in the branches leading to AMP (IA) and GMP (IG), appear primarily controlled b y their respective endproducts alone. However, the possibility that the levels of the first enzyme of Abbreviations: succino-ACR, 5'-phosphoribosyl 5-amino-4-imidazole-N-succinocarboxa m i d e ; ACR, 5 ' - p h o s p h o r i b o s y l - 5 - a m i n o - 4 - i m i d a z o l e c a r b o x a m i d e .
Biochim. Biophys. Acta, 23 ° (1971) 3 4 9 - 3 6 I
350
D.P. NIERLICH, B. MAGASANIt~
GMP formation (IG) might be increased by the presence of substrate, as well as repressed by endproduct, was discussed.
INTRODUCTION Biosynthetic pathways are in general regulated by two specific control mechanisms: endproduct inhibition, controlling the activity of a pathway's first enzyme 2, and endproduct repression, serving to adjust the levels of the pathway's enzymes by controlling the rate of de novo enzyme synthesis a. Extensive study of the regulation of purine biosynthesis in bacteria has provided a view of an elegant system of controls by which the first of these, endproduct inhibition, regulates not only the initial reaction leading to IMP synthesis ~, but also the reactions involved in the synthesis and interconversion of the final products, AMP and GMP 5 7. On the other hand little is known about the control by repression of the enzymes of the pathway v, and detailed studies in bacteria have been restricted to only a few enzymes 7-12. In this work we present studies with six purine biosynthetic enzymes, chosen from different parts of the pathway*. The formation of these enzymes was measured under a variety of conditions of purine limitation and excess. It was hoped that in this way this work would not only provide more information on the regulation of purine biosynthesis, but also more generally indicate the types of controls involved in the regulation of a branched pathway. MATERIALS AND METHODS Chemicals
Beyond those previously listed 4, the chemicals used and their supplies are as follows : adenine, b-histidine-HC1 : California Corp. for Biochemical Research ; guanine (synthetic): Mann Research Laboratories; thiamine-HCl: Nutritional Biochemicals Corp. ; lithium acetyl phosphate: Cambridge Biochemical Co. Tile authors are indebted to Dr. John Buchanan for a gift of 5'-phosphoribosyl 5-amino-4-imidazole-N-succinocarboxamide, to Dr. Thayer French for 4'-phosphoribosyl formylglycinamide, and to Dr. Standish H a r t m a n for 5'-phosphoribosyl glycinamide. Cells and cell extracts Aerobacter aerogenes wild type strain lO33 and two purine mutants derived from
it, P D - I and P-I4, were used in this study 13. Cultures were grown in i-1 quantities in 2-1 flasks, shaken vigorously on a New Brunswick rotary shaker at 37 °. The basic mineral medium used TM was supplemented with 4 ° #g/ml of L-histidine-HC1 (to assure that the histidine supply would not limit protein synthesis during purine starvation of tile auxotrophs), 0.025 /,g/ml thiamine HC1 (required for strain PD-I), 2 mg/ml glucose, and adenine and guanine as indicated in the text. Where a high pH-high ammonia medium is indicated, the standard medium has been adjusted to pH 7.8 by increasing the proportion of dibasic potassium phosphate, and NH4C1 added to a final concentration of 8 mg/ml. * A preliminary report of this work has been given 1. Biochim. Biophys. Acta, 230 (1971) 349-36I
FEEDBACK REPRESSION IN PURINE BIOSYNTHESIS
351
Cultures were inoculated with 5-ml quantities of the appropriate freshly prepared overnight culture, grown with 20 #g/ml guanine. After 14 h the cultures were chilled and the cells were harvested b y centrifugation. They were then washed twice by centrifugation and resuspension in 15o ml of 0.03 M potassium phosphate buffer, p H 7-5; finally, they were resuspended in the same buffer, using io ml for each gram wet weight of cells. Cell extracts were prepared by treatment for 6 rain in a Raytheon Io-kcycle sonic oscillator. These extracts were then centrifuged at 29000 × g for I h. Each preparation was dialyzed for 6 h against two 2o-vol quantities of the extraction buffer. Such preparations contained 13-18 mg/ml protein. The extracts were stored in ice, under which conditions the enzymes were shown to be stable until tested in the course of several days.
Assay of 5'-P-ribosyl-z-P~ amidotransferase (ribosylamine 5'-phosphate:pyrophosphate phosphoribosyltransferase (glutamate-amidating), EC 2.4.2.I4) The assay used was a modification of the procedure of HARTMAN AND BUCHANAN 14, which we have described in detail 4. In these experiments to each reaction mixture (volume 0.35 ml) 4/,moles of lithium acetyl phosphate were added. This serves, with the acyl phosphokinase present in the crude bacterial extracts, as an ATP regenerating system.
Assay of 5'-P-ribosyl glycinamide synthetase (5'-phosphoribosylamine :glycine ligase (ADP), EC 6.3.I.3) The assay for 5'-P-ribosyl glycinamide synthetase has been previously described 1~. 5'-P-ribosyl glycinamide formed in a first incubation is in turn measured enzymatically by a modification of a technique described by WARREN AND BUCHANANTM. As above, 4/,moles of lithium acetyl phosphate were added to each incubation vessel.
Assay of 5'-P-ribosyl formylglycinamide amidotransferase (5'-phosphoribosyl-formylglycinamide :L-glutamine amido-ligase (ADP), EC 6.3.5.3) This enzyme was measured using the modification described b y FRENCH et al. 1~ of a procedure originally described by LEVENBERG AND BUCHANANTM. To each vessel 2.4 #moles of lithium acetyl phosphate were added. The pigeon liver enzyme fraction required for this procedure was the kind gift of Dr. T. French. An increase of I.O unit of absorbance in the assay was taken to indicate the formation of 22,35 nmoles of 5'-P-ribosyl formylglycinamide.
Assay of adenylosuccinate lyase (adenylosuccinate AMP-lyase, EC 4,3.2.2) This enzyme catalyses two reactions in purine biosynthesis, the removal of furnarate from 5-amido-4-imidazole-N-succino carboxamide ribonucleotide as well as from adenylosuceinate 19. The former activity was measured, i.e. the conversion of 5'-phosphoribosyl 5-amino-4-imidazole-N-succinocarboxamide (succino-ACR) to 5'phosphoribosyl-5-amino-4-imidazolecarboxamide (ACR). The ACR produced was measured by a modification of the Bratton and Marshall technique described by GOTS AND GOLLUB2°. This procedure distinguishes between succino-ACR and ACR b y virtue of the relative instability of the diazonium complex of the former at room temperature. The reaction mixture contained in o.35 ml the following: 20 nmoles succino-ACR; 30 #moles potassium phosphate buffer, p H 7.0; and enzyme. After IO rain incubation
Biochim. Biophys. Acta, 23o (1971) 349-361
352
D . P . NIERLICH, B. MAGASANIK
at 37 °, o.o5 ml of 15 % trichloroacetic acid in I M HC1 was added. The vessels were then briefly cooled and the following operations performed at room temperature. The mixtures were centrifuged for 15 min at 80o x g and then the following were added, mixing so as not to disturb the pellet: 0.05 ml NaNO2, o.I %; 0.05 ml ammonium sulfamate, 0.5 %; 0.05 ml N-I-napthylethylenediamine dihydrochloride, o.I %. The latter was added exactly 8 rain after addition of the ammonium sulfamate. After a centrifugation as above, the absorbance of the supernatant was determined. An increase of i.o absorbance unit at 54 ° nm was taken to indicate the formation of 20.8 nmoles ACR 21.
Assay of ade~Lvlosuccinate synthetase (AMP:L-aspartate lyase (GDP), EC 6.3.4.4) This enzyme was assayed by a procedure patterned after one (Assay I) described by LIEBERMAN22. The reaction mixtures contained: IOO/,moles glycine buffer, pH 8.0, 0.3/,mole IMP, 1/,mole aspartic acid (neutralized), o.oi/,mole GTP, o.o 4 #mole ATP, 4/,moles lithium acetyl phosphate, 4/,moles MgC12, and water to give o.69 ml. The temperature of the mixture was brought to 24 ° and o.oi ml of enzyme was added. The initial rate of absorbance change at 28o nm was followed and found proportional to the amount of extract added, although the reactions did not continue linearly after the first 5 min. Tile activities observed in the linear range were very low, from 0.0o2 to o.oi absorbance unit/min. An increase of I.O unit at 280 nm was taken to be equivalent to the formation of o.o51/,mole of adenylosuccinate in this system (based on the data given by CARTER AND COHEN23).
Assay of I M P dehydrogenase ( I M P : N A D ÷ oxidoreductase, EC 1.2.I.I4) The assay for this enzyme followed that described by MAGASANIKet al34 in which the formation of xanthosine-5'-P is followed by measuring the increase in absorbance at 290 nm. The reaction mixture contained: ioo #moles Tris-HC1, pH 8.4; I /~mole IMP; 0.66 #mole NAD+; 3.3 /~moles glutathione; 20/,moles KC1; water and enzyme to a final volume of I.O ml. The reactions were started by adding enzyme to the reaction mixture, prewarmed to 24 °, and following the initial rate of increase of absorbance at that same temperature. An increase of I.O absorbance unit at 290 nm was taken to indicate the formation of 0.208/zmole of xanthosine-5'-P in this system 25.
Protein determinations Protein was estimated by the LOWRY el al. 26 modification of the Folin phenol method. Bovine serum albumin Fraction V (Armour) was used as standard. All of the enzyme assay procedures gave results in proportion to the length of the incubation period, over the times indicated. However, in three of the assay procedures the results were not strictly proportional to the amount of extract added. For this reason, in the cases of 5'-P-ribosyl-I-P 2 amidotransferase, 5'-P-ribosyl glycinamide synthetase, and adenylosuccinate lyase, assays were performed with varying amounts of enzyme, and the data obtained after plotting the results and extrapolating the curve to infinite enzyme dilution. The corrections were not greater than IO %. For the data presented in Tables I and II, all the assays were performed on one set of extracts. In all cases multiple assays were performed on each extract, varying either the time of incubation and amount of extract, or both. An indication of the
Biochim. Biophys. Acta, 23o (1971) 349 361
353
FEEDBACK REPRESSION IN PURINE BIOSYNTHESIS
overall reproducibility of the measurements is gained b y comparing the values shown in Tables I and I I for Extracts I I a and I I b , two cultures grown with identical purine supplement, but varying two supposedly unrelated parameters : the ammonia concentration and medium p H ( i i a was obtained from the standard medium, while I I b was grown in the high p H - h i g h ammonia medium). The values are in good agreement. RESULTS
The activities of six enzymes of purine biosynthesis were measured in extracts of cells grown in media with different purine supplements. The enzymes studied are indicated in Fig. i with their roles in purine biosynthesis. Three of the enzymes studied, 5'-P- ribosyl-l-P2 amidotransferase 5'-P-ribosyl-l-P2 I
5'-P-ribosy[
5'-P-ribosyl glycinarnlde synthetase ornino
,~
5'-P-rJbosyl glycinarnide
1r
e. ~T
5'-P-r~ bosyl forrnylglycinarnlde arnidot ransferose 5'-P-ribosyl 5'-P-ribosyl forrny]glycinarnide ~ formylglycinornidine ]2
~
D
7 (~- ~ "-*- --~ --¢" histidirte
/ ~
---ll- iluccino ACR
"---- I ACR
VIII
/ ---i-
IX
/
/
~,suc;inoie ,, A
' .t~
I,,/_,__
l/'""'"'" -----)-IMP
X ~ I M P
\
mgilt~le "xMP
,
IIG
>GMP
Fig. i. P a t h w a y of purine biosynthesis in bacteria, in outline, s h o w i n g e n z y m e s studied. The p a t h s of exogenous adenine and guanine utilization a n d the p a t h w a y of histidine biosynthesis are indicated s,~, i0.
5 ' - P - r i b o s y l - I - P 2 amidotransferase (I), 5'-P-ribosyl glycinamide synthetase (II), and
5'-P-ribosyl formylglycinamide amidotransferase (IV), catalyse reactions early in the common pathway to IMP. The enzyme adenylosuccinate lyase (VIII-IIA) catalyses two reactions. One, the conversion of succino-ACR to ACR, is a step in IMP formation and the other, the formation of AMP from adenylosuccinate, is the last step in AMP synthesis. The remaining two enzymes studied, adenylosuccinate synthetase (IA) and IMP dehydrogenase (IG), catalyse the first steps in the branches of the pathway leading from IMP to AMP and to GMP, respectively. In these experiments, enzyme levels were measured on extracts of cells grown in .Biochim. Biophys. Acta, 23 ° (1971) 349 361
354
D.P.
N I E R L I C H , B. MAGASANIK
flask culture into the stationary phase. This procedure provides a sensitive means for observing control by repression of these enzymes. It has been shown for example that when a guanine-requiring mutant is starved of guanine, the growth of the cells is restricted, and protein synthesis is reduced to a relatively low, linear rate 9. Under these same conditions an enzyme of the blocked pathway (IMP dehydrogenase) is also formed linearly, but now with a greatly increased differential rate of synthesis. In this way the levels of these enzymes may mount over several hours, resulting in greatly increased specific activities. In contrast, growth of cells in the presence of an excess of the product of the pathway may repress the synthesis of the enzymes. Thus when growth finally ceases due to a limitation of other growth factors, the enzymes' specific activities are relatively low. The specific activities of the six enzymes studied are presented in Tables I and II. The data show the effect of purine limitation or supplementation on the final specific activities of the enzymes. To allow interpretation of the data in a systematic way, we have arranged the two tables from top to bottom according to decreasing supplementation of the growth media. That is, in the center of Tables I and II (Extract 7) are given the specific activities of the enzymes of the wild type strain Io33 in which the intracellular levels of the postulated repressors, the purine nucleotides, are maintained by the endogenous paths of synthesis. Above this in Tables I and II are data obtained for cultures supplemented with an excess of adenine and/or guanine, and below this are data for cultures of the two purine-requiring mutants used in this study, grown with limiting quantities of the purines. The two mutants used, strains PD-I and P-I 4 are both derived from the wild type strain lO33. PD-I is blocked at an early step* in the pathway leading to IMP, and will grow on any one of the products of the pathway, since it possesses the enzymes to interconvert these compounds 5 (Fig. I). Strain P-I 4 is a specific guanine requirer, lacking the enzyme XMP aminase. This guanine requirement cannot be met by adenine, in that the conversion of adenine to guanine involves first the formation of IMP by one of several routes, followed by the formation of GMP along the normal biosynthetic pathway 6. Thus with this mutant one can test specifically for the influence of a guanine limitation, independent of the degree of adenine supplementation. We assume in these studies that the intracellular purine ribonucleotides are the effective repressors. The free bases themselves are not normally found in these cells nor do they occur on the known biosynthetic pathways as intermediates. The ribonucleotides, on the other hand, which are the endproducts of the purine pathway, do not enter the cells from the medium when provided, whereas the free bases are readily utilized and converted to the ribonucleotide level. It must be added that since it is known that a portion of the adenine provided to cells is deaminated directly and enters the cells as hypoxantine 28,29, in those cases where adenine is implicated in the control of an enzyme, the role of the hypoxanthine derivatives cannot be excluded.
Steps prior to the formation of I M P Inspection of Tables I and I I shows readily that the levels of the six enzymes are highly dependent on the level of purine in the growth medium. The levels are low in * T h e e x a c t l o c a t i o n of t h e m u t a t i o n is n o t k n o w n b u t i t is p r o b a b l e t h a t i t l a c k s e i t h e r t h e e n z y m e c a t a l y z i n g r e a c t i o n I I [ or V (Fig. I). I t is p r o b a b l e t h a t i t is b l o c k e d b e f o r e e n z y m e V1 (ref. 27), a n d i t d o e s p o s s e s s I, [ I a n d IV.
Biochim. Biophys Acta, 23 ° (1971) 349-361
FEEDBACK
REPRESSION
IN PURINE
BIOSYNTHESIS
355
the supplemented media (Extracts 1-6), intermediate where the medium is unsupplemented (Extract 7), and, in most instances, elevated where a purine limitation is imposed (Extracts 8-ii), Table I presents the data for 5'-P-ribosyl-I-P 2 amidotransferTABLE
I
EFFECT OF P U R I N E S U P P L E M E N T A T I O N
Extract
Additions* Guanine
ON L E V E L S OF P U R I N E BIOSYNTHETIC E N Z Y M E S
Strain
Enzyme specific activity (nmoles/min per mg protein)
Adenine
5'-P-ribosylz.-P2 amidotransferase
5'-P-ribosylglycinamide synthetase
5'-P-ribosyl forrnylglycinamide amidotransferase
1 2 3 4 5 6
X X X X o o
X o o o X X
PD-I lO33 P-I 4 PD-I lO33 PD-I
0.8 1.5 0.6 2. 5 1.5 2. 4
I.I 1.9 i.o 4-3 2.3 3.2
0. 5 I.i 0, 7 1.4 I .I 1. 4
7
o
o
lO33
7.5
7-5
2.9
8 9 IO I Ia I tb
L L o L L
X o L o o
P-I 4 P-I 4 PD-I PD-I PD-I
-5.2 24.0 29.9 28.2
I.I 8.0 rS.I 15. 3 16. 7
-4.7 16. 3 19. 4 20. I
L, limiting o, n o t a d d e d . TABLE
(6 / , g / m l
adenine
X,
excess
(4 ° / ~ g / m l a d e n i n e
and/or
guanine)
II
EFFECT OF P U R I N E S U P P L E M E N T A T I O N
Extract
or guanine);
Additions* Guanine
ON L E V E L S OF P U R I N E BIOSYNTHETIC E N Z Y M E S
Strain Adenine
Enzyme specific activity (nmoles/min per mg protein) Succino-AMP Succino-AMP 11~lP dehysynthetase lyase drogenase
I 2 3 4 5 6
X X X X o o
X o o o X X
PD-I lO33 P-I 4 PD-I lO33 PD-I
0. 3 I.I 1.2 i.o I.I 0.8
I.I 1. 9 2. 4 2.1 2. 3 2.2
1.6 0. 4 0. 4 0. 7 1. 5 2. 4
7
o
o
lO33
1.7
3 .8
1.6
S 9 IO I ta lib
L L o L L
X o L o o
P-I 4 P-I 4 PD-I PD-I PD-I
1.5 2.5 3.o 4.4 3.4
5.7 8,5 9.o 8.5 11.6
--79.2 34.o 6.0 6.2
* L, limiting o, n o t a d d e d .
(6 / * g / i n l a d e n i n e
or guanine);
X,
excess
(4 ° / , g / m l
adenine
Biochim. Biophys. Mcta,
and/or
guanine)
23o (1971) 349-361
356
D.P. NIERLICH, B. MAGASANIK
ase (I), 5'-P-ribosylglycinamide synthetase (II) and 5'-P-ribosyl formylglycinamide amidotransferase (IV), three enzymes which appear to be very similarly controlled. The results of the assays of 5'-P-ribosyl glycinamide synthetase are representative. The activity of this enzyme was found to be 7.5 units in the wild type strain Io33 where the nucleotide pools are nlaintained by endogenous synthesis (Extract 7). Addition to the medium of 4 ° keg/ml adenine and 4 ° ktg/ml guanine (i5-2o/~g/ml of either purine was sufficient in the basic medium to satisfy the purine requirement and repress enzyme synthesis) leads to a sevenfold repression of the enzyme (Extract I). On the other hand, addition of an excess of only one of the two products, adenine or guanine, leads to a lesser repression of the enzyme level (Extracts 2-6.) The fact t h a t supplementation by adenine and guanine leads to greater enzyme repression than by either one alone m a y be explained in two ways. First this m a y reflect a dual control over the synthesis of this enzyme. Second, it is possible that through the interconversion reactions, the dual supplement leads to a higher endogenous level of that single purine directly responsible for the control. Inspection of the data obtained by purine limitation (Extracts 8--11) indicates that an adenine derivative, and not one of guanine is most likely responsible for the regulation of this enzyme. Although a general purine limitation leads to a derepression of the enzyme (Extracts Io and I I), a specific guanine limitation alone has no effect on the enzyme level (compare extract 9 with Extract 7), while tile importance of the adenine supplement is revealed in the fact that excess adenine-limiting guanine results in full repression (compare Extracts 8 and I). The conclusion that a derivative of adenine is responsible for the control of 5'-P-ribosyl glycinamide synthetase is also applicable to the other two enzymes concerned with early steps in the pathway, 5'-P-ribosyl-I-P 2 amidotransferase and 5'-P-ribosyl formylglycinamide amidotransferase. This m a y be seen by inspection of the date in Table I, but even more easily in Fig. 2 where these data are shown graphically. Fig. 2a shows the relation of the activity of 5'-P-ribosyl-I-P 2 amidotransferase to that of 5'-P-ribosyl formylglycinamide amidotransferase; the points on the graph each represent the activities of the two enzymes in a different cell extract. It can be seen from the fact that a straight line can be drawn through the points in the figure that these two enzymes maintain the same ratio of activities under all growth conditions; that is, they are coordinate 3°. Fig. 2b shows a graph of 5'-P-ribosyl-I-P 2 amidotransferase against 5'-P-ribosyl glyeinamide synthetase, whose control was described in detail above. In this case a smooth curve fits the points very well. This behavior indicates that the two enzymes whose control appear coordinate, are regulated in qualitatively the same manner as 5'-P-ribosyl glycinamide synthetase but are more responsive to regulation than this latter enzyme. A similar situation has been observed in studies of the regulation of two pyrimidine biosynthetic enzymes ~1.
Adenylosuccinate synthetase (IA) and lyase ( V I I I and IIA) Table I I shows the activities of these two enzymes in the cell extracts. Here the response to purine excess and purine limitation is generally similar to that of 5'-Pribosyl glycinamide synthetase, but of a distinctly smaller amplitude. Here again combined adenine and guanine supplementation leads to the greatest enzyme repression (Extract I); supplementation with an excess of only one of the purines gives a
Biochim. Biophys. Acta, 23o (1971) 349-36~
FEEDBACK REPRESSION IN PURINE BIOSYNTHESIS
357
slight repression (Extracts 2-6); the wild type strain in unsupplemented medium possesses intermediate enzyme levels (Extract 7); and a general purine limitation, obtained by starving strain PD-I of either adenine or guanine, produces derepression (Extracts io,ii). To test further if guanine plays an independent role in the regulation I
I
I
20'
0 _go
c ~o
0
0
0
I0 20 5 L P - P i b o s y I - 1 - P 2 AMIDOTRANSFERASE
I
3
I
3O
t
20 0
c
"E
a.
P I I lO 20 5 L p - r [ b o sy1-1-P2 AMIDOTRANSFERASE
I 30
Fig. 2. R e l a t i v e specific activities of t h r e e p u r i n e b i o s y n t h e t i c e n z y m e s in cell-flee e x t r a c t s . D a t a , in n m o l e s / m i n per m g protein, are from T a b l e I. a. 5 ' - P - r i b o s y l f o r m y l g l y c i n a m i d e a m i d o t r a n s f e r a s e a n d 5 - ' P - r i b o s y l - I - P 2 a m i d o t r a n s f e r a s e , b. 5 -P-ribosyl g l y c i n a m i d e s y n t h e t a s e a n d 5 -P-ribosylI-P2 a m i d o t r a n s f e r a s e .
of these two enzymes, measurements were carried out on the guanine-requiring mutant P-I4, in which the specific effects of guanine limitation and adenine supplementation can be tested. For the second enzyme of adenine synthesis, adenylosuccinate lyase, a distinctly different result is obtained than that already described for the early enzymes. Here the condition of excess adenine-limiting guanine (Extract 8) leads to an intermediate enzyme level, near that of the wild type grown in unsupplemented medium (Extract 7). In the case where adenine is provided by endogenous synthesis, and guanine is limiting (Extract 9), a derepression is seen relative to that in which both adenine and guanine are provided by endogenous synthesis. It therefore seems that for enzyme repression both adenine and guanine must be present ; that is, a dual control appears to exist for adenylosuceinate lyase. It should be evident from the data inTable II that the adenylosuecinate syntheBiochirn. Biophys. Acta, 230 (1971) 349-361
35 ~
D . P . NIERLICH, B. MAGASAN1K
tase and the lyase, if not coordinate in their control, are at least quite similarly regulated. The question, however, is complicated because of the general difficulty experienced in measuring the first of these enzymes in crude extracts. The measurements are performed by following the production of adenylosuccinate, and the accumulation of this compound is influenced by the presence of the second enzyme. However, in spite of the problem of measurement, there appears to be at least one significant difference in the control of these enzymes. While the lyase appears to be subject to dual control, and both adenine and guanine limitations lead to a partial derepression, in the case of adenylosuccinate synthetase, the condition excess adenine-limiting guanine leads to repression (Extract 8). Therefore it appears that this enzyme is controlled by adenine.
I M P dehydrogenase The data for this enzyme, the first in the conversion of IMP to GMP, are also given in Table II. This enzyme is repressed in cultures of the wild type and the mutants by excess guanine but not by excess adenine (Extracts 2-6). Therefore, it seems reasonable to conclude that a guanine derivative is responsible for its control. This departure from the pattern of control observed with the other enzymes can be further seen in the very great derepression obtained, 2oo-fold, when a specific guanine limitation is imposed (Extract 9). DISCUSSION
We have shown here that while tile synthesis of tile enzymes of tile purine pathway is subject to regulation by the supply of endproducts, adenine and guanine, there is no single unitary control. In fact each segment of the pathway seems uniquely regulated to accommodate the special function of the enzymes within it. This conclusion becomes evident on listing the various responses we have observed here, with additional ca.ses others have reported: (a) The synthesis of the three early enzymes of the pathway studied (5'-P-ribosyl-I-P2 amidotransferase, 5'-P-ribosyl glycinamide synthetase and 5'-P-ribosyl formylglycinamide amidotransferase), are similar and possibly coordinate in control. These respond primarily to the supplementation of tile medium with adenine. (b) The synthesis of the enzyme adenylosuccinate lyase, serving both a step prior to the formation of IMP and a step in the conversion of IMP to AMP, appears equally sensitive to supplementation by both adenine and guanine. (c) The enzyme IMP cyclohydrolase (studied in the same series of mutants by LEVIN AN[) MAGASANIKS), forming part of an enzyme complex which is responsible for the two steps (IX and X) following that catalyzed by adenylosuccinate lyasO °, is responsive to guanine supplementation, and is not repressed by adenine during guanine starvation. (d) The synthesis of the first enzyme of GMP biosynthesis, IMP dehydrogenase, responds primarily to guanine supplementation. This same control is probable as well for the second enzyme of GMP biosynthesis, XMP aminase, since it and the first enzyme have been shown to be under coordinate control in Escherichia coli al,az (e) The first enzyme of AMP synthesis, adenylosuccinate synthetase, m a y be controlled by adenine alone, although our experiments do not exclude the possibility that its control is the same dual control as that of adenylosuccinate lyase. These controls are summarized in Fig. 3The dual control over the synthesis of adenylosuccinate lyase is similar to that tJiochim, t3iophys, dcla, 230 (~97 I) 349 36~
F E E D B A C K R E P R E S S I O N IN P U R I N E B I O S Y N T H E S I S
359
of several of the enzymes responsible for the synthesis of the branched chain amino acids in Salmonella 32. These enzymes contribute to the formation of valine, leucine, and isoleucine, and are only completely repressed when the three amino acids are present in excess. This so-called "multivalent" control is seen here in the fact that adenyloA
A and G ~ H l s h d i n e
G
Fig. 3. Control over the synthesis of the enzymes of the purine pathway. A and G are meant to indicate adenine and guanine and their ribonucleotide derivatives. See text. succinate lyase is only maximally repressed when both adenine and guanine are present in excess, and only fully derepressed when both are limiting. Since this enzyme carries out a reaction in the pathway common to both the formation of AMP and GMP, and a second reaction in the branch leading specifically to AMP, such a control would be clearly advantageous to the cell. The specificity and apparent utility of these controls is further seen in the control of IMP cyclohydrolase, which, as mentioned above, carries out a reaction in IMP formation following that of adenylosuccinate ]yase, but unlike tile latter enzyme is apparently controlled by guanine. This enzyme, in addition to its role in de novo IMP formation, can be seen as catalyzing a step in tile conversion of AMP to GMP by way of the histidine pathway (Fig. I). A control somewhat similar to that of adenylosuccinate lyase was observed with the three early enzymes studied. Here an excess of the two purines led to maximal repression, which might be taken to indicate a dual control. However, it was not possible to demonstrate independently an influence of guanine on the synthesis; i.e. a guanine limitation, when adenine was present in excess, led to repression. Although this does not rule out the possibility of a dual control of some direct type, it does suggest a simpler explanation. That is that a guanine supplement increases the repression given by adenine, by sparing adenine utilization. For this reason we envision adenine derivatives as playing the principal role in the regulation of these enzymes. The enzyme IMP dehydrogenase seems regulated by the pool of guanine derivatives. The enzyme is repressed by excess guanine, but not by adenine. However, there are some anomalous features of its regulation that need explanation. First, whereas generally speaking, the greatest repression of the other purine enzymes is observed with combined adenine and guanine supplementation, this was not the case with IMP dehydrogenase. Here, on the contrary, the addition of adenine to guaninecontaining media actually raised the enzyme levels (Table II). Secondly, when the general purine auxotroph (strain PD-I) was grown on limiting amounts of purine, the extent of derepression observed was highly dependent on whether that purine was adenine or guanine; a much higher enzyme level was in fact observed when adenine was the limiting substance. There are a number of possible explanations. One would be that adenine interferes with the utilization of guanine, such that the intracellular Biochirn. Biophys. Acta, 230 (1971) 34q-361
360
D.P.
N I E R L I C H , B. MAGASANIK
pools of GMP are lower with adenine present than without it. This possibility derives from the observation that adenine is readily deaminated to hypoxanthine by bacterial cultures and that hypoxanthine and guanine compete for a common pyrophosphorylase in their conversion to nucleoside monopbosphates~8, 29. Such a competition has been used to explain an increase in the levels of IMP dehydrogenase in cultures of Salmonella, brought about by hypoxanthine, when the enzymes had been repressed by 8-azaguanine, a guanine analog aa. A more interesting possibility, however, from the standpoint of our knowledge of the operation of these controls is that tile increase in IMP dehydrogenase may be attributed to induction by IMP. This compound is the precursor to the GMP branch of tile purine pathway, and would be formed in the conversion of adenine to guanine ribonucleotides, known to take place. Although enzyme induction by a precursor in biosynthetic pathways has not been generally observed, Gorini has reported some effect on enzyme levels by glutamic acid, tile precursor to the arginine pathway, in studies of the regulation of ornithine transcarbamylase, an arginine biosynthetic enzyme a4. It must be added also that the induction of the guanine biosynthetic enzymes has been observed in cultures of Escherichia coli treated with 5-amino-4-imidazolecarboxamide ribonucleoside n. The effect of the latter compound was, however, considered to be indirect, since this compound seemed to interfere with guanine utilization. ACKNOWLEDGEMENT
This work was supported in part by a grant, GM-o7446, from the National Institutes of Health, U.S. Public Health Service. REFERENCES I D. P. NIERLICH AND B. MAGASANIK, Federation Proc., 22 (1963) 476. 2 H. E. UMBARGER, in D. M. BONNER, Control Mechanisms in Cellular Processes, The Ronald Press, N e w York, 1961, p. 67. 3 H. J. VOGEL, in D. M. BONNER, Control Mechanisms in Cellular Processes, The Ronald Press, N e w Y o r k , 196I, p. 23. 4 D. P. NIERLICH AND B. MAGANANIK, J. Biol. Chem., 240 (1965) 358. 5 B. ~IAGASANIK AND D. [~ARIBIAN, J. Biol. Chem., 235 (196o) 2672. 6 B. ~'~IAGASANIK, in I. E. GUNSALUS AND R. Y. STANIER, The Bacteria, Vol. 3, Academic Press, N e w Y o r k , I962, p. 295. 7 R. L. BLAKLE¥ AND E . VITOLS, Ann. Rev. Biochem., 37 (1968) 2Ol. 8 A. P. LEVIN AND B. MAGASANIK, J. Biol. Chem., 236 (1961) 184. 9 A. P. LEVlN AND B. MAGASANIK, ./r. Biol. Chem., 236 (1961) 181o. 1o J. S. GUTS, F. R. DALAL AND S. R. SHUMAS, J. Bacteriol., 99 (1969) 441I I H . K. KURAMITSU, S. UDAKA AND H. S. -'X~OYED, J. Biol. Chem., 239 (1964) 3425 . 12 H. J. J. NIJCAMP AND P. G. D E HAAN, Biochim. Biophys. Acta, 145 (1967) 31. 13 D. USHIBA AND B. MAGASANIK, Proc. Soc. E:@tl. Biol. 3led., 80 (1952) 626. 14 S. C. HARTMAN AND J. M. BUCHANAN, J. Biol. Chem., 233 (1958) 451. 15 D. P. NIERLICH AND B. MAGASANIK, J . Biol. Chem., 240 (1965) 366. 16 L. WARREN AND J. M. BUCHANAN, J. Biol. Chem., 229 (1957) 613. 17 T. C. FRENCH, I. B. DAWID, R. A. DAY AND J. M. BUCHANAN, J. Biol. Chem., 238 (1963) 2171. 18 B. LEVENBERG AND J. M. BUCHANAN, J. Biol. Chem., 224 (1957) lOl 9. 19 1{. \xv'. MILLER, L. N. LUKENS AND J. M. BUCHANAN, J. Am. Chem. Sue., 79 (1957) 15132o J. S. GuTs AND E. G. GOLLUB, Proc. Natl. Acad. Sei. U.S., 43 (1957) 826. 21 J. G. FLAKS, L. WARREN AND J. ~I. BUCHANAN, J . Biol. Chem., 228 (I957) 215. 22 1. LIEBERMAN, J. Biol. Chem., 223 (1956 ) 327 . 23 C. E. CARTER AYD L. H . COHEN, J. Am. Chem. Soc., 77 (1955) 49924 B. MAGASANIK, H. S. MOYED AND L. B. GEHRING, J. Biol. Chem., 226 (1957) 339. 25 H. S. MOYED AND B. MAGASANIK, J . Biol. Chem., 226 (1957) 351 .
Biochim. Biophys. Acta, 230 (1971) 3 4 9 - 3 6 i
FEEDBACK REPRESSION IN PURINE BIOSYNTHESIS
361
26 O. H LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. S. RANDALL, J . Biol. Chem., 193 (1951) 265 . 27 M. S. BROOKE AND ]3. MAGASANIK, J. Bacteriol., 68 (1954) 727 . 28 S. FRIEDMAN AND J. S. GOTS, J. Biol. Chem., 2Ol (1953) 125. 29 g F. ZIMMERMAN AND ]3. MAGASANIK, J. Biol. Chem., 239 (1964) 2933 ° F. JACOB, D. PERRIN, C. SANCHEZ AND J. MONOD, Compl. Rend., 25 ° (196o) 1727. 31 J. R. BECKWlTH, A. B. PARDEE, R. AUSTRIAN AND F. JACOB, J. ,~lol. Biol., 5 (1962) 618. 32 M. FREUNDLICH, R. O. BURNS AND H. E. UMBARGER, Proc. Natl. Acad. Sci. U.S., 48 (1962) 18o4. 33 A. P. LEVlN, D o c t o r a l D i s s e r t a t i o n , H a r v a r d U n i v e r s i t y , 1959. 34 L. GORINI, Bacteriol. Rev., 27 (1963) 182.
Biochim. Biophys. Acta, 23 ° (1971) 349-361