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Metabolite-induced Enzymatic Inactivation of Glutamine Synthetase in E. Coli
METABOLITE-INDUCED INACTIVATION
ENZYMATIC
OF GLUTAMINE
SYNTHETASE
I N E. C O L I H. HOLZER, D. MECKE, K. WULFF, K. LIESS and L. HEILMEYER,JR. Biochemisches Institut der Universit~it Freiburg im Breisgau, Germany
INTRODUCTION
WHEN studying the effect of glucose on the synthesis of different enzymes in yeast, we found (1"2) a remarkable behavior of malate dehydrogenase. In contrast to other enzymes as isocitrate lyase, malate synthase, and alcohol dehydrogenase, where addition of glucose to the nutrient medium only inhibits the further synthesis of these enzymes by "repression", malate dehydrogenase was found to be inactivated very rapidly (Fig. 1). When the increase of the cell mass is taken into account, the product P = cell mass × specific activity is found to remain constant after addition of glucose in the cases of isocitrate lyase, malate synthase, and !~÷ Gtucose
NDH 04-PDH
JCL
A
ADHxl03
~ i : ~ "
MsF ]2
~/////~~, ........ .................... • I,latate Synthase(MS) .c 2,0 (1.
a
).1
.c_ E
.....
E
t1.1 E .'~' 0
Dehydrogenose 1
2
3
4
l
5
B hours FIG. 1 Kinetics of the effect of glucose on the specific activity of different enzymes in yeast cells.
211
212
H. HOLZER, D. MECKE, K. W U L F F , K. LIESS, L. HEILMEYER, JR.
alcohol dehydrogenase. For malate dehydrogenase, however, this product shows a strong decline. Consequently not only an inhibition of the synthesis of this enzyme takes place, but also a rapid inactivation. We looked, in yeast and in E. coli, for further enzymes where the addition of a metabolite causes not only repression of synthesis but also inactivation. In yeast, we found no other example for such an inactivation. But the rapid drop in specific activity of the mitochondrial respiratory enzymes in yeast upon the addition of glucose, observed by Utter, ~a) may be due to an inactivation by glucose. Out of several repressible enzymes studied in E. coli, ~4"~ only one enzyme was found to be inactivated: glutamine synthetase. This lecture will report on repression and inactivation of this enzyme. I. Repression of the Synthesis o f Glutamine Synthetase When yeast cells are cultivated with ammonium as N-source only low specific activity of glutamine synthetase is found3 6) This is also true for E. coli ~7) (Table 1). Since glutamate and ammonium together give rise TABLE 1 Specific Activity of Glutamine Synthetase from E. coli After 90-rain Incubation with Different Nitrogen Sources (Details see ~7)) N-Source in the medium
Glutamine synthetase (units per nag protein)
Glutamic acid + NH4 + NH4 + L-Glutamlc acid L-Glutamine L-Aspartic acid L-Asparagine L-Arginine L-Methionine L-Ornithine L-Proline DL-Threonine L-Valine Urea No N-source
7 7 91 85 70 48 78 89 75 100 35 130 43 27
to the same low specific activity as ammonium alone, this cannot be explained by induction of the enzyme synthesis by glutamate, but only by its repression by ammonium. Replacement of the ammonium in an ammonium-grown culture by another N-source, e.g. glutamate, results in an increase of specific activity, as shown in Fig. 2, due to "derepression". Chloramphenicol instantly stops the further rise of specific activity. This demonstrates the existence of a de novo synthesis. In accordance
INACTIVATION OF GLUTAMINE SYNTHETASE IN E. COLI
213
with that, p-fluorophenylalanine, 5-methyltryptophan, and p u r o m y c i n inhibit this "derepression" too. (¢)
e
,80"
~
60"
:~ 40" u
~ZO== '10
20
30
40 rnin
50
60
=
FIG. 2 Derepression of glutamine synthetase in E. coli. (For details cf.(7).)
II. Inactivation and Reactivation of Giutamine Synthetase When ammonium salts are added to derepressed cells, a rapid drop of the specific activity of glutamine synthetase is observed. As shown in Fig. 3, the activity goes down within 1-2 min to about 15-20% of the initial value. This cannot be explained by repression, because a"dilution" of the original enzyme quantity by the growth of the cell mass would
1
>,100
~ 5o o
o
o
37°
o
lOO
zOO
--
.%---o
360 ;" 6oo seconds
FiG. 3 Inactivation of glutamine synthetase in E. coli by 2 × 10 -~ M N H 4 + at 37 °. (For details cf3r).)
halve the specific activity only in the course of about 50 min, the generation time under our growth conditions. A rapid turnover of the enzyme cannot explain the phenomenon either, because there is no drop in specific activity after protein synthesis has been stopped by chloramphenicol
214 H. HOLZER, D. MECKE, K. WULFF, K. LIESS, L. HEILMEYER, JR. (Fig. 2). T h e only remaining possibility to explain this effect is an inactivation of the e n z y m e by the addition of ammonium to the medium. T h e reaction is very specific and very sensitive. (7) A concentration of NH4 + as low as 10-rM lowers the specific activity within 5 min by about 30%. Li ÷, K ÷, Rb ÷ and Cs ÷ show no effect. T h e anion of the ammonium salt added has no influence. Substituted NH3, e.g. hydrazine, hydroxylamine, trimethylamine, have no effect either. Several other enzymes which we tested are not influenced during the inactivation o f glutamine synthetase b y ammonium. (8) In addition to the synthesis of glutamine, expressed by: glutamate + NH3 + A T P ~ glutamine + A D P + Pi, the e n z y m e also catalyzes a transfer of the y-glutamyl residue o f glutamine to hydroxylamine: glutamine + H 2 N O H ~ y - g l u t a m y l - N H O H + NH3. Table 2 shows the glutamine synthetase activity of the e n z y m e to be TABLE 2 Glutamine Synthetase and Glutamine Transferase Before and After Inactivation with NH4+ (For details cUr)) Specificactivity (units per nagprotein) Treatment Glutamate culture Glutamate culture, then short time inactivation by NH4+
Transferase fr) 535
Synthetase (s) 180
282
6.4
T/S 3.0
44
preferentially cut down by the inactivation by ammonium, whereas the glutamyl transferase activity is affected to a much lesser extent. W e suppose that the ammonium inactivation causes a change in the catalytic site o f the e n z y m e which is needed only t~or the synthetase reaction, and not for the transferase reaction. I f we suppose the reaction mechanism to follow the scheme developed by Meister <9) (Fig. 4), we can assume that a special part o f the catalytic site is influenced which participates in the A T P - d e p e n d e n t conversion of glutamate into T-glutamylphosphate. Since the inactivation merely consists in a minute structural change in the catalytic site of the enzyme, we checked whether a reactivation of the e n z y m e can be detected which is independent of a d e n o v o synthesis of
INACTIVATION OF GLUTAMINE SYNTHETASE IN E. COLI
215
the enzyme. The experiment given in Table 3 demonstrates a reactivation of this kind. When cells inactivated with ammonium sulfate over a short period are incubated in the presence of chloramphenicol, a regeneration of the enzymatic activity is observed. Thus a reactivation of the enzyme previously inactivated by NH4 ÷ is possible without protein synthesis. Gtu
AT P ~
P; ~
~ v'Gtu-P ~, ADP
~
~
GtuNH 2
NH3
NH20~ ~P
Synthetase Reaction 1
l
Transferase Reaction
~t'GtuNHOH
FIG. 4 Scheme of synthetase and transferase reactions. (y-Glu-P = y-glutamylphosphate.)
p-Fluorophenylalanine does not inhibit the. reactivation either (Table 4). Another inhibitor of protein synthesis, 5-methyltryptophan, does affect the reactivation. We found, however, (1°) that in intact cells 5-methyltryptophan as well as ammonium induces an inactivation of the enzyme. Therefore 5-methyltryptophan probably inhibits the reactivation not by TABLE 3 Change in Specific Activity of Glutamine Synthetase in E. coil After Resuspension of Repressed and Inactivated Cells in Glutamate-containing Medium With and Without Addition of Chloramphenicol (For details cf. (7~) Specific activity (units per mg protein)
Treatment
At the beginning of the experiment
60 min in NH4+-free glutamate medium without chloramphenicol
with chloramphenicol
Culture in glutamate medium, then short time inactivation by NH4 +
6.4
210
180
Culture in NH4 + medium
5.0
117
10
inhibition of protein synthesis but by an inactivation which is superimposed on the reactivation. 2,4-Dinitrophenol prevents the reactivation. Thus the reactivation process can be supposed to be independent of protein synthesis, but dependent on ATP.
216
H. H O L Z E R , D. M E C K E , K. W U L F F ,
K. LIESS, L. H E I L M E Y E R , JR.
TABLE 4 Effect o f Different T o x i c S u b s t a n c e s o n the Reactivation of G l u t a m i n e S y n t h e t a s e ~°~ Specific activity (units per nag protein) Inhibitor (added during reactivation)
Before inactivation (glutamate grown cells)
A f t e r 10 min inactivation (by 2 × l0 -4 M N H4 + )
After a n o t h e r 10 min reactivation (--NH4 + + 10 -z M glutamate)
113
27
159
113
27
135
5.4 x 10 -4 M F - P h e * 9.2 x 10 -4 M CH3-Try*
72 72 72
10 10 10
-10 -2 M 2 , 4 - D N P *
60 60
5 5
-
6.2 × 10 -4 M chloramphenicol --
1 8 8
133 3.7 274 15
* F - P h e = p-fluorophenylalanine, C H 3 - T r y = 5-methyltryptophan, D N P = dinitrophenol.
The experiments reported so far on inactivation and reactivation can be summarized in the following scheme: glutamine synthetase a ~
(NH4 +) (ATP)
~ glutamine s y n t h e t a s e b.
The denominations a and b for active and inactive enzyme respectively have been taken from the glycogen phosphorylase nomenclature. As the experiments to be described in section III will show, probably not ammonium itself, but some product in the cell derived from ammonium, is the metabolite inducing the inactivation. Out of the inactivation reaction the question arises whether the "repression" of glutamine synthetase described in section I is not merely simulated by inactivation of the enzyme. The experiment given in Table 3, however, proves that besides the inactivation a repression of the synthesis does exist. Cells with low specific activity of the enzyme have been prepared in two ways: (1) by growth with ammonium in the medium; (2) by short-time inactivation of derepressed cells by ammonium. Upon incubation of such cells in a growth medium containing chloramphenicol, the rapidly inactivated cells exhibit a revival of the high specific activity, while the cells grown with ammonium show no reactivation. Thus growth on a m m o n i u m - i n accordance with the existence of " r e p r e s s i o n " prevents the synthesis of the enzyme protein with the result that neither glutamine synthetase a nor b is present. If only inactivation existed, one
INACTIVATION OF G L U T A M I N E SYNTHETASE IN E. COLI
217
would expect even after growth with ammonium the presence of glutamine synthetase b which can be reactivated in the presence of chloramphenicol. It should, however, be kept in mind that "repression" is not the only possible explanation of the experimental results. They are also consistent with a slow degradation of glutamine synthetase b to peptides and/or amino acids during growth with ammonium. At the moment we cannot distinguish between this mechanism and" repression". III. Inactivation of Glutamine Synthetase in a Cell-free System ( c f s) a n d Ol))
To demonstrate an inactivation of glutamine synthetase in a cell-free extract, we first added ammonium sulfate to a crude extract obtained by grinding E. coil cells with aluminium oxide powder. The enzyme is completely stable under these conditions; no trace of inactivation by the added ammonium can be observed. Upon the addition of A T P and Mg z+, however, rapid inactivation occurs even in the absence of ammonium. As shown by Table 5, both A T P and Mg ~+ are necessary for this effect. TABLE 5 Inactivation of Glutamine Synthetase in the Crude Extract from E. coli Specific activity after 30 min at 37 ° (units per mg protein) Crude Crude Crude Crude
extract extract + A T P extract + Mg 2+ extract + A T P + Mg 2+
66.5 61.0 75.0 3.6
TABLE 6 Glutamine Synthetase and other Enzymes in Crude Extract from E. coli (The numbers represent units per mg protein; for further details cf.ts).)
cO t-.
o _
Control 45 min incubated at 37 ° without addition 45 rain incubated at 37 ° with A T P + Mg ~+
40.5
35.2
6.3
770
77
269
43
39.6
7.2
815
86.5
295
40.2
6.6
750
5.8
100
262
218
H. HOLZER, D. MECKE, K. W U L F F , K. LIESS, L. HEILMEYER, JR.
Ammonium salts have no influence. Table 6 shows that among several enzymes tested only glutamine synthetase is inactivated. The enzyme specificity of this inactivation effect is therefore identical with the specificity we observed after the addition of ammonium sulfate to intact cells. If instead of a crude extract, glutamine synthetase, which had been partially purified by ammonium sulfate fractionation and dialysis, is used, ATP and Mg z+ do not cause inactivation any more. Therefore during purification a factor has been removed which is necessary for the inactivation. This factor, present in the crude extract from E. coli, is stable against heating (Table 7). It is lost during dialysis. As well as
TABLE 7 Influence of Different Additions on the Inactivation of Partially Purified Glutamine Synthetase (% Inactivation in 15 rain at 37°; for further details cf3 s)) Control (purified synthetase + A T P + Mg 2+) + crude extract from E. coli + boiled crude extract from E. coli + dialysed, boiled crude extract fromE, coli + boiled crude yeast extract
<5% 60% 50% <5% 55%
in E. coli, this factor necessary for the inactivation is contained in yeast. So it must be a substance of low molecular weight which is heat-stable and not specific for E. coli: probably a metabolite. We did not undertake the effort of purifying this factor from boiled extracts of E. coli or yeast, but we tested several intermediates of N-metabolism whom we expected TABLE 8 Inactivation of Glutamine Synthetase by Different Amino Acids in Percent of the Values with Glutamine (The incubation mixture contained partially purified glutamine synthetase + ATP + Mga+; amino acid concentration 10 -3 M; incubation time 10 rain.) Glutamine Glutamate NH4 + Tryptophan Methionine
100 80 < 5 50-60 40-50
Asparagine Threonine Serine Other amino acids* Without addition
10 10 10 < 5 < 5
*Alanine, aspartic acid, arginine, D-glutamic acid + ammonium, L-a-methylglutamic acid + ammonium, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, tyrosine, valine.
INACTIVATION OF GLUTAMINE SYNTHETASE IN E, COLI
2]9
to influence the glutamine synthetase reaction in a biologically reasonable way. As shown b y Table 8, glutamine is particularly efficient; the effect o f glutamic acid is perhaps due to its enzymatic conversion to glutamine. T h e results obtained so far are summarized in the following equation (glutamine was taken as metabolite inducing the inactivation, because this compound exhibited the strongest effect o f all the substances we have investigated as yet): glutamine synthetase a
glutamine, ATP, Mg2+ ~ (ATP .... ?) ~
glutamine synthetase b
F o r further experiments glutamine synthetase was purified by the procedure of Woolfolk and Stadtman: (12) precipitation with streptomycin, fractionation with acid and heat treatment. T h e purified e n z y m e behaves uniformly in the ultracentrifuge, agar gel electrophoresis, and chromatography on DEAE-cellulose. In contrast to the only partially purified enzyme, the pure e n z y m e is not inactivated by incubation with glutamine, A T P and Mg 2+. During the purification
N
300
I 0"--
~200
E 0
10o. C
10
20 time (minutes)
30
FIG. 5 Glutamine induced enzymatic inactivation of glutamine synthetase from E. coli.
x @ ©
(cf.01).) x -- glutamine synthetase + glutamine + ATP + Mg2+ • = same plus "acid fraction", © = same plus boiled "acid fraction".
220
H. HOLZER, D. MECKE, K. W U L F F , K. LIESS, L. HEILMEYER, JR.
another factor has therefore been lost which is necessary for the inactivation. A protein fraction removed by the acid fractionation was shown to contain the missing factor. From Fig. 5 one can see that there is no inactivation in the presence of glutamine, ATP and Mg2+ alone, but it takes place upon addition of the "acid fraction". With this test system the factor necessary for the inactivation could be purified. As yet we have reached about a 10-fold purification3 TM The behavior of this factor during purification, heat treatment and dialysis shows that it is a protein, for which we use the name "inactivating enzyme". The purified "inactivase" is free of glutamine synthetase. The following scheme summarizes our present knowledge on the inactivation and reactivation of glutamine synthetase: inactivating enzyme, glutamine, glutamine synthetase a
ATP, Mg~÷ ~
(ATP.... ?)
glutamine ' synthetase b.
DISCUSSION
We cannot give a definite answer yet to the question whether the inactivation observed in intact cells upon the addition of ammonium salts is identical with the effect observed in cell-free extracts upon the addition of glutamine. But there are two observations which support the identity of both reactions. (a) The inactivation in the cell-free extract as well as in intact cells mainly affects the glutamine synthetase activity of the enzyme, and much less the transferase activity38~ (b) Out of five different enzymes only glutamine synthetase was found to be inactivated in the cell-free extract (upon addition of glutamine) as well as in intact cells (upon addition of NH4÷). If one accepts the identity of the inactivation processes in intact cells and in the cell-free extract, the question arises: "Why in intact cells only ammonium inactivates and not glutamine, but in the cell-free extract only glutamine and not ammonium?" A possible explanation would be that glutamine, in contrast to ammonium, does not easily enter the cells. The experiment, the results of which appear in Table 9, supports this possibility. Low concentrations of glutamine cause no change of activity because of the permeability barrier. High concentrations of glutamine break through the permeability barrier and produce the same inactivation as low concentrations of ammonium.
I N A C T I V A T I O N O F G L U T A M I N E S Y N T H E T A S E I N E. COLI
221
' TABLE 9 Inactivation o f Glutamine Synthetase in E. coli after Addition of Glutamine or NH4 + to the G r o w t h Medium Specific activity 10 min after addition of glutamine or NH4 + (units per mg protein) Without addition 10 -4 M NH4 + 10 -4 M glutamine 10 -3 M glutamine 10 -2 M glutamine
104 2.3 47 8.5 4.7
Only a minute structural change of glutamine synthetase during the enzymatic inactivation is indicated by the following results: (a) There is little change of transferase activity under conditions where the synthetase activity is almost completely inactivated. (b) In vivo inactivated glutamine synthetase (which can be tested by the transferase reaction) behaves exactly like active glutamine synthetase during the purification by streptomycin precipitation, acid fractionation, heat denaturation, and chromatography on DEAE-cellulose34) (c) In the analytical ultracentrifuge active glutamine synthetase shows the same sedimentation rate as the inactivated enzyme obtained by ammonium sulfate treatment of intact cells, t4) (d) In agar gel electrophoresis both forms of the enzyme behave in the same way. (4) We could not find any support for a fixation of I4C from uniformly 14Clabeled glutamine into the protein of glutamine synthetase. Experiments on the incorporation of 15N-amide nitrogen of glutamine and of the Tphosphorus of T_32P-ATP are in preparation. In Salmonella typhimurium upon addition of ammonium sulfate to cells grown on glutamate as N-source an inactivation of glutamine synthetase is observed as in E. coli. In Lactobacillus arabinosus and in Saccharomyces cerevisiae we have not found an inactivation ofglutamine synthetase. The feedback inhibition of glutamine synthetase by glutamine described for Lactobacillus arabinosus 03) could be reproduced by us. This inhibition is reversed by dialysis. On the contrary, the inactivation of glutamine synthetase from E. coli described here is not reversible by dialysis. Thus the effect in Lactobacillus arabinosus must be a real feedback inhibition, not an inactivation. Figure 6 shows schematically the position of glutamine synthetase in metabolism. The glutamine synthesized from glutamate and NH3 is needed for the synthesis of proteins and for the introduction of nitrogen
222
H. HOLZER, D. MECKE, K. WULFF, K. LIESS, L. HEILMEYER, JR. R • pr ess i o n
(?)
Protein Synthesis
Nucteotldes,Histidine I NH3
.~ ~
~ GLutom~
I= Tryptophsugars, NAo.Amln°-[ Corbarny[phosphat .,j GLutornyl Transfer (?)
Cumutotive
Feedback
FIG. 6 Regulation of glutamine synthetase in E. coil
into different metabolites. Probably also the glutamyl residue can be transferred from glutamine to a biological acceptor by the aid of glutamine synthetase. This assumption is favored by the well-known transfer of the glutamyl residue to the unphysiological acceptor hydroxylamine Cglutamyl transferase reaction"). A physiological acceptor, however, has not been found so far. Accumulation of metabolites synthesized with the aid of the amide group of glutamine reversibly inhibits the activity of glutamine synthetase by "cumulative feedback" as discovered by Woolfolk and Stadtman. t12~ The enzymatic inactivation of glutamine synthetase by glutamine described in this paper is a second mechanism preventing an excessive supply of glutamine. This additional mechanism may be necessary, because glutamine is a starting material for the synthesis not only of metabolites of low molecular weight, but also for protein synthesis and perhaps the synthesis of other polymers by glutamyl transfer reactions. Polymers fixed in cellular structures are unable to regulate their own synthesis by feedback which involves dissociable binding to the active or allosteric site of an enzyme. Therefore the precursor of the polymer, namely glutamine, must take over the regulatiorL Another possibility of feedback control by glutamine is realized in Lactobacillus arabinosus (cf.(la)). Here the accumulating glutamine causes no irreversible enzymatic inactivation of the synthetase, but a reversible feedback inhibition. Besides the rapidly operating "fine control" of glutamine synthetase by feedback and enzymatic inactivation, a "coarse control" (for the definition of "fine" and "coarse control" see ~4) and {~5)) is achieved by repression of the synthesis of the enzyme. This is true for E. coli (7"~2} and for yeast, (6) where a compound originating from ammonium, possibly glutamine, is the corepressor. In H E L A cells {16~addition of glutamine to the growth medium leads to a low specific activity of glutamine synthetase. Here too a repression of enzyme synthesis by glutamine is probable.
I N A C T I V A T I O N O F G L U T A M I N E S Y N T H E T A S E I N E. COLI
223
"Fine control" by feedback and enzymatic inactivation affects the activity of the enzyme within a few minutes or even seconds. In the contrary, "coarse control" by repression is only efficient with the rate of dilution of the existing enzyme quantity by growth of the cell mass. As compared to feedback, regulation of enzyme activity by "metabolite-dependent enzymatic inactivation" is an "expensive" mechanism because of the necessity of an additional enzyme. For reasons of economy one can understand that this kind of regulatory mechanism is realized only in the case of a few key-enzymes. We found the following examples in the literature: (1) (2) (3) (4)
mammalian glycogen phosphorylase ;~1r.18) 6-phosphofructokinase ~19)in yeast; mammalian glycogen synthetase (for a summary cf.~°)); glutamine synthetase in E. coli.
Glycogen phosphorylase, glycogen synthetase and 6-phosphofructokinase are key-enzymes of carbohydrate metabolism, glutamine synthetase is a key-enzyme of nitrogen metabolism. SUMMARY
1. Addition of ammonium salts to E. coli cells grown on a medium containing no ammonium salts produces a 80-90% inactivation of glutamine synthetase within 1 to 2 min. The enzyme's ability to transfer the T-glutamyl residue of glutamine to hydroxylamine (glutamyl transferase) is only slightly affected by this inactivation. 2. Addition of ammonium salts to the medium causes not only inactivation of glutamine synthetase, but also repression of the synthesis of the enzyme. 3. Glutamine synthetase inactivated by the addition of ammonium ion to intact cells is reactivated after the ammonium ion is washed out. The reactivation does not involve de novo protein synthesis, but probably requires ATP. 4. An inactivation of glutamine synthetase activity with only a small effect on the glutamyl transferase activity is demonstrated also in a cellfree system. This inactivation requires ATP, Mg ~+, glutamine (or certain other amino acids) and a specific protein ("inactivating enzyme"). The inactivating enzyme has been purified about 10-fold and separated from glutamine synthetase. 5. Preliminary results support the view that the inactivation produced by ammonium ion in intact cells is identical with the enzymatic inactivation induced by glutamine in the cell-free extract.
224
H. HOLZER, D. MECKE, K. WULFF, K. LIESS, L. HEILMEYER, JR.
6. A p o s s i b l e biological f u n c t i o n o f the " m e t a b o l i t e - i n d u c e d e n z y m a t i c i n a c t i v a t i o n o f g l u t a m i n e s y n t h e t a s e " is d i s c u s s e d . E n z y m e r e g u l a t i o n t h r o u g h e n z y m a t i c i n a c t i v a t i o n a n d r e a c t i v a t i o n s e e m s to b e r e s t r i c t e d to a f e w key e n z y m e s o f m e t a b o l i s m .
ACKNOWLEDGMENTS S u p p o r t e d b y the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t a n d t h e B u n d e s m i n i s t e r i u m f'tir w i s s e n s c h a f t l i c h e F o r s c h u n g . REFERENCES 1. I. WITT,R. KRONAUand H. HOLZER,Repression von Alkoholdehydrogenase, Malatdehydrogenase, Isocitratlyase und Malatsynthase in Hefe durch Glucose, Biochim. Biophys. ,4cta 118, 522-537 (1966). 2. I. WITT, R. KRONAUand H. HOLZER, Isoenzyme der Malatdehydrogenase und ihre Regulation in Saccharomyces cerevisiae, Biochim. Biophys. ,4cta, 128, 63-73 (1966). 3. M. F. UTTER,E. A. BUELL a~d C. BERNOFSKY,Alterations in the respiratory enzymes of the mitochondria of growing and resting yeast in Aspects o f Yeast Metabolism, (A. K. MILLS, ed.; SIR HANSKREBS,Consulting Ed.), Blackwell Scientific Publications, Oxford (1966). 4. K. LIESS, Dissertation in preparation for the Naturwissenschaftlich-mathematische Fakult~t der Universit~it Freiburg im Breisgau. 5. CH. GARTNER,unpublished results. 6. G. KOHLHAW,W. DRAGERTand H. HOLZER, Parallel-Repression der Synthese yon Glutaminsynthetase und DPN-abh~ingiger Glutamatdehydrogenase in Hefe, Biochem. Z. 341,224-238 (1965). 7. D. MECKE and H. HOLZER, Repression und Inaktivierung yon Glutaminsynthetase in E. coil durch NH4+, Biochim. Biophys. ,4cta 122, 341-351 (1966). 8. n . MECKE, K. WULFF and H. HOLZER, Metabolit-induzierte Inaktivierung yon Glutaminsynthetase aus E. coil im zellfreien System, Biochim. Biophys. Acta, 128, 559-567 (1966). 9. A. MEISTER, Giutamine synthesis, pp. 443-468 in The Enzymes 6, 2nd Ed. (P. D. BOYER,H. LARDYand K. MYRBXCK,eds.) Academic Press, New York (1962). 10. L. HEILMEYER,JR, D. MECKEand H. HOLZER,unpublished results. 11. D. MECKE, K. WULFF, K. LIESS and H. HOLZER, Characterization of a glutamine synthetase inactivating enzyme from E. coli, Biochem. Biophys. Res. Communs. 24, 452-458 (1966). 12. C. A. WOOLFOLKand E. R. STAOTMAN,Cumulative feedback inhibitionin the multiple end product regulation of glutamine synthetase activity in E. coil, Biochem. Biophys. R es. Communs. 17, 313-319 (1964). 13. J. M. RAVEL, J. S. HUMPHREYS and W. SHIVE, Control of glutamine synthesis in Lactobacillus arabinosus,,4rchives Biochem. Biophys. 111,720-726 (1965). 14. J. M. ASHWORTand H. L. KORNBERG,Fine control of the glyoxylate cycle by allosteric inhibitionof isocitrate lyase, Biochim. Biophys..4 cta 73, 517-519 (1963). 15. H. A. KREeS, Meeting of the Nobel-Laureates at Lindan (Germany), July 1963. 16. R. DE MARS,The inhi'bition by glutamine of glutamyl transferase formation in cultures of human cells, Biochim. Biophys. ,4 cta 27, 435-436 (1958). 17. G. T. COR1and A. A. GREEN, Crystalline muscle phosphorylase, II. Prosthetic group, J. Biol. Chem. 151, 31-38 (1943). 18. E. G. KREBSand E. H. FISCHER,Molecular properties and transformations of glycogen phosphorylase in animal tissues,,4 dvances in Enzymology 24, 263-290 (1962).
INACTIVATION OF GLUTAMINE SYNTHETASE IN E. COLI
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19. E. VINUELA, M. L. SALAS,M. SALASarid A. SOLS,Two interconvertible forms of yeast phosphofructokinase with different sensitivity to endproduct inhibition, Biochem. Biophys. Res. Communs. 15, 243-249 (1964). 20. S. H. GOLDEMBERG, Reactions of glycogen synthesis and glycolysis, pp. 292-317 in D-Glucose und verwandte Verbindungen in Medizin und Biologie (H. BARTELHEIMER, W. HEVDE and W. THORN, eds.), Veflag F. Enke, Stuttgart (1966).
ENZYMATIC
OF GLUTAMINE
SYNTHETASE
I N E. C O L I H. HOLZER, D. MECKE, K. WULFF, K. LIESS and L. HEILMEYER,JR. Biochemisches Institut der Universit~it Freiburg im Breisgau, Germany
INTRODUCTION
WHEN studying the effect of glucose on the synthesis of different enzymes in yeast, we found (1"2) a remarkable behavior of malate dehydrogenase. In contrast to other enzymes as isocitrate lyase, malate synthase, and alcohol dehydrogenase, where addition of glucose to the nutrient medium only inhibits the further synthesis of these enzymes by "repression", malate dehydrogenase was found to be inactivated very rapidly (Fig. 1). When the increase of the cell mass is taken into account, the product P = cell mass × specific activity is found to remain constant after addition of glucose in the cases of isocitrate lyase, malate synthase, and !~÷ Gtucose
NDH 04-PDH
JCL
A
ADHxl03
~ i : ~ "
MsF ]2
~/////~~, ........ .................... • I,latate Synthase(MS) .c 2,0 (1.
a
).1
.c_ E
.....
E
t1.1 E .'~' 0
Dehydrogenose 1
2
3
4
l
5
B hours FIG. 1 Kinetics of the effect of glucose on the specific activity of different enzymes in yeast cells.
211
212
H. HOLZER, D. MECKE, K. W U L F F , K. LIESS, L. HEILMEYER, JR.
alcohol dehydrogenase. For malate dehydrogenase, however, this product shows a strong decline. Consequently not only an inhibition of the synthesis of this enzyme takes place, but also a rapid inactivation. We looked, in yeast and in E. coli, for further enzymes where the addition of a metabolite causes not only repression of synthesis but also inactivation. In yeast, we found no other example for such an inactivation. But the rapid drop in specific activity of the mitochondrial respiratory enzymes in yeast upon the addition of glucose, observed by Utter, ~a) may be due to an inactivation by glucose. Out of several repressible enzymes studied in E. coli, ~4"~ only one enzyme was found to be inactivated: glutamine synthetase. This lecture will report on repression and inactivation of this enzyme. I. Repression of the Synthesis o f Glutamine Synthetase When yeast cells are cultivated with ammonium as N-source only low specific activity of glutamine synthetase is found3 6) This is also true for E. coli ~7) (Table 1). Since glutamate and ammonium together give rise TABLE 1 Specific Activity of Glutamine Synthetase from E. coli After 90-rain Incubation with Different Nitrogen Sources (Details see ~7)) N-Source in the medium
Glutamine synthetase (units per nag protein)
Glutamic acid + NH4 + NH4 + L-Glutamlc acid L-Glutamine L-Aspartic acid L-Asparagine L-Arginine L-Methionine L-Ornithine L-Proline DL-Threonine L-Valine Urea No N-source
7 7 91 85 70 48 78 89 75 100 35 130 43 27
to the same low specific activity as ammonium alone, this cannot be explained by induction of the enzyme synthesis by glutamate, but only by its repression by ammonium. Replacement of the ammonium in an ammonium-grown culture by another N-source, e.g. glutamate, results in an increase of specific activity, as shown in Fig. 2, due to "derepression". Chloramphenicol instantly stops the further rise of specific activity. This demonstrates the existence of a de novo synthesis. In accordance
INACTIVATION OF GLUTAMINE SYNTHETASE IN E. COLI
213
with that, p-fluorophenylalanine, 5-methyltryptophan, and p u r o m y c i n inhibit this "derepression" too. (¢)
e
,80"
~
60"
:~ 40" u
~ZO== '10
20
30
40 rnin
50
60
=
FIG. 2 Derepression of glutamine synthetase in E. coli. (For details cf.(7).)
II. Inactivation and Reactivation of Giutamine Synthetase When ammonium salts are added to derepressed cells, a rapid drop of the specific activity of glutamine synthetase is observed. As shown in Fig. 3, the activity goes down within 1-2 min to about 15-20% of the initial value. This cannot be explained by repression, because a"dilution" of the original enzyme quantity by the growth of the cell mass would
1
>,100
~ 5o o
o
o
37°
o
lOO
zOO
--
.%---o
360 ;" 6oo seconds
FiG. 3 Inactivation of glutamine synthetase in E. coli by 2 × 10 -~ M N H 4 + at 37 °. (For details cf3r).)
halve the specific activity only in the course of about 50 min, the generation time under our growth conditions. A rapid turnover of the enzyme cannot explain the phenomenon either, because there is no drop in specific activity after protein synthesis has been stopped by chloramphenicol
214 H. HOLZER, D. MECKE, K. WULFF, K. LIESS, L. HEILMEYER, JR. (Fig. 2). T h e only remaining possibility to explain this effect is an inactivation of the e n z y m e by the addition of ammonium to the medium. T h e reaction is very specific and very sensitive. (7) A concentration of NH4 + as low as 10-rM lowers the specific activity within 5 min by about 30%. Li ÷, K ÷, Rb ÷ and Cs ÷ show no effect. T h e anion of the ammonium salt added has no influence. Substituted NH3, e.g. hydrazine, hydroxylamine, trimethylamine, have no effect either. Several other enzymes which we tested are not influenced during the inactivation o f glutamine synthetase b y ammonium. (8) In addition to the synthesis of glutamine, expressed by: glutamate + NH3 + A T P ~ glutamine + A D P + Pi, the e n z y m e also catalyzes a transfer of the y-glutamyl residue o f glutamine to hydroxylamine: glutamine + H 2 N O H ~ y - g l u t a m y l - N H O H + NH3. Table 2 shows the glutamine synthetase activity of the e n z y m e to be TABLE 2 Glutamine Synthetase and Glutamine Transferase Before and After Inactivation with NH4+ (For details cUr)) Specificactivity (units per nagprotein) Treatment Glutamate culture Glutamate culture, then short time inactivation by NH4+
Transferase fr) 535
Synthetase (s) 180
282
6.4
T/S 3.0
44
preferentially cut down by the inactivation by ammonium, whereas the glutamyl transferase activity is affected to a much lesser extent. W e suppose that the ammonium inactivation causes a change in the catalytic site o f the e n z y m e which is needed only t~or the synthetase reaction, and not for the transferase reaction. I f we suppose the reaction mechanism to follow the scheme developed by Meister <9) (Fig. 4), we can assume that a special part o f the catalytic site is influenced which participates in the A T P - d e p e n d e n t conversion of glutamate into T-glutamylphosphate. Since the inactivation merely consists in a minute structural change in the catalytic site of the enzyme, we checked whether a reactivation of the e n z y m e can be detected which is independent of a d e n o v o synthesis of
INACTIVATION OF GLUTAMINE SYNTHETASE IN E. COLI
215
the enzyme. The experiment given in Table 3 demonstrates a reactivation of this kind. When cells inactivated with ammonium sulfate over a short period are incubated in the presence of chloramphenicol, a regeneration of the enzymatic activity is observed. Thus a reactivation of the enzyme previously inactivated by NH4 ÷ is possible without protein synthesis. Gtu
AT P ~
P; ~
~ v'Gtu-P ~, ADP
~
~
GtuNH 2
NH3
NH20~ ~P
Synthetase Reaction 1
l
Transferase Reaction
~t'GtuNHOH
FIG. 4 Scheme of synthetase and transferase reactions. (y-Glu-P = y-glutamylphosphate.)
p-Fluorophenylalanine does not inhibit the. reactivation either (Table 4). Another inhibitor of protein synthesis, 5-methyltryptophan, does affect the reactivation. We found, however, (1°) that in intact cells 5-methyltryptophan as well as ammonium induces an inactivation of the enzyme. Therefore 5-methyltryptophan probably inhibits the reactivation not by TABLE 3 Change in Specific Activity of Glutamine Synthetase in E. coil After Resuspension of Repressed and Inactivated Cells in Glutamate-containing Medium With and Without Addition of Chloramphenicol (For details cf. (7~) Specific activity (units per mg protein)
Treatment
At the beginning of the experiment
60 min in NH4+-free glutamate medium without chloramphenicol
with chloramphenicol
Culture in glutamate medium, then short time inactivation by NH4 +
6.4
210
180
Culture in NH4 + medium
5.0
117
10
inhibition of protein synthesis but by an inactivation which is superimposed on the reactivation. 2,4-Dinitrophenol prevents the reactivation. Thus the reactivation process can be supposed to be independent of protein synthesis, but dependent on ATP.
216
H. H O L Z E R , D. M E C K E , K. W U L F F ,
K. LIESS, L. H E I L M E Y E R , JR.
TABLE 4 Effect o f Different T o x i c S u b s t a n c e s o n the Reactivation of G l u t a m i n e S y n t h e t a s e ~°~ Specific activity (units per nag protein) Inhibitor (added during reactivation)
Before inactivation (glutamate grown cells)
A f t e r 10 min inactivation (by 2 × l0 -4 M N H4 + )
After a n o t h e r 10 min reactivation (--NH4 + + 10 -z M glutamate)
113
27
159
113
27
135
5.4 x 10 -4 M F - P h e * 9.2 x 10 -4 M CH3-Try*
72 72 72
10 10 10
-10 -2 M 2 , 4 - D N P *
60 60
5 5
-
6.2 × 10 -4 M chloramphenicol --
1 8 8
133 3.7 274 15
* F - P h e = p-fluorophenylalanine, C H 3 - T r y = 5-methyltryptophan, D N P = dinitrophenol.
The experiments reported so far on inactivation and reactivation can be summarized in the following scheme: glutamine synthetase a ~
(NH4 +) (ATP)
~ glutamine s y n t h e t a s e b.
The denominations a and b for active and inactive enzyme respectively have been taken from the glycogen phosphorylase nomenclature. As the experiments to be described in section III will show, probably not ammonium itself, but some product in the cell derived from ammonium, is the metabolite inducing the inactivation. Out of the inactivation reaction the question arises whether the "repression" of glutamine synthetase described in section I is not merely simulated by inactivation of the enzyme. The experiment given in Table 3, however, proves that besides the inactivation a repression of the synthesis does exist. Cells with low specific activity of the enzyme have been prepared in two ways: (1) by growth with ammonium in the medium; (2) by short-time inactivation of derepressed cells by ammonium. Upon incubation of such cells in a growth medium containing chloramphenicol, the rapidly inactivated cells exhibit a revival of the high specific activity, while the cells grown with ammonium show no reactivation. Thus growth on a m m o n i u m - i n accordance with the existence of " r e p r e s s i o n " prevents the synthesis of the enzyme protein with the result that neither glutamine synthetase a nor b is present. If only inactivation existed, one
INACTIVATION OF G L U T A M I N E SYNTHETASE IN E. COLI
217
would expect even after growth with ammonium the presence of glutamine synthetase b which can be reactivated in the presence of chloramphenicol. It should, however, be kept in mind that "repression" is not the only possible explanation of the experimental results. They are also consistent with a slow degradation of glutamine synthetase b to peptides and/or amino acids during growth with ammonium. At the moment we cannot distinguish between this mechanism and" repression". III. Inactivation of Glutamine Synthetase in a Cell-free System ( c f s) a n d Ol))
To demonstrate an inactivation of glutamine synthetase in a cell-free extract, we first added ammonium sulfate to a crude extract obtained by grinding E. coil cells with aluminium oxide powder. The enzyme is completely stable under these conditions; no trace of inactivation by the added ammonium can be observed. Upon the addition of A T P and Mg z+, however, rapid inactivation occurs even in the absence of ammonium. As shown by Table 5, both A T P and Mg ~+ are necessary for this effect. TABLE 5 Inactivation of Glutamine Synthetase in the Crude Extract from E. coli Specific activity after 30 min at 37 ° (units per mg protein) Crude Crude Crude Crude
extract extract + A T P extract + Mg 2+ extract + A T P + Mg 2+
66.5 61.0 75.0 3.6
TABLE 6 Glutamine Synthetase and other Enzymes in Crude Extract from E. coli (The numbers represent units per mg protein; for further details cf.ts).)
cO t-.
o _
Control 45 min incubated at 37 ° without addition 45 rain incubated at 37 ° with A T P + Mg ~+
40.5
35.2
6.3
770
77
269
43
39.6
7.2
815
86.5
295
40.2
6.6
750
5.8
100
262
218
H. HOLZER, D. MECKE, K. W U L F F , K. LIESS, L. HEILMEYER, JR.
Ammonium salts have no influence. Table 6 shows that among several enzymes tested only glutamine synthetase is inactivated. The enzyme specificity of this inactivation effect is therefore identical with the specificity we observed after the addition of ammonium sulfate to intact cells. If instead of a crude extract, glutamine synthetase, which had been partially purified by ammonium sulfate fractionation and dialysis, is used, ATP and Mg z+ do not cause inactivation any more. Therefore during purification a factor has been removed which is necessary for the inactivation. This factor, present in the crude extract from E. coli, is stable against heating (Table 7). It is lost during dialysis. As well as
TABLE 7 Influence of Different Additions on the Inactivation of Partially Purified Glutamine Synthetase (% Inactivation in 15 rain at 37°; for further details cf3 s)) Control (purified synthetase + A T P + Mg 2+) + crude extract from E. coli + boiled crude extract from E. coli + dialysed, boiled crude extract fromE, coli + boiled crude yeast extract
<5% 60% 50% <5% 55%
in E. coli, this factor necessary for the inactivation is contained in yeast. So it must be a substance of low molecular weight which is heat-stable and not specific for E. coli: probably a metabolite. We did not undertake the effort of purifying this factor from boiled extracts of E. coli or yeast, but we tested several intermediates of N-metabolism whom we expected TABLE 8 Inactivation of Glutamine Synthetase by Different Amino Acids in Percent of the Values with Glutamine (The incubation mixture contained partially purified glutamine synthetase + ATP + Mga+; amino acid concentration 10 -3 M; incubation time 10 rain.) Glutamine Glutamate NH4 + Tryptophan Methionine
100 80 < 5 50-60 40-50
Asparagine Threonine Serine Other amino acids* Without addition
10 10 10 < 5 < 5
*Alanine, aspartic acid, arginine, D-glutamic acid + ammonium, L-a-methylglutamic acid + ammonium, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, tyrosine, valine.
INACTIVATION OF GLUTAMINE SYNTHETASE IN E, COLI
2]9
to influence the glutamine synthetase reaction in a biologically reasonable way. As shown b y Table 8, glutamine is particularly efficient; the effect o f glutamic acid is perhaps due to its enzymatic conversion to glutamine. T h e results obtained so far are summarized in the following equation (glutamine was taken as metabolite inducing the inactivation, because this compound exhibited the strongest effect o f all the substances we have investigated as yet): glutamine synthetase a
glutamine, ATP, Mg2+ ~ (ATP .... ?) ~
glutamine synthetase b
F o r further experiments glutamine synthetase was purified by the procedure of Woolfolk and Stadtman: (12) precipitation with streptomycin, fractionation with acid and heat treatment. T h e purified e n z y m e behaves uniformly in the ultracentrifuge, agar gel electrophoresis, and chromatography on DEAE-cellulose. In contrast to the only partially purified enzyme, the pure e n z y m e is not inactivated by incubation with glutamine, A T P and Mg 2+. During the purification
N
300
I 0"--
~200
E 0
10o. C
10
20 time (minutes)
30
FIG. 5 Glutamine induced enzymatic inactivation of glutamine synthetase from E. coli.
x @ ©
(cf.01).) x -- glutamine synthetase + glutamine + ATP + Mg2+ • = same plus "acid fraction", © = same plus boiled "acid fraction".
220
H. HOLZER, D. MECKE, K. W U L F F , K. LIESS, L. HEILMEYER, JR.
another factor has therefore been lost which is necessary for the inactivation. A protein fraction removed by the acid fractionation was shown to contain the missing factor. From Fig. 5 one can see that there is no inactivation in the presence of glutamine, ATP and Mg2+ alone, but it takes place upon addition of the "acid fraction". With this test system the factor necessary for the inactivation could be purified. As yet we have reached about a 10-fold purification3 TM The behavior of this factor during purification, heat treatment and dialysis shows that it is a protein, for which we use the name "inactivating enzyme". The purified "inactivase" is free of glutamine synthetase. The following scheme summarizes our present knowledge on the inactivation and reactivation of glutamine synthetase: inactivating enzyme, glutamine, glutamine synthetase a
ATP, Mg~÷ ~
(ATP.... ?)
glutamine ' synthetase b.
DISCUSSION
We cannot give a definite answer yet to the question whether the inactivation observed in intact cells upon the addition of ammonium salts is identical with the effect observed in cell-free extracts upon the addition of glutamine. But there are two observations which support the identity of both reactions. (a) The inactivation in the cell-free extract as well as in intact cells mainly affects the glutamine synthetase activity of the enzyme, and much less the transferase activity38~ (b) Out of five different enzymes only glutamine synthetase was found to be inactivated in the cell-free extract (upon addition of glutamine) as well as in intact cells (upon addition of NH4÷). If one accepts the identity of the inactivation processes in intact cells and in the cell-free extract, the question arises: "Why in intact cells only ammonium inactivates and not glutamine, but in the cell-free extract only glutamine and not ammonium?" A possible explanation would be that glutamine, in contrast to ammonium, does not easily enter the cells. The experiment, the results of which appear in Table 9, supports this possibility. Low concentrations of glutamine cause no change of activity because of the permeability barrier. High concentrations of glutamine break through the permeability barrier and produce the same inactivation as low concentrations of ammonium.
I N A C T I V A T I O N O F G L U T A M I N E S Y N T H E T A S E I N E. COLI
221
' TABLE 9 Inactivation o f Glutamine Synthetase in E. coli after Addition of Glutamine or NH4 + to the G r o w t h Medium Specific activity 10 min after addition of glutamine or NH4 + (units per mg protein) Without addition 10 -4 M NH4 + 10 -4 M glutamine 10 -3 M glutamine 10 -2 M glutamine
104 2.3 47 8.5 4.7
Only a minute structural change of glutamine synthetase during the enzymatic inactivation is indicated by the following results: (a) There is little change of transferase activity under conditions where the synthetase activity is almost completely inactivated. (b) In vivo inactivated glutamine synthetase (which can be tested by the transferase reaction) behaves exactly like active glutamine synthetase during the purification by streptomycin precipitation, acid fractionation, heat denaturation, and chromatography on DEAE-cellulose34) (c) In the analytical ultracentrifuge active glutamine synthetase shows the same sedimentation rate as the inactivated enzyme obtained by ammonium sulfate treatment of intact cells, t4) (d) In agar gel electrophoresis both forms of the enzyme behave in the same way. (4) We could not find any support for a fixation of I4C from uniformly 14Clabeled glutamine into the protein of glutamine synthetase. Experiments on the incorporation of 15N-amide nitrogen of glutamine and of the Tphosphorus of T_32P-ATP are in preparation. In Salmonella typhimurium upon addition of ammonium sulfate to cells grown on glutamate as N-source an inactivation of glutamine synthetase is observed as in E. coli. In Lactobacillus arabinosus and in Saccharomyces cerevisiae we have not found an inactivation ofglutamine synthetase. The feedback inhibition of glutamine synthetase by glutamine described for Lactobacillus arabinosus 03) could be reproduced by us. This inhibition is reversed by dialysis. On the contrary, the inactivation of glutamine synthetase from E. coli described here is not reversible by dialysis. Thus the effect in Lactobacillus arabinosus must be a real feedback inhibition, not an inactivation. Figure 6 shows schematically the position of glutamine synthetase in metabolism. The glutamine synthesized from glutamate and NH3 is needed for the synthesis of proteins and for the introduction of nitrogen
222
H. HOLZER, D. MECKE, K. WULFF, K. LIESS, L. HEILMEYER, JR. R • pr ess i o n
(?)
Protein Synthesis
Nucteotldes,Histidine I NH3
.~ ~
~ GLutom~
I= Tryptophsugars, NAo.Amln°-[ Corbarny[phosphat .,j GLutornyl Transfer (?)
Cumutotive
Feedback
FIG. 6 Regulation of glutamine synthetase in E. coil
into different metabolites. Probably also the glutamyl residue can be transferred from glutamine to a biological acceptor by the aid of glutamine synthetase. This assumption is favored by the well-known transfer of the glutamyl residue to the unphysiological acceptor hydroxylamine Cglutamyl transferase reaction"). A physiological acceptor, however, has not been found so far. Accumulation of metabolites synthesized with the aid of the amide group of glutamine reversibly inhibits the activity of glutamine synthetase by "cumulative feedback" as discovered by Woolfolk and Stadtman. t12~ The enzymatic inactivation of glutamine synthetase by glutamine described in this paper is a second mechanism preventing an excessive supply of glutamine. This additional mechanism may be necessary, because glutamine is a starting material for the synthesis not only of metabolites of low molecular weight, but also for protein synthesis and perhaps the synthesis of other polymers by glutamyl transfer reactions. Polymers fixed in cellular structures are unable to regulate their own synthesis by feedback which involves dissociable binding to the active or allosteric site of an enzyme. Therefore the precursor of the polymer, namely glutamine, must take over the regulatiorL Another possibility of feedback control by glutamine is realized in Lactobacillus arabinosus (cf.(la)). Here the accumulating glutamine causes no irreversible enzymatic inactivation of the synthetase, but a reversible feedback inhibition. Besides the rapidly operating "fine control" of glutamine synthetase by feedback and enzymatic inactivation, a "coarse control" (for the definition of "fine" and "coarse control" see ~4) and {~5)) is achieved by repression of the synthesis of the enzyme. This is true for E. coli (7"~2} and for yeast, (6) where a compound originating from ammonium, possibly glutamine, is the corepressor. In H E L A cells {16~addition of glutamine to the growth medium leads to a low specific activity of glutamine synthetase. Here too a repression of enzyme synthesis by glutamine is probable.
I N A C T I V A T I O N O F G L U T A M I N E S Y N T H E T A S E I N E. COLI
223
"Fine control" by feedback and enzymatic inactivation affects the activity of the enzyme within a few minutes or even seconds. In the contrary, "coarse control" by repression is only efficient with the rate of dilution of the existing enzyme quantity by growth of the cell mass. As compared to feedback, regulation of enzyme activity by "metabolite-dependent enzymatic inactivation" is an "expensive" mechanism because of the necessity of an additional enzyme. For reasons of economy one can understand that this kind of regulatory mechanism is realized only in the case of a few key-enzymes. We found the following examples in the literature: (1) (2) (3) (4)
mammalian glycogen phosphorylase ;~1r.18) 6-phosphofructokinase ~19)in yeast; mammalian glycogen synthetase (for a summary cf.~°)); glutamine synthetase in E. coli.
Glycogen phosphorylase, glycogen synthetase and 6-phosphofructokinase are key-enzymes of carbohydrate metabolism, glutamine synthetase is a key-enzyme of nitrogen metabolism. SUMMARY
1. Addition of ammonium salts to E. coli cells grown on a medium containing no ammonium salts produces a 80-90% inactivation of glutamine synthetase within 1 to 2 min. The enzyme's ability to transfer the T-glutamyl residue of glutamine to hydroxylamine (glutamyl transferase) is only slightly affected by this inactivation. 2. Addition of ammonium salts to the medium causes not only inactivation of glutamine synthetase, but also repression of the synthesis of the enzyme. 3. Glutamine synthetase inactivated by the addition of ammonium ion to intact cells is reactivated after the ammonium ion is washed out. The reactivation does not involve de novo protein synthesis, but probably requires ATP. 4. An inactivation of glutamine synthetase activity with only a small effect on the glutamyl transferase activity is demonstrated also in a cellfree system. This inactivation requires ATP, Mg ~+, glutamine (or certain other amino acids) and a specific protein ("inactivating enzyme"). The inactivating enzyme has been purified about 10-fold and separated from glutamine synthetase. 5. Preliminary results support the view that the inactivation produced by ammonium ion in intact cells is identical with the enzymatic inactivation induced by glutamine in the cell-free extract.
224
H. HOLZER, D. MECKE, K. WULFF, K. LIESS, L. HEILMEYER, JR.
6. A p o s s i b l e biological f u n c t i o n o f the " m e t a b o l i t e - i n d u c e d e n z y m a t i c i n a c t i v a t i o n o f g l u t a m i n e s y n t h e t a s e " is d i s c u s s e d . E n z y m e r e g u l a t i o n t h r o u g h e n z y m a t i c i n a c t i v a t i o n a n d r e a c t i v a t i o n s e e m s to b e r e s t r i c t e d to a f e w key e n z y m e s o f m e t a b o l i s m .
ACKNOWLEDGMENTS S u p p o r t e d b y the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t a n d t h e B u n d e s m i n i s t e r i u m f'tir w i s s e n s c h a f t l i c h e F o r s c h u n g . REFERENCES 1. I. WITT,R. KRONAUand H. HOLZER,Repression von Alkoholdehydrogenase, Malatdehydrogenase, Isocitratlyase und Malatsynthase in Hefe durch Glucose, Biochim. Biophys. ,4cta 118, 522-537 (1966). 2. I. WITT, R. KRONAUand H. HOLZER, Isoenzyme der Malatdehydrogenase und ihre Regulation in Saccharomyces cerevisiae, Biochim. Biophys. ,4cta, 128, 63-73 (1966). 3. M. F. UTTER,E. A. BUELL a~d C. BERNOFSKY,Alterations in the respiratory enzymes of the mitochondria of growing and resting yeast in Aspects o f Yeast Metabolism, (A. K. MILLS, ed.; SIR HANSKREBS,Consulting Ed.), Blackwell Scientific Publications, Oxford (1966). 4. K. LIESS, Dissertation in preparation for the Naturwissenschaftlich-mathematische Fakult~t der Universit~it Freiburg im Breisgau. 5. CH. GARTNER,unpublished results. 6. G. KOHLHAW,W. DRAGERTand H. HOLZER, Parallel-Repression der Synthese yon Glutaminsynthetase und DPN-abh~ingiger Glutamatdehydrogenase in Hefe, Biochem. Z. 341,224-238 (1965). 7. D. MECKE and H. HOLZER, Repression und Inaktivierung yon Glutaminsynthetase in E. coil durch NH4+, Biochim. Biophys. ,4cta 122, 341-351 (1966). 8. n . MECKE, K. WULFF and H. HOLZER, Metabolit-induzierte Inaktivierung yon Glutaminsynthetase aus E. coil im zellfreien System, Biochim. Biophys. Acta, 128, 559-567 (1966). 9. A. MEISTER, Giutamine synthesis, pp. 443-468 in The Enzymes 6, 2nd Ed. (P. D. BOYER,H. LARDYand K. MYRBXCK,eds.) Academic Press, New York (1962). 10. L. HEILMEYER,JR, D. MECKEand H. HOLZER,unpublished results. 11. D. MECKE, K. WULFF, K. LIESS and H. HOLZER, Characterization of a glutamine synthetase inactivating enzyme from E. coli, Biochem. Biophys. Res. Communs. 24, 452-458 (1966). 12. C. A. WOOLFOLKand E. R. STAOTMAN,Cumulative feedback inhibitionin the multiple end product regulation of glutamine synthetase activity in E. coil, Biochem. Biophys. R es. Communs. 17, 313-319 (1964). 13. J. M. RAVEL, J. S. HUMPHREYS and W. SHIVE, Control of glutamine synthesis in Lactobacillus arabinosus,,4rchives Biochem. Biophys. 111,720-726 (1965). 14. J. M. ASHWORTand H. L. KORNBERG,Fine control of the glyoxylate cycle by allosteric inhibitionof isocitrate lyase, Biochim. Biophys..4 cta 73, 517-519 (1963). 15. H. A. KREeS, Meeting of the Nobel-Laureates at Lindan (Germany), July 1963. 16. R. DE MARS,The inhi'bition by glutamine of glutamyl transferase formation in cultures of human cells, Biochim. Biophys. ,4 cta 27, 435-436 (1958). 17. G. T. COR1and A. A. GREEN, Crystalline muscle phosphorylase, II. Prosthetic group, J. Biol. Chem. 151, 31-38 (1943). 18. E. G. KREBSand E. H. FISCHER,Molecular properties and transformations of glycogen phosphorylase in animal tissues,,4 dvances in Enzymology 24, 263-290 (1962).
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19. E. VINUELA, M. L. SALAS,M. SALASarid A. SOLS,Two interconvertible forms of yeast phosphofructokinase with different sensitivity to endproduct inhibition, Biochem. Biophys. Res. Communs. 15, 243-249 (1964). 20. S. H. GOLDEMBERG, Reactions of glycogen synthesis and glycolysis, pp. 292-317 in D-Glucose und verwandte Verbindungen in Medizin und Biologie (H. BARTELHEIMER, W. HEVDE and W. THORN, eds.), Veflag F. Enke, Stuttgart (1966).