ARCHIVES OF BIOCHEMISTRY Vol. 191, No. 2, December,
AND BIOPHYSICS pp. 602-612, 1978
Cascade
Control of E. Co/i Glutamine
II. Metabolite EDGAR
Regulation
G. ENGLEMAN*’
of the Enzymes AND
Synthetase
in the Cascade
SHARRON
H. FRANCIS
Section on Enzymes, Laboratory of Biochemistry, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 Received
April
28,1978;
revised
July
31,1978
Enzymes and regulatory proteins involved in the cascade control of glutamine synthetase activity of Escherichia coli have been separated from one another and the effects of numerous metabolites on each step in the cascade have been determined. The adenylyl transferase (ATase) -catalyzed adenylylation of glutamine synthetase, which requires the presence of the unmodified form of the regulatory protein PI1 is enhanced by glutamine and is inhibited by either a-ketoglutarate (a-KG) or the uridylylated form (PII’ UMP) of the regulatory protein. PII’UMP and a-KG act synergistically to inhibit this activity. In contrast, the PII. UMP-dependent, ATase-catalyzed deadenylylation of glutamine synthetase requires a-KG and ATP and is inhibited by glutamine or PI1 and synergistically by glutamine plus PII. The capacity of uridylyl transferase (UTase) to catalyze the uridylylation of PI1 is dependent on the presence of o-KG and ATP and is inhibited by glutamine. The deuridylylation of PII’UMP by the uridylyl removing enzyme (UR) is enhanced by glutamine but is unaffected by a-KG. However, CMP, UMP, and CoA all inhibit activity at lo-@ M. High concentrations of ATase inhibit both UR and UTase activities, presumably by binding the regulatory protein. Of more than 50 substances that alter the activity of at least one enzyme in the cascade, only a-KG and glutamine affect the activity at every step. This accounts for the observation that glutamine synthetase activity in viva is very sensitive to the intracellular ratio of o-KG to glutamine.
uiuo to the intracellular ratio of a-ketoglutarate to glutamine (5). These metabolites, as well as various nucleotides, amino acids, and divalent cations also affect the steady state level of adenylylation in vitro under conditions that allow adenylylation and deadenylylation reactions to occur simultaneously (6). The effects of these ligands on individual steps in the cascade have not been rigorously studied, in part, due to the difficulty of separating cascade enzymes from one another. Thus, the present work represents an effort to identify the site(s) of metabolite control of the cascade system. To this end, the cascade enzymes and the two forms of the regulatory protein have been separated from one another, and each has been assayed in the presence of one or more effectors. The results indicate that although many metabolites affect the activity of at
One important mechanism for the regulation of glutamine synthetase activity in Escherichia coli is the covalent attachment and removal of AMP from a specific tyrosyl residue in each of the enzyme’s 12 identical subunits (l-4). Adenylylation of a subunit converts it to a form inactive in the presence of Mg2+ (1). The enzyme’s activity is thus controlled by the average number of adenylylated subunits per molecule which can vary from zero to 12. The cascade which controls the state of adenylylation of glutamine synthetase is under strict metabolite control, with particular sensitivity in ’ Present Address: Department of Pathology, Stanford University School of Medicine, Stanford, California 94305. * Present Address: Department of Physiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232. To whom all correspondence should be addressed. 602 0003-9861/78/1912-0602$02.00/O Copyright
0 1978 by Academic
All rights of reproduction
F’reea,
Inc.
in any form reserved.
METABOLITE
REGULATION
OF
GLUTAMINE
least one enzyme in the cascade, only the reciprocal effects of glutamine and CY-KG” are seen at every step. Moreover, PII, PII. UMP, and ATase all affect cascade steps in addition to the ones for which they are required, and in two instances, these effects are synergistic with the effects of (YKG or glut-amine. Finally, we report that the UR enzyme activity, which catalyzes the deuridylylation of PII and thereby initiates the first step in the adenylylation of GS, is very sensitive to a group of effecters including coenzyme A and certain nucleotide monophosphates which have little or no effect on the other cascade components but affect UR activity at concentrations as low
8s lo-’
M.
MATERIALS
AND
METHODS
Enzyme assays. GS was assayed by the y-glutamyl transferase assay (7). One unit of activity is equal to 1.0 ~01 of y-glutamyl hydroxamate formed per min at 37’C. The state of adenylylation was determined by the ratio of y-glutemyl transferase activities in the presence of Mn’+ alone and of Mn*+ + Me (7). Adenylylation of GS by ATase was assayed as previously described (8). A two-step calorimetric assay consisted of (a) adenylylation by ATase of unadenylylated GS followed by (b) an assay of the remaining unadenylylated GS, using the y-glutamyl transferase assay described above. A decrease in A.w ,,,,, compared to a control incubation without ATase indicates the presence of ATase activity in the fraction tested. One unit of activity is defined as that amount of ATase necessary to decrease the G&catalyzed formation of y-glutamyl hydroxamate in the presence of Mn” and Me by 1.0 ~01 (AM, 0.36) per min. To assess the metabolite effects on adenylylation, the assay contained fixed levels of ATase and GS, subsaturating concentrations of ATP and Mg*+, and no glutamine. A 40+1 reaction mix contained 50 mu O-methyl imidazale, pH 7.6, 15 units of ATase, 50 pg of unadenylylated GS, and either 2.5 mu ATP plus 25 mM Mg*+ (“high” ATP) or 0.25 mu ATP plus 2.5 mu Mg’+ (“low” ATP), and effector in the concentrations indicated. Aliquota were withdrawn after various periods of incubation and assayed for the G&catalyzed y-glutamyl transferase activity. PI1 activity was measured as previously described (8). A relative PI1 activity of one represents the amount of PII required to produce a rate of adenylyl3Abbreviations used: ATase, adenylyl transferase; a-KG, a-ketoglutarate; GS, glutamine synthetase; PIT, unmodified regulatory protein, PII. UMP, uridylylated regulatory protein, UR, uridylyl removing enzyme; UTase, uridylyl transferase.
SYNTHETASE
IN
Escherichia
coli
603
ation of GS of 0.6 adenylyl group/molecule/min under the conditions described. For purposes of assessing the effects of metabolites on PII-supported adenylylation, the assay was modified as follows. A 40 pl reaction mix contained 59 mM 2-methyl imidasole, pH 7.6,40 units of PI1 (a mixture of PI1 and PII’UMP which was at least 90% PII), 15 units of ATase, 50 pg of adenylylated GS, and either 2.5 mu ATP plus 25 mM Mg’+ or 0.25 rruu ATP plus 2.5 mM Me, and effector in the concentrations indicated. The rate of adenylylation of GS was followed by determining the decrease in yglutamyl transferase activity in the presence of Mn*’ + Me (7). Initial rates of adenylylation were not always measurable in the presence of added effecters, and such qualitative data are presented in table form. PII. UMP activity was measured as previously described (9). One unit of PII. UMP is the amount required to stimulate the removal of 1 pmol of [‘4C]AMP from [5’-W]AMP-GS per min under standard assay conditions. The effects of metabohtes on PII.UMP activity were assayed in either of two methods. 1) Effecters were added to a 109+l reaction mixture containing 50 mu %-methyl imidazole, pH 7.6, 160 pg [5’-W]AMP-GS, 27 units of ATase, 130 units of PII.UMP, 2.5 mM ATP, 20 mM Mg’+, 0.03 mM (Y-KG, and 20 mM potassium phosphate. After 10 min at 37”C, 0.15 ml of 6% HClO, was added and the mixtures were centrifuged. Following this, 0.15 ml of the supernatant was dissolved in 10 ml of Aquasol (New England Nuclear Corp.) and counted on a scintillation spectrophotometer. 2) PII’UMP was also measured by coupling the deadenylylation reaction to a colorimetric y-glutamyl transferase assay at pH 8.0 developed by Dr. S. Rhee (National Heart and Lung Institute, Bethesda, Md.) for measurement of unadenylylated glutamine synthetase. Effecters were incubated at 26°C with a 0.5~ml mixture containing 50 mM 2methyl imidazole, pH 7.6, 60 pg of adenylylated GS, 130 units of PII’ UMP, 15 units of ATase, 28.5 mM potassium phosphate, 15 mu Me, 1mM dithiothreitol, and various concentrations of ATP and (U-KG. After 10 min, a l-ml mixture (“transferase mix”) was added which contained 150 mM 2-methyl imidazole, 0.15 mM ADP, 30 mM sodium arsenate, 66 mM NHzOH’HC~, 225 mM glutamine, 150 mM KCl, and 0.6 mM Mn*+. After incubation for 5 min at 26”C, the reaction was stopped and the color developed by the addition of 0.5 ml of a solution containing equal volumes of 24% trichloracetic acid, 6 N HCl, and 10% FeCL.GH20 in 0.02 N HCl. Following vigorous mixing and centrifugation for 10 min at 10,06Og, the absorbance was read at 540 nm using a Gilford 300 N spectrophotometer. An increase in Amnm compared to a control incubation without PI1 UMP reflects the formation of unadenylylated GS due to the action of PII. UMP. UTase was measured as previously described (10) by incubating 75 units of PI1 and enzyme at 37°C in a
604
ENGLEMAN
50-d reaction mixture (“UTase reaction mixture”) containing 50 mM %-methyl imidazole, pH 7.6, 100 mM KCl, 10 mM MgClz, 0.1 mM ATP, 0.2 mu UTP, 5 mM a-Kg, and 1 mM dithiothreitol. After 5 min at 37’C, a 5-~1 aliquot was withdrawn and the increase in PII. UMP activity measured as described above. One unit of activity is defined as that amount of UTase necessary to increase the formation of PI1 UMP by 1 ymol per min under these conditions. To determine the effects of metabolites on UTase activity, two methods were used. 1) Effectors were incubated with 275 units of UTase in the above “UTase reaction mixture” at 37°C for 5 min, after which 5-~1 aliquots were removed and assayed for PII.UMP activity with the first of the two methods described above. 2) UTase was also measured by coupling the uridylylation reaction to the alternate PII’UMP assay (described above). In this method, effecters were incubated at 26“C in a 35-d mixture containing 50 mM 2-methyl imidazole, pH 7.6, 1 mM UTP, 10 mM MgCL, 33 mu KCl, 75 units PII, 150 units UTase, and various concentrations of ATP and (U-KG. After 5 min a 709~1 mixture was added which contained 75 mM 2-methyl imidazole, pH 7.2,30 mM a-KG, 1.5 mM dithiothreitol, 1.5 mM ATP 25 mM MgCl2, 45 mM potassium phosphate, 15 units ATase and 60 pg of adenylylated GS. After 5 min at 26’C, 1.0 ml of the pH 8.0 y-glutamyl “transferase mixture” (see above) was added and incubated at 26’C for measurement of unadenylylated GS. UR activity was measured as previously described (lo), except that the buffer was P-methyl imidazole, pH 7.6. To determine the effects of metabolites on UR activity, a 30-d reaction mixture containing 130 units of PII’UMP, 40 mM a-methyl imidazole, pH 7.6, 100 mM KCl, 1 mM MnClz, and an amount of UR enzyme which catalyzed 50% inactivation of the PII. UMP in the absence of effecters were incubated at 37°C with and without effecters. After 10 min, 70 ).d of deadenylylation reaction mixture were added and the residual PI1 UMP activity measured. Whenever metabolite effects were being determined, control assays were run with effector present but a key enzyme omitted. For example, in determining the effect of glutamine on UR activity, a control reaction mixture was assayed with glutamine added and UR enzyme omitted. In this way, the effect of glutamine on deadenylylation could be distinguished from effects on UR activity. Protein concentrations. Protein concentration was determined either by the method of Lowry (11) or, for protein solutions greater than 10 mg/ml, by the biuret method (12).
AND FRANCIS
supported adenylylation reaction in the presence of a “low” (0.25 mu) ATP concentration. In the absence of PI1 and glutamine, adenylylation activity is perceptible but very low. The addition of up to 1.0 mu glut-amine failed to enhance this “intrinsic” ATase activity, and the addition of PI1 results in a g-fold increase in activity. However, the addition of PI1 plus glutamine yields synergistic enhancement of adenylylation. In a manner analogous to the glutamine effect; a-KG has no effect on adenylylation in the absence of PII; however, in the presence of PII, adenylylation is inhibited by o-Kg in a dose-dependent manner. Figure 1 depicts the dose-dependent effects of o-KG and glutamine on PII-supported adenylylation. A mixed inhibition pattern for a-KG with respect to glutamine is seen. Whe both effecters are present, their molar ratio determined the rate of adenylylation. Table IB summarizes the effects of o-KG, glutamine, and PI1 on the adenylylation reaction in the presence of “high” ATP
I
2
4 l/(Gltmnine]
6 x 10"
6 IW'I
FIG. 1. The effects of a-ketoglutarate and glutamine on PII-supported adenylylation of glutamine synthetase. The adenylylation activity was assayed as described in Materials and Methods in the presence of varying concentrations of glutamine and a-ketoglutarRESULTS ate. The concentrations of ATP and Mg*+ were 0.25 and 2.5 mM, respectively. V represents the rate of Effecters of Adenylylation adenylylation as measured by the decrease in formaTable IA summarizes the effects of LY- tion of y-glutamyl hydroxamate expressed in micromoles per min. ketoglutarate and glutamine on the Me-
METABOLITE
REGULATION TABLE
OF
GLUTAMINE
SYNTHETASE
IA
TABLE
EFFECTSOF PII, GLUTAMINE,AND (YKETOGLUTARATEONADENYLYLATIONOF GLUTAMINE~YNTHETASEATLow ATP CONCENTRATIONS PI1 (units)
Gll;ty mM
a-Ketoglutarate bhf)
0 0 0 0 40 40 40 40 0 0 0 40 40 40 40 40 40 40
0 0.1 0.5 1.0 0 0.1 0.5 1.0 0 0 0 0 0 0 0.1 1.0 0.1 1.0
0 0 0 0 0 0 0 0 0.1 1.0 1.0 0.05 0.2 1.0 0.2 0.2 1.0 1.0
IN Escherichia
Rate of adenylylation SO.03 SO.03 SO.03 SO.03 0.27 0.54 0.97 1.19 SO.03 SO.03 SO.03 0.27 0.17 SO.03 0.22 0.69 0.06 0.27
a Assays were performed in the presence of 0.25 mM ATP and 2.5 mM Mg*+. The rate of adenylylation is expressed as the decrease in formation of y-glutamyl hydroxamate in micromoles per mm as determined in the y-glutamyl transferase assay (7).
concentration (2.5 mu). In contrast to the result at low ATP, there is significant ATase activity in the absence of PI1 and glutamine, and the addition of 1.0 mu glutamine enhances this activity more than 13-fold. At 2.5 mu ATP, the addition of 40 PI1 units to the assay mixture increases the adenylylation rate by g-fold from 0.17 to 1.11 ~mol/minute. The addition of 0.05 mu glutamine enhances the PII-supported activity by 2-fold, but in the absence of PII, 0.1 mM glutamine enhances the adenylylation by 2-fold. At high ATP concentrations the stimulation of adenylylation may be related only to the interaction of glutamine with ATase. At low ATP (0.25 mM), PI1 plus glutamine augmented activity synergistically but at higher ATP concentration (2.5 mM), glutamine and PI1 do not appear to be synergistic. At the higher ATP concentration, the LXKG inhibition of adenylylation remains dependent on the presence of PII. In the
605
coli
IB
EFFECTSOFPII,PII.UMP, GLUTAMINE,ANDW KETOGLUTARATEONADENYLYLATIONOF GLUTAMINE~YNTHETASEATHIGHATP CONCENTRATIONS PI1 (units)
Glutamine bM)
0 0 0 40 40 40 0 0 40 40 40 40 40 0 0 0 40 40 40 40 40 40
0 0.1 1.0 0 0.01 0.05 0 0 0 0 0 0 0.05 0 0 0 0 0 0 0 0.05 0.05
cr-Ketoglutarate (rnM) 0 0 0 0 0 0 0.1 1.0 0.01 0.03 0.05 0.1 0.05 0 0 0 0 0 0 0.05 0 0.05
PII. UM P (units) 0 0 0 0 0 0 0 0 0 0 0 0 0 130 325 650 130 325 650 325 325 325
Rate of adenylylation 0.17 0.36 2.22 1.11 1.11 2.16 0.17 0.17 1.11 0.67 0.42 0.19 0.83 0.17 0.11 so.03 1.0 0.61 0.14 SO.03 0.69 0.19
a Reaction mixtures were as in Materials and Methods, except for concentrations of ATP and a-ketoglutarate which were 2.5 and 25 mru, respectively.
absence of PII, 1.0 mu a-KG has no effect on the adenylylation rate (Table IB). However, in the presence of PII, 0.03 mu (Y-KG inhibits adenylylation by 40%. This concentration of (Y-KG is an order of magnitude lower than that required (0.2 mu) for a similar effect at low ATP. These results suggest, paradoxically, that synergism exists between both ATP and a-KG and between ATP and glutamine. Hennig and Ginsburg (18) have shown that in the absence of PI1 the glutamine promotion of adenylylation is inhibited by the presence of o-KG. The addition of PII. UMP results in significant inhibition of both intrinsic ATase activity and PI1 supported adenylylation (Table IB), and PII. UMP and (Y-KG synergistically inhibit the PII. ATase supported adenylylation. Figure 2 demonstrates the effects of PII, (U-KG, and the
606
ENGLEMAN
combination of PII. UMP and a-KG on adenylylation. Several metabolites and cofactors have been reported to affect either glutamine synthetase activity or ATase activity in the absence of the regulatory protein (1,13,14). The capacity of these and a number of other compounds to affect the ATase-catalyzed adenylylation in the presence and absence of PI1 was examined and the results are summarized in Table II. Only those substances affecting ATase activity had any effect on PII-supported adenylylation, and these effects were similar to
.021
mM o-KG 1M
300
effects of a-ketoglutarate, PII, and PII. UMP on the adenylylation reaction. The adenylylation activity was assayed as described in Materials and Methods in the presence of varying concentrations of a-ketoglutarate and PII’UMP. Glutamine was absent. TABLE
II
EFFECTSOFAMINOACIDS,GLYCOLYTICANDKREBS CYCLEINTERMEDIATESONADEN~YLATIONOF GLUTAMINE SYNTHETASE Effect
on adenylylation mine synthetase”
PI1 absent L-Glutamine L-Methionine L-Tryptophan a-Ketoglutarate Coenmne A
+ + + 0 -
FRANCIS
those observed on ATase alone, except in the case of glutamine and o-KG. Thus, Lmethionine and L-tryptophan enhanced both intrinsic and PII-supported adenylylation. Phosphoenolpyruvate, coenzyme A, and UTP (not shown) inhibited both activities. The concentration of these effecters required to demonstrate an effect was at least one order of magnitude greater than that needed for glutamine or a-KG. In contrast, compounds previously shown to influence glutamine synthetase activity directly (i.e., glycine, histidine, and alanine) at concentrations up to 10 mu had no demonstrable effect on the adenylylation reaction. Other ligands which had no effect on adenylylation at 10 rnkl concentrations included fructose 1,6-diphosphate, fructose lphosphate, 3-phosphoglycerate, and NAD. Ba2+, Ca2+, Cd2+, Co2+, Hg2+, Ni2+, and Zn2+ in concentrations up to 1 mu failed to support the ATase-catalyzed adenylylation reaction with or without PI1 present. Under the same assay conditions, Mn2+ supported both activities at approximately half of the velocity reached in the presence of Mg2+. In the presence of 2.5 mu ATP, the optimal Mn2+ concentration was 10 mu.
600 Units Pn.UMP
FIG. 2. The
Effector
AND
(lo-4 (lo-3 (lo-3 (l@ (lo-’
M)* hi)
M) M) M)
of gluta-
PI1 present + + + -
(lo-5 (lo-’ (@ (lo-5 (lo-’
I&
M)
Effecters of Deadenylylation
Catalysis of deadenylylation by ATase requires PII. UMP, inorganic phosphate, ATP, and a-KG (8). The requirement for a-KG and ATP in the presence of a constant concentration of PII. UMP are illustrated in Fig. 3. The concentration of ATP required for activity varies inversely with the level of a-KG and vice versa. With 1 mu o-KG, as little as 10m6 M ATP will support the reaction, compared to the 10V4 M ATP requirement for adenylylation. One possible mechanism to explain the synergism between ATP and a-KG in the deadenylylation reaction is diagrammed below:
M)
M)
M)
D Assays were performed as described in Materials and Methods in the presence of 2.5 mM ATP and 25 mM Mg*+. * Concentration given is that which results in approximately a 50% change in the activity, either inhibition (-) or enhancement (+).
AMP.GS&GS
+ AMP K2
METABOLITE
REGULATION
.3 I
/”
OF
GLUTAMINE
/Y Ir”
2 O-
l/o-KG l/V 1’o-
4 I$ 1
x
ki”
A
1 mM a-KG
10 mM a-KG
I 50 l/[ATP]
I
I
100 x lo-, M-’
150
FIG. 3. The role of ATP and a-ketoglutarate in the PI1 UMP supported deadenylylation of glutamine synthetase. The rate of deadenylylation (V) was measured as described in Materials and Methods. The concentrations of ATP and cY-ketoglutarate were varied in the presence of constant levels of PII. UMP and adenylyltransferase. Points for the two secondary plots (see inset) were obtained directly from the primary plots. These form straight lines as predicted by a hypothetical reaction scheme (see text).
It is proposed that ATP (“T”) and a-KG (“K”) can bind independently to the ATase enzyme (“E”) and that the binding of one effector enhances the binding of the other by a factor ((w). The rate expression derived from this mechanism by P. B. Chock (National Heart & Lung Institute, Bethesda, Md.) is:
SYNTHETASE
IN Escherichia
coli
607
lines as predicted and therefore, are consistent with the proposed reaction scheme. The reciprocal effects of glutamine and a-KG on the deadenylylation reaction are shown in Fig. 4. A mixed pattern of inhibition by glutamine with respect to a-KG is seen, analogous to the inhibition by a-KG of the glutamine-enhanced, PII-dependent adenylylation reaction. The concentration of glutamine required to inhibit deadenylylation, however, is at least lo-fold greater than the concentration of glutamine that causes enhancement of adenylylation. Figure 5 shows that glutamine is a potent inhibitor of deadenylylation, whereas PI1 inhibits deadenylylation only weakly. However, the combination of PI1 and glutamine results in synergistic inhibition in a manner analogous to the inhibition of PI1 supported adenylylation by PII. UMP plus a-KG. A summary of the effects of other compounds on deadenylylation is given in Table III. Many of the amino acids and metabolites tested are inhibitory, but none are as potent as glutamine. Alanine, glycine, and histidine at 10 mu concentrations have no effect on either adenylylation or deadenylylation, whereas L-tryptophan and Lmethionine promote adenylylation (Table II) and inhibit deadenylylation (Table III), and coenzyme A inhibited both reactions. No positive effecters were found. Various metals were tested for an effect in the deadenylylation reaction in the presence of 1 to
IO-
a Kl
-+a&
K2
[Kl
+(I+%)} from which one obtains the dissociation constants for (w-KG (0.1 mu) and ATP (0.01 mu) and the enhancement factor (Y = 20. The equation predicts that plots of the slope uersus l/[&-KG] and the Y intercept uersus l/[a-KG] will be straight lines. Both the slope and the Y intercept are directly obtained from the plot of 1/V versus l[ATP] (Fig. 3). As seen in the inset to Fig, 3, the secondary plots do result in straight
FIG. 4. The reciprocal effects of glutamine and aketoglutarate on PII. UMP-supported deadenylylation. The rate of deadenylylation (V) was measured as described in Materials and Methods. The concentrations of a-ketoglutarate and glutamine were varied in the presence of constant levels of all other reactants.
608
ENGLEMAN Po
Concentr.mon
(units)
Effecters of Uridylyl
A
15 Glutamine
Concentration
AND FRANCIS
bnM)
Transferase
Utase requires ATP, UTP, Mg?, and (YKG. The ATP requirement varies inversely with the level of a-KG and, as depicted in Fig. 6, the synergism between ATP and (YKG in the UTase reaction is analogous to the synergistic effect of ATP and o-KG on the deadenylylation reaction catalyzed by ATase. A similar reaction scheme is, therefore, proposed for the uridylylation reaction in which ATP (“T”) and a-KG (“K”) bind randomly to the UTase enzyme (“E”) and the binding of one effector enhances the binding of the other by a factor (a). As predicted by the rate equation derived from this mechanism, the two secondary plots result in straight lines (Fig. 6, inset). The calculated dissociation constants for a-KG and ATP are 4 and 0.07 mu, respectively, with an enhancement factor (a) of 156. Inhibition of UTase activity by gluta-
FIG. 5. Synergistic effects of glutamine and PI1 in inhibiting PII. UMP-supported deadenylylation. Deadenylylation activity was determined by following the removal of [14C]AMP from glutamine synthetase (see Materials and Methods). A relative activity of 1.0 represents base-line deadenylylation activity in the absence of PI1 and glutamine. These latter effecters were added alone or mixed in the amounts indicated. TABLE
III
EFFECTS OF VARIOUS METABOLITES ON DEADENYLYLATION OF GLUTAMINE SYNTHETASE
Effector
Effect ;;t-leledenyly-
L-Glutamine - (lo+ M)* L-Methionine - (lo-’ M) - (5 X lo-3 M) L-Tryptophan a-Ketoglutarate + (required) - (lo-’ M) Fructose 1,6diphosphate - (5 X lo-3 M) 3-Phosphoglycerate - (5 X lo-3 M) Coenzyme A a Deadenylylation was assayed as described in Materials and Methods by following the release of [Y!]AMP from [‘4C]AMP-glutamine synthetase. * Concentration given is that which results in approximately a 50% change in the activity, either inhibition (-) or enhancement (+).
10 mu MC. At 1.5 mu concentrations, Me, Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Ni2’, and Zn2+ had little or no effect on the deadenylylation reaction.
I
50
2w
loo l/(ATP]
x lL+.
I
303
I
403
I
Il.4 ‘,
FIG. 6. Synergistic effects of ATP and a-ketoglutarate on the uridylyl transferase reaction. The rate of uridylylation (V) was measured by coupling the uridylylation reaction to the alternate PII’UMP assay (described in Materials and Methods) which utilizes a calorimetric y-glutamyl transferase assay at pH 8.0. The concentrations of ATP and a-ketoglutarate were varied in the presence of constant levels of uridylyltransferase and PII. Points for the two secondary plots (see inset) were obtained directly from the primary double reciprocal plots. These form straight lines as predicted by a hypothetical reaction scheme (see text).
METABOLITE
REGULATION
OF
GLUTAMINE
SYNTHETASE
IN Escherichia
coli
609
mine is demonstrated in Fig. 7. When the levels of glutamine and o-KG are varied, the inhibition pattern is noncompetitive, suggesting that these two effecters bind at separate sites on UTase. Other substances known to effect glutamine synthetase and ATase had no effect on UTase. These included all of the amino acids, metabolites, cofactors, and nucleotides tested on the UR enzyme and listed in Table IV. Effecters of Mn”-dependent Removing Enzyme
Uridylyl
The UR enzyme is active in the presence of Mn” alone, but the activity is enhanced by glutamine (Fig. 8). Glutamine (5 m14 has no demonstrable effect on UR activity at the enzyme’s optimum pH of 8.6; however, at pH 7.6, there was significant stimulation of UR activity by 0.5 mu glutamine.4 In contrast, up to 5 mu (U-KG had no effect on the UR activity at either pH 7.6 or 8.6. The conversion of PII. UMP to PI1 is, therefore, the only step in the glutamine synthetase cascade that is not affected by a-KG. As summarized in Table IV, several cofactors and nucleotides (most of which had no effect on the other cascade steps) were potent inhibitors of UR activity. CMP, UMP, TMP, and coenzyme A were the most potent inhibitors with significant effects at 10m6 M at pH 7.6. Acetyl-CoA and other cofactors and nucleotides were less inhibitory. As was found for the stimulation of UR activity by glutamine, all of the inhibitors of UR activity were much more effective at pH 7.6 than at pH 8.6 (Table IV) and the presence of glutamine did not alter the potency of these inhibitors (data not shown). High concentrations of ATase inhibit both UR and UTase activities in a concentration-dependent manner (Table V). This 4 Glutamine failed to stimulate the deuridylylation reaction in a recent study in which a different enzyme preparation and a different UR assay were used (Dr. Charles Huang, National Heart and Lung Institute, personalcommunication).The explanation for the disparity between this result and our own is not yet hOWll.
FIG. 7. Inhibition of UTase activity by glutamine. The rate of uridylylation (V) was measured by coupling the uridylylation reaction to the alternate PII. UMP assay described in Materials and Methods. The concentrations of o-ketoglutarate and glutamine were varied in the presence of constant levels of all other reactants.
inhibition is presumably due to competition for the binding of PI1 and PIT. UMP. DISCUSSION
Our current knowledge of the complex system that regulates glutamine synthetase activity in E. coli is summarized in Fig. 9, A and B, which depict the six cascade reactions. As shown in Fig. 9A, inactivation of glutamine synthetase activity is initiated by the action of the UR enzyme which catalyzes the conversion (deuridylylation) of PII. UMP to PII. The latter, presumably by direct action, stimulates the capacity of ATase to catalyze the adenylylation of glutamine synthetase, thus converting it from a Mp-dependent form with a pH optimum of 8.0 to the less active Mn2’-dependent form having a pH optimum of 6.9. Figure 9B depicts the cascade leading to the activation of glutamine synthetase. This cascade is initiated by the action of UTase, which catalyzes the uridylylation of PII, converting it to the modified form
610
ENGLEMAN
AND
FRANCIS
TABLE IV REGULATIONOFTHEMANCANESE-SUPPORTEDURIDYLYLREMOVINGENZYME" Effector
pH 8.6
Metabolites r.-Glutamine cY-Ketoglutarate Fructose 1,6diphosphate, phosphoenolpyruvate, phoglycerate Cofactors CoA AC-CoA DPN, DPNH TPN, TPNH Nucleotides CMP, LJMP, TMP, deoxy-CMP, deoxy-UMP AMP, IMP, GMP, CDP, TDP, UDP ADP, GDP, IDP, ATP, CTP, GTP, ITP, TTP, UDPG
3-phos-
UTP,
a Concentrations given results in 50% effect. concentrations tested with no effect (0).
I
I
0.5
1
1
15
2
either
I
25
3
Glutamine ImMl
FIG. 8. Glutamine enhancement of uridylyl removing activity was assayed as described in Materials and Methods. TABLE
V
INHIBITION OF URIDYLYLREMOVINGAND TRANSFERASEACTIVITIESBYADENYLYL TRANSFERASE" ATase
(units)
0 3 7.5 15 22
URIDYLYL
% Inhibition UR
Utase
0 5 33 40 -
0 10 37 48 75
n UTase and UR activities were measured as described in Materials and Methods. ATase was dialyzed in 2-methyl imidazole, EDTA, dithiothreitol buffer prior to addition into the assay.
CDPG, inhibition
pH 7.6
0 (10-L) 0 (5 X lo-$M) - (lo-3M)
+ (5 X lo-4M) 0 (5 X 10-3M) - (10-4M)
-
-
(lo+M) (10-3M) (10-3M) (10-4M)
- (lo-5M) - (10-4M) - (lo-’ M) (-)
or enhancement
(lo-6M) (l@M) (10-4M) (10-5M)
- (10-6M) - (10-5M) - (lo-*M) (+),
or are the highest
PII. UMP, whose interaction with ATase stimulates the deadenylylation of glutamine synthetase, and thereby converts it back to the more active Mg2+-dependent form. The fine modulation of the uridylylation and adenylylation systems by the various effecters listed in Tables II, III, and IV was the subject of the current study and is discussed below. Each cascade step has both unique and common effecters. The initial steps (uridylylation of PI1 and deuridylylation of PII. UMP) are affected by lower levels of their metabolite effecters than the later steps. Several end products of glutamine metabolism are capable of inhibiting glutamine synthetase activity directly. Individually, they are rather poor inhibitors but, collectively, their inhibition is greatly enhanced by their cumulative feedback effects (14-16). At relatively high concentrations, several of these same metabolites also inhibit the deadenylylation reaction and enhance the adenylylation reaction; however, they have little or no effect on the UTase and UR activities. Thus, these compounds probably do not exert a major influence on the state of adenylylation of glutamine synthetase in uivo. E. coli glutamine synthetase activity varies with the ratio of glutamine to cllKG whether studied under steady state conditions, in vitro (6), or in continuous culture
METABOLITE
REGULATION
OF
GLUTAMINE
SYNTHETASE
IN Escherichia
coli
611
/iDP
UTase
FIG. 9. Cascades involved (adenylylation) of glutamine
in the regulation of glutamine synthetase synthetase; B, activation (deadenylylation)
in uiuo (5). Although these metabolites have no direct effects on glutamine synthetase activity, they affect almost every step in the cascade in a reciprocal manner. Moreover, marked effects are observed at concentrations of low4 and10F5M. These effects are amplified not only by their reciprocal nature, but also by their actions on consecutive steps in the cascade and by their synergism with other effecters. It is not surprising, therefore, that the ratio of glutamine to a-KG plays such a dominant role in determining the level of GS activity in E. coli. The mixed pattern of inhibition observed when a-KG and glutamine are varied simultaneously in either the adenylylation or deadenylylation reaction suggests that in the presence of the regulatory protein, glutamine and a-KG bind to separate sites on ATase (17). However, other studies with ATase alone demonstrated classical competitive inhibition between (YKG and glutamine in the absence of PI1 (18, 19). The presence of the regulatory protein in the assay may alter the interaction of these metabolites with ATase, or
activity: A, inactivation of glutamine synthetase.
the metabolites could interact directly with the regulatory protein to produce these effects. In the uridylylation reaction, the pattern of inhibition by glutamine is purely noncompetitive with respect to a-KG (17) (see Fig. 7), indicating that UTase also has separate binding sites for glutamine and o-KG. In contrast, the Mn2+-dependent UR activity (which might exist as a complex with UTase) is enhanced by glutamine, but neither in the absence or presence of glutamine is its activity effected by concentrations of a-ketoglutarate as high as 10 mu. UTase and ATase appear to have much in common. Each protein catalyzes a “forward” reaction involving covalent attachment in phosphodiester linkage of a nucleotide monophosphate to specific tyrosyl residues (8, 20). Moreover, both activities are subject to the reciprocal effects of glutamine and a-KG, and the kinetic evidence indicates that both proteins contain separate binding sites for these effecters. The two reactions directed toward activation of GS (uridylylation of PI1 and deadenylyla-
612
ENGLEMAN
tion of GS) require ATP and (U-KG, which act synergistically to promote both reactions. The ATase and UTase are similar in size, both are sensitive to sulfhydryl reagents, and both specifically bind the two forms of the regulatory protein, i.e., PI1 and PII.UMP. The ATase and UTase may be evolutionarily related in structure since their function, size, and regulation are similar in so many ways. The Mn2+-dependent UR activity is unique both in its insensitivity to a-KG and its potent inhibition by a variety of nucleotide monophosphates and cofactors, most of which have little or no effect on the other cascade enzymes. A physiologic role of CMP and UMP in the regulation of the GS cascade is supported by the fact that the concentrations of these effecters in E. coli extracts is sufficient to render the UR enzyme inactive (10). The failure until now to discover these or any other effecters of the UR activity is partly explained by their greater potency at pH 7.6 than pH 8.6 (optimum for the assay). For example, 0.5 mu glutamine enhances the deuridylylation reaction at pH 7.6, but up to 5 mu glutamine has no effect at pH 8.6. Inhibitors such as CMP, UMP, and coenzyme A are lo-fold more potent at pH 7.6 than pH 8.6 (see Table IV). PI1 and PII. UMP inhibit their reciprocal reactions, and these effects are synergistic with the effects of (w-KG and glutamine, respectively. The PII. UMP effect is more striking than the PI1 effect. This difference may explain the observation of Brown et al. (9) that in a mixture of PII. UMP and PII, stimulation of deadenylylation by PII. UMP appeared to be much greater than stimulation of adenylylation by PII. The inhibitory effects of high concentrations of ATase on the uridylylation and deuridylylation activities are not surprising since ATase, UTase, and UR must compete for the binding of the regulatory protein. This competition may play an important role in control of the glutamine synthetase cascade. The effects of metabolite control on the UTase and UR activities may be considerably altered if the substrate is a complex of the regulatory protein and ATase rather than a free molecule of the regulatory protein.
AND
FRANCIS ACKNOWLEDGMENTS
The authors wish to thank Dr. Earl Stadtman Dr. P. Boon Chock for invaluable suggestions discussions during the course of this investigation. would also like to thank Dr. Stuart Adler for his of the E. coli PII. UMP.
and and We gift
REFERENCES H. S., SHAPIRO, B. M., AND STADTMAN, 1. KINCDON, E. R. (1967) Proc. Nat. Acad. Sci. USA 58, 1703-1710. 2. KINGDON, H. S., SHAPIRO, B. M., AND STADTMAN, E. R. (1967) Proc. Nat. Acad. Sci. USA 58, 642-648. 3. MECKE, D., WULFF, K., LIESS, K., AND HOLZER, H. (1966) Biochem. Biophys. Res. Commun. 158,514-525. SHAPIRO, B. M., AND STADTMAN, E. R. (1968) J.
Biol. Chem. 243,3769-3771.
a. 9. 10. 11.
12.
13. 14.
SENIOR, P. J. (1975) J. Bacterial. 123, 408-418. SEGAL, A., BROWN, M. S., AND STADTMAN, E. R. (1972) Arch. Biochem. Biophys. 101, 319-327. STADTMAN, E. R. (1969) in The Role of Nucleotides for the Function and Conformation of Enzymes (KaIckar, H. M., KIenow, H., and Ottensen, M., eds.), pp. 111-137, Munksgaard, Copenhagen. ADLER, S. T., PURICH, D., AND STADTMAN, E. R. (1975) J. Biol. Chem. 18,6264-6272. BROWN, M. S., SEGAL, A., AND STADTMAN, E. R. (1971) Proc. Nat. Acad. Sci. USA 68,2949-2953. FRANCIS, S. F., AND ENGLEMAN, E. G. (1978) Arch. Biochem. Biophys. 191,590-601. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. LAYNE, E. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, NC., eds.), Vol. 3, pp. 450-451, Academic Press, New York. WOOLFOLK, C. A., AND STADTMAN, E. R. (1967) Arch. Biochem. Biophys. 118,736-755. KINGDON, H. S., AND STADTMAN, E. R. (1967) J.
Bacterial. 15. SEGAL,
94,949-957.
A., AND
STADTMAN,
E. R. (1972)
Arch.
Biochem. Biophys. 152,356-366. 16. WOOLFOLK,
C. A., AND STADTMAN,
E. R. (1964)
Biochem. Biophys. Res. Commun. 17, 313-319. 17. DIXON, M., AND WEBB, E. C. (1964) Enzymes, 2nd ed., pp. 315-359, Academic Press, New York. S. B., AND GINSBURG, A. (1971) Arch. 18. HENNIG, Biochem. Biophys. 144,611-627. H. (1969) in Proceedings of the Alfred 19. HOLZER, Benson Symposium I. (Kalckar H. M., Klenow, H., Munsch-Petersen, A., Ottesen, M., and Thaysen, J. H., eds.), pp. 94-110, Munksgaard, Copenhagen. 20. SHAPIRO, B. M., AND STADTMAN, E. R. (1968) J.
Biol. Chem. 248, 376-377.
AND BIOPHYSICS pp. 602-612, 1978
Cascade
Control of E. Co/i Glutamine
II. Metabolite EDGAR
Regulation
G. ENGLEMAN*’
of the Enzymes AND
Synthetase
in the Cascade
SHARRON
H. FRANCIS
Section on Enzymes, Laboratory of Biochemistry, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 Received
April
28,1978;
revised
July
31,1978
Enzymes and regulatory proteins involved in the cascade control of glutamine synthetase activity of Escherichia coli have been separated from one another and the effects of numerous metabolites on each step in the cascade have been determined. The adenylyl transferase (ATase) -catalyzed adenylylation of glutamine synthetase, which requires the presence of the unmodified form of the regulatory protein PI1 is enhanced by glutamine and is inhibited by either a-ketoglutarate (a-KG) or the uridylylated form (PII’ UMP) of the regulatory protein. PII’UMP and a-KG act synergistically to inhibit this activity. In contrast, the PII. UMP-dependent, ATase-catalyzed deadenylylation of glutamine synthetase requires a-KG and ATP and is inhibited by glutamine or PI1 and synergistically by glutamine plus PII. The capacity of uridylyl transferase (UTase) to catalyze the uridylylation of PI1 is dependent on the presence of o-KG and ATP and is inhibited by glutamine. The deuridylylation of PII’UMP by the uridylyl removing enzyme (UR) is enhanced by glutamine but is unaffected by a-KG. However, CMP, UMP, and CoA all inhibit activity at lo-@ M. High concentrations of ATase inhibit both UR and UTase activities, presumably by binding the regulatory protein. Of more than 50 substances that alter the activity of at least one enzyme in the cascade, only a-KG and glutamine affect the activity at every step. This accounts for the observation that glutamine synthetase activity in viva is very sensitive to the intracellular ratio of o-KG to glutamine.
uiuo to the intracellular ratio of a-ketoglutarate to glutamine (5). These metabolites, as well as various nucleotides, amino acids, and divalent cations also affect the steady state level of adenylylation in vitro under conditions that allow adenylylation and deadenylylation reactions to occur simultaneously (6). The effects of these ligands on individual steps in the cascade have not been rigorously studied, in part, due to the difficulty of separating cascade enzymes from one another. Thus, the present work represents an effort to identify the site(s) of metabolite control of the cascade system. To this end, the cascade enzymes and the two forms of the regulatory protein have been separated from one another, and each has been assayed in the presence of one or more effectors. The results indicate that although many metabolites affect the activity of at
One important mechanism for the regulation of glutamine synthetase activity in Escherichia coli is the covalent attachment and removal of AMP from a specific tyrosyl residue in each of the enzyme’s 12 identical subunits (l-4). Adenylylation of a subunit converts it to a form inactive in the presence of Mg2+ (1). The enzyme’s activity is thus controlled by the average number of adenylylated subunits per molecule which can vary from zero to 12. The cascade which controls the state of adenylylation of glutamine synthetase is under strict metabolite control, with particular sensitivity in ’ Present Address: Department of Pathology, Stanford University School of Medicine, Stanford, California 94305. * Present Address: Department of Physiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232. To whom all correspondence should be addressed. 602 0003-9861/78/1912-0602$02.00/O Copyright
0 1978 by Academic
All rights of reproduction
F’reea,
Inc.
in any form reserved.
METABOLITE
REGULATION
OF
GLUTAMINE
least one enzyme in the cascade, only the reciprocal effects of glutamine and CY-KG” are seen at every step. Moreover, PII, PII. UMP, and ATase all affect cascade steps in addition to the ones for which they are required, and in two instances, these effects are synergistic with the effects of (YKG or glut-amine. Finally, we report that the UR enzyme activity, which catalyzes the deuridylylation of PII and thereby initiates the first step in the adenylylation of GS, is very sensitive to a group of effecters including coenzyme A and certain nucleotide monophosphates which have little or no effect on the other cascade components but affect UR activity at concentrations as low
8s lo-’
M.
MATERIALS
AND
METHODS
Enzyme assays. GS was assayed by the y-glutamyl transferase assay (7). One unit of activity is equal to 1.0 ~01 of y-glutamyl hydroxamate formed per min at 37’C. The state of adenylylation was determined by the ratio of y-glutemyl transferase activities in the presence of Mn’+ alone and of Mn*+ + Me (7). Adenylylation of GS by ATase was assayed as previously described (8). A two-step calorimetric assay consisted of (a) adenylylation by ATase of unadenylylated GS followed by (b) an assay of the remaining unadenylylated GS, using the y-glutamyl transferase assay described above. A decrease in A.w ,,,,, compared to a control incubation without ATase indicates the presence of ATase activity in the fraction tested. One unit of activity is defined as that amount of ATase necessary to decrease the G&catalyzed formation of y-glutamyl hydroxamate in the presence of Mn” and Me by 1.0 ~01 (AM, 0.36) per min. To assess the metabolite effects on adenylylation, the assay contained fixed levels of ATase and GS, subsaturating concentrations of ATP and Mg*+, and no glutamine. A 40+1 reaction mix contained 50 mu O-methyl imidazale, pH 7.6, 15 units of ATase, 50 pg of unadenylylated GS, and either 2.5 mu ATP plus 25 mM Mg*+ (“high” ATP) or 0.25 mu ATP plus 2.5 mu Mg’+ (“low” ATP), and effector in the concentrations indicated. Aliquota were withdrawn after various periods of incubation and assayed for the G&catalyzed y-glutamyl transferase activity. PI1 activity was measured as previously described (8). A relative PI1 activity of one represents the amount of PII required to produce a rate of adenylyl3Abbreviations used: ATase, adenylyl transferase; a-KG, a-ketoglutarate; GS, glutamine synthetase; PIT, unmodified regulatory protein, PII. UMP, uridylylated regulatory protein, UR, uridylyl removing enzyme; UTase, uridylyl transferase.
SYNTHETASE
IN
Escherichia
coli
603
ation of GS of 0.6 adenylyl group/molecule/min under the conditions described. For purposes of assessing the effects of metabolites on PII-supported adenylylation, the assay was modified as follows. A 40 pl reaction mix contained 59 mM 2-methyl imidasole, pH 7.6,40 units of PI1 (a mixture of PI1 and PII’UMP which was at least 90% PII), 15 units of ATase, 50 pg of adenylylated GS, and either 2.5 mu ATP plus 25 mM Mg’+ or 0.25 rruu ATP plus 2.5 mM Me, and effector in the concentrations indicated. The rate of adenylylation of GS was followed by determining the decrease in yglutamyl transferase activity in the presence of Mn*’ + Me (7). Initial rates of adenylylation were not always measurable in the presence of added effecters, and such qualitative data are presented in table form. PII. UMP activity was measured as previously described (9). One unit of PII. UMP is the amount required to stimulate the removal of 1 pmol of [‘4C]AMP from [5’-W]AMP-GS per min under standard assay conditions. The effects of metabohtes on PII.UMP activity were assayed in either of two methods. 1) Effecters were added to a 109+l reaction mixture containing 50 mu %-methyl imidazole, pH 7.6, 160 pg [5’-W]AMP-GS, 27 units of ATase, 130 units of PII.UMP, 2.5 mM ATP, 20 mM Mg’+, 0.03 mM (Y-KG, and 20 mM potassium phosphate. After 10 min at 37”C, 0.15 ml of 6% HClO, was added and the mixtures were centrifuged. Following this, 0.15 ml of the supernatant was dissolved in 10 ml of Aquasol (New England Nuclear Corp.) and counted on a scintillation spectrophotometer. 2) PII’UMP was also measured by coupling the deadenylylation reaction to a colorimetric y-glutamyl transferase assay at pH 8.0 developed by Dr. S. Rhee (National Heart and Lung Institute, Bethesda, Md.) for measurement of unadenylylated glutamine synthetase. Effecters were incubated at 26°C with a 0.5~ml mixture containing 50 mM 2methyl imidazole, pH 7.6, 60 pg of adenylylated GS, 130 units of PII’ UMP, 15 units of ATase, 28.5 mM potassium phosphate, 15 mu Me, 1mM dithiothreitol, and various concentrations of ATP and (U-KG. After 10 min, a l-ml mixture (“transferase mix”) was added which contained 150 mM 2-methyl imidazole, 0.15 mM ADP, 30 mM sodium arsenate, 66 mM NHzOH’HC~, 225 mM glutamine, 150 mM KCl, and 0.6 mM Mn*+. After incubation for 5 min at 26”C, the reaction was stopped and the color developed by the addition of 0.5 ml of a solution containing equal volumes of 24% trichloracetic acid, 6 N HCl, and 10% FeCL.GH20 in 0.02 N HCl. Following vigorous mixing and centrifugation for 10 min at 10,06Og, the absorbance was read at 540 nm using a Gilford 300 N spectrophotometer. An increase in Amnm compared to a control incubation without PI1 UMP reflects the formation of unadenylylated GS due to the action of PII. UMP. UTase was measured as previously described (10) by incubating 75 units of PI1 and enzyme at 37°C in a
604
ENGLEMAN
50-d reaction mixture (“UTase reaction mixture”) containing 50 mM %-methyl imidazole, pH 7.6, 100 mM KCl, 10 mM MgClz, 0.1 mM ATP, 0.2 mu UTP, 5 mM a-Kg, and 1 mM dithiothreitol. After 5 min at 37’C, a 5-~1 aliquot was withdrawn and the increase in PII. UMP activity measured as described above. One unit of activity is defined as that amount of UTase necessary to increase the formation of PI1 UMP by 1 ymol per min under these conditions. To determine the effects of metabolites on UTase activity, two methods were used. 1) Effectors were incubated with 275 units of UTase in the above “UTase reaction mixture” at 37°C for 5 min, after which 5-~1 aliquots were removed and assayed for PII.UMP activity with the first of the two methods described above. 2) UTase was also measured by coupling the uridylylation reaction to the alternate PII’UMP assay (described above). In this method, effecters were incubated at 26“C in a 35-d mixture containing 50 mM 2-methyl imidazole, pH 7.6, 1 mM UTP, 10 mM MgCL, 33 mu KCl, 75 units PII, 150 units UTase, and various concentrations of ATP and (U-KG. After 5 min a 709~1 mixture was added which contained 75 mM 2-methyl imidazole, pH 7.2,30 mM a-KG, 1.5 mM dithiothreitol, 1.5 mM ATP 25 mM MgCl2, 45 mM potassium phosphate, 15 units ATase and 60 pg of adenylylated GS. After 5 min at 26’C, 1.0 ml of the pH 8.0 y-glutamyl “transferase mixture” (see above) was added and incubated at 26’C for measurement of unadenylylated GS. UR activity was measured as previously described (lo), except that the buffer was P-methyl imidazole, pH 7.6. To determine the effects of metabolites on UR activity, a 30-d reaction mixture containing 130 units of PII’UMP, 40 mM a-methyl imidazole, pH 7.6, 100 mM KCl, 1 mM MnClz, and an amount of UR enzyme which catalyzed 50% inactivation of the PII. UMP in the absence of effecters were incubated at 37°C with and without effecters. After 10 min, 70 ).d of deadenylylation reaction mixture were added and the residual PI1 UMP activity measured. Whenever metabolite effects were being determined, control assays were run with effector present but a key enzyme omitted. For example, in determining the effect of glutamine on UR activity, a control reaction mixture was assayed with glutamine added and UR enzyme omitted. In this way, the effect of glutamine on deadenylylation could be distinguished from effects on UR activity. Protein concentrations. Protein concentration was determined either by the method of Lowry (11) or, for protein solutions greater than 10 mg/ml, by the biuret method (12).
AND FRANCIS
supported adenylylation reaction in the presence of a “low” (0.25 mu) ATP concentration. In the absence of PI1 and glutamine, adenylylation activity is perceptible but very low. The addition of up to 1.0 mu glut-amine failed to enhance this “intrinsic” ATase activity, and the addition of PI1 results in a g-fold increase in activity. However, the addition of PI1 plus glutamine yields synergistic enhancement of adenylylation. In a manner analogous to the glutamine effect; a-KG has no effect on adenylylation in the absence of PII; however, in the presence of PII, adenylylation is inhibited by o-Kg in a dose-dependent manner. Figure 1 depicts the dose-dependent effects of o-KG and glutamine on PII-supported adenylylation. A mixed inhibition pattern for a-KG with respect to glutamine is seen. Whe both effecters are present, their molar ratio determined the rate of adenylylation. Table IB summarizes the effects of o-KG, glutamine, and PI1 on the adenylylation reaction in the presence of “high” ATP
I
2
4 l/(Gltmnine]
6 x 10"
6 IW'I
FIG. 1. The effects of a-ketoglutarate and glutamine on PII-supported adenylylation of glutamine synthetase. The adenylylation activity was assayed as described in Materials and Methods in the presence of varying concentrations of glutamine and a-ketoglutarRESULTS ate. The concentrations of ATP and Mg*+ were 0.25 and 2.5 mM, respectively. V represents the rate of Effecters of Adenylylation adenylylation as measured by the decrease in formaTable IA summarizes the effects of LY- tion of y-glutamyl hydroxamate expressed in micromoles per min. ketoglutarate and glutamine on the Me-
METABOLITE
REGULATION TABLE
OF
GLUTAMINE
SYNTHETASE
IA
TABLE
EFFECTSOF PII, GLUTAMINE,AND (YKETOGLUTARATEONADENYLYLATIONOF GLUTAMINE~YNTHETASEATLow ATP CONCENTRATIONS PI1 (units)
Gll;ty mM
a-Ketoglutarate bhf)
0 0 0 0 40 40 40 40 0 0 0 40 40 40 40 40 40 40
0 0.1 0.5 1.0 0 0.1 0.5 1.0 0 0 0 0 0 0 0.1 1.0 0.1 1.0
0 0 0 0 0 0 0 0 0.1 1.0 1.0 0.05 0.2 1.0 0.2 0.2 1.0 1.0
IN Escherichia
Rate of adenylylation SO.03 SO.03 SO.03 SO.03 0.27 0.54 0.97 1.19 SO.03 SO.03 SO.03 0.27 0.17 SO.03 0.22 0.69 0.06 0.27
a Assays were performed in the presence of 0.25 mM ATP and 2.5 mM Mg*+. The rate of adenylylation is expressed as the decrease in formation of y-glutamyl hydroxamate in micromoles per mm as determined in the y-glutamyl transferase assay (7).
concentration (2.5 mu). In contrast to the result at low ATP, there is significant ATase activity in the absence of PI1 and glutamine, and the addition of 1.0 mu glutamine enhances this activity more than 13-fold. At 2.5 mu ATP, the addition of 40 PI1 units to the assay mixture increases the adenylylation rate by g-fold from 0.17 to 1.11 ~mol/minute. The addition of 0.05 mu glutamine enhances the PII-supported activity by 2-fold, but in the absence of PII, 0.1 mM glutamine enhances the adenylylation by 2-fold. At high ATP concentrations the stimulation of adenylylation may be related only to the interaction of glutamine with ATase. At low ATP (0.25 mM), PI1 plus glutamine augmented activity synergistically but at higher ATP concentration (2.5 mM), glutamine and PI1 do not appear to be synergistic. At the higher ATP concentration, the LXKG inhibition of adenylylation remains dependent on the presence of PII. In the
605
coli
IB
EFFECTSOFPII,PII.UMP, GLUTAMINE,ANDW KETOGLUTARATEONADENYLYLATIONOF GLUTAMINE~YNTHETASEATHIGHATP CONCENTRATIONS PI1 (units)
Glutamine bM)
0 0 0 40 40 40 0 0 40 40 40 40 40 0 0 0 40 40 40 40 40 40
0 0.1 1.0 0 0.01 0.05 0 0 0 0 0 0 0.05 0 0 0 0 0 0 0 0.05 0.05
cr-Ketoglutarate (rnM) 0 0 0 0 0 0 0.1 1.0 0.01 0.03 0.05 0.1 0.05 0 0 0 0 0 0 0.05 0 0.05
PII. UM P (units) 0 0 0 0 0 0 0 0 0 0 0 0 0 130 325 650 130 325 650 325 325 325
Rate of adenylylation 0.17 0.36 2.22 1.11 1.11 2.16 0.17 0.17 1.11 0.67 0.42 0.19 0.83 0.17 0.11 so.03 1.0 0.61 0.14 SO.03 0.69 0.19
a Reaction mixtures were as in Materials and Methods, except for concentrations of ATP and a-ketoglutarate which were 2.5 and 25 mru, respectively.
absence of PII, 1.0 mu a-KG has no effect on the adenylylation rate (Table IB). However, in the presence of PII, 0.03 mu (Y-KG inhibits adenylylation by 40%. This concentration of (Y-KG is an order of magnitude lower than that required (0.2 mu) for a similar effect at low ATP. These results suggest, paradoxically, that synergism exists between both ATP and a-KG and between ATP and glutamine. Hennig and Ginsburg (18) have shown that in the absence of PI1 the glutamine promotion of adenylylation is inhibited by the presence of o-KG. The addition of PII. UMP results in significant inhibition of both intrinsic ATase activity and PI1 supported adenylylation (Table IB), and PII. UMP and (Y-KG synergistically inhibit the PII. ATase supported adenylylation. Figure 2 demonstrates the effects of PII, (U-KG, and the
606
ENGLEMAN
combination of PII. UMP and a-KG on adenylylation. Several metabolites and cofactors have been reported to affect either glutamine synthetase activity or ATase activity in the absence of the regulatory protein (1,13,14). The capacity of these and a number of other compounds to affect the ATase-catalyzed adenylylation in the presence and absence of PI1 was examined and the results are summarized in Table II. Only those substances affecting ATase activity had any effect on PII-supported adenylylation, and these effects were similar to
.021
mM o-KG 1M
300
effects of a-ketoglutarate, PII, and PII. UMP on the adenylylation reaction. The adenylylation activity was assayed as described in Materials and Methods in the presence of varying concentrations of a-ketoglutarate and PII’UMP. Glutamine was absent. TABLE
II
EFFECTSOFAMINOACIDS,GLYCOLYTICANDKREBS CYCLEINTERMEDIATESONADEN~YLATIONOF GLUTAMINE SYNTHETASE Effect
on adenylylation mine synthetase”
PI1 absent L-Glutamine L-Methionine L-Tryptophan a-Ketoglutarate Coenmne A
+ + + 0 -
FRANCIS
those observed on ATase alone, except in the case of glutamine and o-KG. Thus, Lmethionine and L-tryptophan enhanced both intrinsic and PII-supported adenylylation. Phosphoenolpyruvate, coenzyme A, and UTP (not shown) inhibited both activities. The concentration of these effecters required to demonstrate an effect was at least one order of magnitude greater than that needed for glutamine or a-KG. In contrast, compounds previously shown to influence glutamine synthetase activity directly (i.e., glycine, histidine, and alanine) at concentrations up to 10 mu had no demonstrable effect on the adenylylation reaction. Other ligands which had no effect on adenylylation at 10 rnkl concentrations included fructose 1,6-diphosphate, fructose lphosphate, 3-phosphoglycerate, and NAD. Ba2+, Ca2+, Cd2+, Co2+, Hg2+, Ni2+, and Zn2+ in concentrations up to 1 mu failed to support the ATase-catalyzed adenylylation reaction with or without PI1 present. Under the same assay conditions, Mn2+ supported both activities at approximately half of the velocity reached in the presence of Mg2+. In the presence of 2.5 mu ATP, the optimal Mn2+ concentration was 10 mu.
600 Units Pn.UMP
FIG. 2. The
Effector
AND
(lo-4 (lo-3 (lo-3 (l@ (lo-’
M)* hi)
M) M) M)
of gluta-
PI1 present + + + -
(lo-5 (lo-’ (@ (lo-5 (lo-’
I&
M)
Effecters of Deadenylylation
Catalysis of deadenylylation by ATase requires PII. UMP, inorganic phosphate, ATP, and a-KG (8). The requirement for a-KG and ATP in the presence of a constant concentration of PII. UMP are illustrated in Fig. 3. The concentration of ATP required for activity varies inversely with the level of a-KG and vice versa. With 1 mu o-KG, as little as 10m6 M ATP will support the reaction, compared to the 10V4 M ATP requirement for adenylylation. One possible mechanism to explain the synergism between ATP and a-KG in the deadenylylation reaction is diagrammed below:
M)
M)
M)
D Assays were performed as described in Materials and Methods in the presence of 2.5 mM ATP and 25 mM Mg*+. * Concentration given is that which results in approximately a 50% change in the activity, either inhibition (-) or enhancement (+).
AMP.GS&GS
+ AMP K2
METABOLITE
REGULATION
.3 I
/”
OF
GLUTAMINE
/Y Ir”
2 O-
l/o-KG l/V 1’o-
4 I$ 1
x
ki”
A
1 mM a-KG
10 mM a-KG
I 50 l/[ATP]
I
I
100 x lo-, M-’
150
FIG. 3. The role of ATP and a-ketoglutarate in the PI1 UMP supported deadenylylation of glutamine synthetase. The rate of deadenylylation (V) was measured as described in Materials and Methods. The concentrations of ATP and cY-ketoglutarate were varied in the presence of constant levels of PII. UMP and adenylyltransferase. Points for the two secondary plots (see inset) were obtained directly from the primary plots. These form straight lines as predicted by a hypothetical reaction scheme (see text).
It is proposed that ATP (“T”) and a-KG (“K”) can bind independently to the ATase enzyme (“E”) and that the binding of one effector enhances the binding of the other by a factor ((w). The rate expression derived from this mechanism by P. B. Chock (National Heart & Lung Institute, Bethesda, Md.) is:
SYNTHETASE
IN Escherichia
coli
607
lines as predicted and therefore, are consistent with the proposed reaction scheme. The reciprocal effects of glutamine and a-KG on the deadenylylation reaction are shown in Fig. 4. A mixed pattern of inhibition by glutamine with respect to a-KG is seen, analogous to the inhibition by a-KG of the glutamine-enhanced, PII-dependent adenylylation reaction. The concentration of glutamine required to inhibit deadenylylation, however, is at least lo-fold greater than the concentration of glutamine that causes enhancement of adenylylation. Figure 5 shows that glutamine is a potent inhibitor of deadenylylation, whereas PI1 inhibits deadenylylation only weakly. However, the combination of PI1 and glutamine results in synergistic inhibition in a manner analogous to the inhibition of PI1 supported adenylylation by PII. UMP plus a-KG. A summary of the effects of other compounds on deadenylylation is given in Table III. Many of the amino acids and metabolites tested are inhibitory, but none are as potent as glutamine. Alanine, glycine, and histidine at 10 mu concentrations have no effect on either adenylylation or deadenylylation, whereas L-tryptophan and Lmethionine promote adenylylation (Table II) and inhibit deadenylylation (Table III), and coenzyme A inhibited both reactions. No positive effecters were found. Various metals were tested for an effect in the deadenylylation reaction in the presence of 1 to
IO-
a Kl
-+a&
K2
[Kl
+(I+%)} from which one obtains the dissociation constants for (w-KG (0.1 mu) and ATP (0.01 mu) and the enhancement factor (Y = 20. The equation predicts that plots of the slope uersus l/[&-KG] and the Y intercept uersus l/[a-KG] will be straight lines. Both the slope and the Y intercept are directly obtained from the plot of 1/V versus l[ATP] (Fig. 3). As seen in the inset to Fig, 3, the secondary plots do result in straight
FIG. 4. The reciprocal effects of glutamine and aketoglutarate on PII. UMP-supported deadenylylation. The rate of deadenylylation (V) was measured as described in Materials and Methods. The concentrations of a-ketoglutarate and glutamine were varied in the presence of constant levels of all other reactants.
608
ENGLEMAN Po
Concentr.mon
(units)
Effecters of Uridylyl
A
15 Glutamine
Concentration
AND FRANCIS
bnM)
Transferase
Utase requires ATP, UTP, Mg?, and (YKG. The ATP requirement varies inversely with the level of a-KG and, as depicted in Fig. 6, the synergism between ATP and (YKG in the UTase reaction is analogous to the synergistic effect of ATP and o-KG on the deadenylylation reaction catalyzed by ATase. A similar reaction scheme is, therefore, proposed for the uridylylation reaction in which ATP (“T”) and a-KG (“K”) bind randomly to the UTase enzyme (“E”) and the binding of one effector enhances the binding of the other by a factor (a). As predicted by the rate equation derived from this mechanism, the two secondary plots result in straight lines (Fig. 6, inset). The calculated dissociation constants for a-KG and ATP are 4 and 0.07 mu, respectively, with an enhancement factor (a) of 156. Inhibition of UTase activity by gluta-
FIG. 5. Synergistic effects of glutamine and PI1 in inhibiting PII. UMP-supported deadenylylation. Deadenylylation activity was determined by following the removal of [14C]AMP from glutamine synthetase (see Materials and Methods). A relative activity of 1.0 represents base-line deadenylylation activity in the absence of PI1 and glutamine. These latter effecters were added alone or mixed in the amounts indicated. TABLE
III
EFFECTS OF VARIOUS METABOLITES ON DEADENYLYLATION OF GLUTAMINE SYNTHETASE
Effector
Effect ;;t-leledenyly-
L-Glutamine - (lo+ M)* L-Methionine - (lo-’ M) - (5 X lo-3 M) L-Tryptophan a-Ketoglutarate + (required) - (lo-’ M) Fructose 1,6diphosphate - (5 X lo-3 M) 3-Phosphoglycerate - (5 X lo-3 M) Coenzyme A a Deadenylylation was assayed as described in Materials and Methods by following the release of [Y!]AMP from [‘4C]AMP-glutamine synthetase. * Concentration given is that which results in approximately a 50% change in the activity, either inhibition (-) or enhancement (+).
10 mu MC. At 1.5 mu concentrations, Me, Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Ni2’, and Zn2+ had little or no effect on the deadenylylation reaction.
I
50
2w
loo l/(ATP]
x lL+.
I
303
I
403
I
Il.4 ‘,
FIG. 6. Synergistic effects of ATP and a-ketoglutarate on the uridylyl transferase reaction. The rate of uridylylation (V) was measured by coupling the uridylylation reaction to the alternate PII’UMP assay (described in Materials and Methods) which utilizes a calorimetric y-glutamyl transferase assay at pH 8.0. The concentrations of ATP and a-ketoglutarate were varied in the presence of constant levels of uridylyltransferase and PII. Points for the two secondary plots (see inset) were obtained directly from the primary double reciprocal plots. These form straight lines as predicted by a hypothetical reaction scheme (see text).
METABOLITE
REGULATION
OF
GLUTAMINE
SYNTHETASE
IN Escherichia
coli
609
mine is demonstrated in Fig. 7. When the levels of glutamine and o-KG are varied, the inhibition pattern is noncompetitive, suggesting that these two effecters bind at separate sites on UTase. Other substances known to effect glutamine synthetase and ATase had no effect on UTase. These included all of the amino acids, metabolites, cofactors, and nucleotides tested on the UR enzyme and listed in Table IV. Effecters of Mn”-dependent Removing Enzyme
Uridylyl
The UR enzyme is active in the presence of Mn” alone, but the activity is enhanced by glutamine (Fig. 8). Glutamine (5 m14 has no demonstrable effect on UR activity at the enzyme’s optimum pH of 8.6; however, at pH 7.6, there was significant stimulation of UR activity by 0.5 mu glutamine.4 In contrast, up to 5 mu (U-KG had no effect on the UR activity at either pH 7.6 or 8.6. The conversion of PII. UMP to PI1 is, therefore, the only step in the glutamine synthetase cascade that is not affected by a-KG. As summarized in Table IV, several cofactors and nucleotides (most of which had no effect on the other cascade steps) were potent inhibitors of UR activity. CMP, UMP, TMP, and coenzyme A were the most potent inhibitors with significant effects at 10m6 M at pH 7.6. Acetyl-CoA and other cofactors and nucleotides were less inhibitory. As was found for the stimulation of UR activity by glutamine, all of the inhibitors of UR activity were much more effective at pH 7.6 than at pH 8.6 (Table IV) and the presence of glutamine did not alter the potency of these inhibitors (data not shown). High concentrations of ATase inhibit both UR and UTase activities in a concentration-dependent manner (Table V). This 4 Glutamine failed to stimulate the deuridylylation reaction in a recent study in which a different enzyme preparation and a different UR assay were used (Dr. Charles Huang, National Heart and Lung Institute, personalcommunication).The explanation for the disparity between this result and our own is not yet hOWll.
FIG. 7. Inhibition of UTase activity by glutamine. The rate of uridylylation (V) was measured by coupling the uridylylation reaction to the alternate PII. UMP assay described in Materials and Methods. The concentrations of o-ketoglutarate and glutamine were varied in the presence of constant levels of all other reactants.
inhibition is presumably due to competition for the binding of PI1 and PIT. UMP. DISCUSSION
Our current knowledge of the complex system that regulates glutamine synthetase activity in E. coli is summarized in Fig. 9, A and B, which depict the six cascade reactions. As shown in Fig. 9A, inactivation of glutamine synthetase activity is initiated by the action of the UR enzyme which catalyzes the conversion (deuridylylation) of PII. UMP to PII. The latter, presumably by direct action, stimulates the capacity of ATase to catalyze the adenylylation of glutamine synthetase, thus converting it from a Mp-dependent form with a pH optimum of 8.0 to the less active Mn2’-dependent form having a pH optimum of 6.9. Figure 9B depicts the cascade leading to the activation of glutamine synthetase. This cascade is initiated by the action of UTase, which catalyzes the uridylylation of PII, converting it to the modified form
610
ENGLEMAN
AND
FRANCIS
TABLE IV REGULATIONOFTHEMANCANESE-SUPPORTEDURIDYLYLREMOVINGENZYME" Effector
pH 8.6
Metabolites r.-Glutamine cY-Ketoglutarate Fructose 1,6diphosphate, phosphoenolpyruvate, phoglycerate Cofactors CoA AC-CoA DPN, DPNH TPN, TPNH Nucleotides CMP, LJMP, TMP, deoxy-CMP, deoxy-UMP AMP, IMP, GMP, CDP, TDP, UDP ADP, GDP, IDP, ATP, CTP, GTP, ITP, TTP, UDPG
3-phos-
UTP,
a Concentrations given results in 50% effect. concentrations tested with no effect (0).
I
I
0.5
1
1
15
2
either
I
25
3
Glutamine ImMl
FIG. 8. Glutamine enhancement of uridylyl removing activity was assayed as described in Materials and Methods. TABLE
V
INHIBITION OF URIDYLYLREMOVINGAND TRANSFERASEACTIVITIESBYADENYLYL TRANSFERASE" ATase
(units)
0 3 7.5 15 22
URIDYLYL
% Inhibition UR
Utase
0 5 33 40 -
0 10 37 48 75
n UTase and UR activities were measured as described in Materials and Methods. ATase was dialyzed in 2-methyl imidazole, EDTA, dithiothreitol buffer prior to addition into the assay.
CDPG, inhibition
pH 7.6
0 (10-L) 0 (5 X lo-$M) - (lo-3M)
+ (5 X lo-4M) 0 (5 X 10-3M) - (10-4M)
-
-
(lo+M) (10-3M) (10-3M) (10-4M)
- (lo-5M) - (10-4M) - (lo-’ M) (-)
or enhancement
(lo-6M) (l@M) (10-4M) (10-5M)
- (10-6M) - (10-5M) - (lo-*M) (+),
or are the highest
PII. UMP, whose interaction with ATase stimulates the deadenylylation of glutamine synthetase, and thereby converts it back to the more active Mg2+-dependent form. The fine modulation of the uridylylation and adenylylation systems by the various effecters listed in Tables II, III, and IV was the subject of the current study and is discussed below. Each cascade step has both unique and common effecters. The initial steps (uridylylation of PI1 and deuridylylation of PII. UMP) are affected by lower levels of their metabolite effecters than the later steps. Several end products of glutamine metabolism are capable of inhibiting glutamine synthetase activity directly. Individually, they are rather poor inhibitors but, collectively, their inhibition is greatly enhanced by their cumulative feedback effects (14-16). At relatively high concentrations, several of these same metabolites also inhibit the deadenylylation reaction and enhance the adenylylation reaction; however, they have little or no effect on the UTase and UR activities. Thus, these compounds probably do not exert a major influence on the state of adenylylation of glutamine synthetase in uivo. E. coli glutamine synthetase activity varies with the ratio of glutamine to cllKG whether studied under steady state conditions, in vitro (6), or in continuous culture
METABOLITE
REGULATION
OF
GLUTAMINE
SYNTHETASE
IN Escherichia
coli
611
/iDP
UTase
FIG. 9. Cascades involved (adenylylation) of glutamine
in the regulation of glutamine synthetase synthetase; B, activation (deadenylylation)
in uiuo (5). Although these metabolites have no direct effects on glutamine synthetase activity, they affect almost every step in the cascade in a reciprocal manner. Moreover, marked effects are observed at concentrations of low4 and10F5M. These effects are amplified not only by their reciprocal nature, but also by their actions on consecutive steps in the cascade and by their synergism with other effecters. It is not surprising, therefore, that the ratio of glutamine to a-KG plays such a dominant role in determining the level of GS activity in E. coli. The mixed pattern of inhibition observed when a-KG and glutamine are varied simultaneously in either the adenylylation or deadenylylation reaction suggests that in the presence of the regulatory protein, glutamine and a-KG bind to separate sites on ATase (17). However, other studies with ATase alone demonstrated classical competitive inhibition between (YKG and glutamine in the absence of PI1 (18, 19). The presence of the regulatory protein in the assay may alter the interaction of these metabolites with ATase, or
activity: A, inactivation of glutamine synthetase.
the metabolites could interact directly with the regulatory protein to produce these effects. In the uridylylation reaction, the pattern of inhibition by glutamine is purely noncompetitive with respect to a-KG (17) (see Fig. 7), indicating that UTase also has separate binding sites for glutamine and o-KG. In contrast, the Mn2+-dependent UR activity (which might exist as a complex with UTase) is enhanced by glutamine, but neither in the absence or presence of glutamine is its activity effected by concentrations of a-ketoglutarate as high as 10 mu. UTase and ATase appear to have much in common. Each protein catalyzes a “forward” reaction involving covalent attachment in phosphodiester linkage of a nucleotide monophosphate to specific tyrosyl residues (8, 20). Moreover, both activities are subject to the reciprocal effects of glutamine and a-KG, and the kinetic evidence indicates that both proteins contain separate binding sites for these effecters. The two reactions directed toward activation of GS (uridylylation of PI1 and deadenylyla-
612
ENGLEMAN
tion of GS) require ATP and (U-KG, which act synergistically to promote both reactions. The ATase and UTase are similar in size, both are sensitive to sulfhydryl reagents, and both specifically bind the two forms of the regulatory protein, i.e., PI1 and PII.UMP. The ATase and UTase may be evolutionarily related in structure since their function, size, and regulation are similar in so many ways. The Mn2+-dependent UR activity is unique both in its insensitivity to a-KG and its potent inhibition by a variety of nucleotide monophosphates and cofactors, most of which have little or no effect on the other cascade enzymes. A physiologic role of CMP and UMP in the regulation of the GS cascade is supported by the fact that the concentrations of these effecters in E. coli extracts is sufficient to render the UR enzyme inactive (10). The failure until now to discover these or any other effecters of the UR activity is partly explained by their greater potency at pH 7.6 than pH 8.6 (optimum for the assay). For example, 0.5 mu glutamine enhances the deuridylylation reaction at pH 7.6, but up to 5 mu glutamine has no effect at pH 8.6. Inhibitors such as CMP, UMP, and coenzyme A are lo-fold more potent at pH 7.6 than pH 8.6 (see Table IV). PI1 and PII. UMP inhibit their reciprocal reactions, and these effects are synergistic with the effects of (w-KG and glutamine, respectively. The PII. UMP effect is more striking than the PI1 effect. This difference may explain the observation of Brown et al. (9) that in a mixture of PII. UMP and PII, stimulation of deadenylylation by PII. UMP appeared to be much greater than stimulation of adenylylation by PII. The inhibitory effects of high concentrations of ATase on the uridylylation and deuridylylation activities are not surprising since ATase, UTase, and UR must compete for the binding of the regulatory protein. This competition may play an important role in control of the glutamine synthetase cascade. The effects of metabolite control on the UTase and UR activities may be considerably altered if the substrate is a complex of the regulatory protein and ATase rather than a free molecule of the regulatory protein.
AND
FRANCIS ACKNOWLEDGMENTS
The authors wish to thank Dr. Earl Stadtman Dr. P. Boon Chock for invaluable suggestions discussions during the course of this investigation. would also like to thank Dr. Stuart Adler for his of the E. coli PII. UMP.
and and We gift
REFERENCES H. S., SHAPIRO, B. M., AND STADTMAN, 1. KINCDON, E. R. (1967) Proc. Nat. Acad. Sci. USA 58, 1703-1710. 2. KINGDON, H. S., SHAPIRO, B. M., AND STADTMAN, E. R. (1967) Proc. Nat. Acad. Sci. USA 58, 642-648. 3. MECKE, D., WULFF, K., LIESS, K., AND HOLZER, H. (1966) Biochem. Biophys. Res. Commun. 158,514-525. SHAPIRO, B. M., AND STADTMAN, E. R. (1968) J.
Biol. Chem. 243,3769-3771.
a. 9. 10. 11.
12.
13. 14.
SENIOR, P. J. (1975) J. Bacterial. 123, 408-418. SEGAL, A., BROWN, M. S., AND STADTMAN, E. R. (1972) Arch. Biochem. Biophys. 101, 319-327. STADTMAN, E. R. (1969) in The Role of Nucleotides for the Function and Conformation of Enzymes (KaIckar, H. M., KIenow, H., and Ottensen, M., eds.), pp. 111-137, Munksgaard, Copenhagen. ADLER, S. T., PURICH, D., AND STADTMAN, E. R. (1975) J. Biol. Chem. 18,6264-6272. BROWN, M. S., SEGAL, A., AND STADTMAN, E. R. (1971) Proc. Nat. Acad. Sci. USA 68,2949-2953. FRANCIS, S. F., AND ENGLEMAN, E. G. (1978) Arch. Biochem. Biophys. 191,590-601. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. LAYNE, E. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, NC., eds.), Vol. 3, pp. 450-451, Academic Press, New York. WOOLFOLK, C. A., AND STADTMAN, E. R. (1967) Arch. Biochem. Biophys. 118,736-755. KINGDON, H. S., AND STADTMAN, E. R. (1967) J.
Bacterial. 15. SEGAL,
94,949-957.
A., AND
STADTMAN,
E. R. (1972)
Arch.
Biochem. Biophys. 152,356-366. 16. WOOLFOLK,
C. A., AND STADTMAN,
E. R. (1964)
Biochem. Biophys. Res. Commun. 17, 313-319. 17. DIXON, M., AND WEBB, E. C. (1964) Enzymes, 2nd ed., pp. 315-359, Academic Press, New York. S. B., AND GINSBURG, A. (1971) Arch. 18. HENNIG, Biochem. Biophys. 144,611-627. H. (1969) in Proceedings of the Alfred 19. HOLZER, Benson Symposium I. (Kalckar H. M., Klenow, H., Munsch-Petersen, A., Ottesen, M., and Thaysen, J. H., eds.), pp. 94-110, Munksgaard, Copenhagen. 20. SHAPIRO, B. M., AND STADTMAN, E. R. (1968) J.
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