ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 196, No. 2, September, pp. 588-597, 1979
Regulation
of the Activity of Mung Bean (Phaseolus aureus) Synthetase by Amino Acids and Nucleotides S. SEETHALAKSHMI
AND
Department of Biochemistry, Indian Institute
Glutamine
N. APPAJI RAO
of Science, Bangalore-
012, India
Received November 15, 1978; revised March 26, 1979 The activity of glutamine synthetase isolated from the germinated seedlings of Phaseolus aureus was regulated by feedback inhibition by alanine, glycine, histidine, AMP, and ADP. When glutamate was the varied substrate, alanine, histidine, and glycine were partial noncompetitive, competitive, and mixed-type inhibitors, respectively. The type of inhibition by these amino acids was confirmed by fractional inhibition analysis. The adenine nucleotides, AMP and ADP, completely inhibited the enzyme activity and were competitive with respect to ATP. Multiple inhibition analyses revealed the presence of separate and nonexclusive binding sites for the amino acids and mutually exclusive sites for adenine nucleotides. Cumulative inhibition was observed with these end products.
Glutamine synthetase (L-glutamate:ammonia ligase, EC 6.3.1.2) occupies a key position in a highly branched metabolic pathway and its activity is under rigorous cellular control. The mammalian and bacterial glutamine synthetases are subject to feedback inhibition by the end products of glutamine metabolism (1). It is known that glutamine serves diverse functions in different cells and hence a study of the regulatory mechanism of this enzyme isolated from different sources is of great significance. Although the enzyme from plant sources has been examined in some detail (2-6), the kinetic features of the regulation of this enzyme activity have not been thoroughly investigated. A study of the regulatory properties of this enzyme isolated from plant sources is of special interest in view of a large number of nitrogenous end products of the secondary metabolism in plants. We have recently reported the purification of glutamine synthetase from mung bean (Phaseohs aweus) seedlings and the alteration of the enzyme-antibody reaction on the addition of the substrates (7). The y-glutamyl transferase reaction catalyzed by this enzyme proceeds by a ping-pong mechanism 0003-9861/79/100588-10$02.00/O copyright 0 1979by Academic Press, Inc. All rights of reproduction in any form reserved.
(8). The present study describes the regulation of the enzyme activity by feedback inhibitors.
MATERIALS
AND METHODS
Reagents including ATP, ADP, AMP, sodium L-glutamate, hydroxylamine hydrochloride, and amino acids were purchased from Sigma Chemical Company, St. Louis, Missouri. All other reagents were of the analytical reagent grade. Mung bean seeds were purchased from the local market. Enzyme assay. Glutamine synthetase was purified to homogeneity and characterized as reported earlier (7, 9). Glutamine synthetase activity was determined by estimating y-glutamylhydroxamate formed in the reaction (10). The reaction mixture (1.0 ml) contained 80 mM imidazole hydrochloride buffer (pH 7.2), 10 mM MgCl*, 10 mM P-mercaptoethanol, 100 mM glutamate, 10mM NHZOH, 10mM ATP, and enzyme. After incubation at 37°C for 15 min, the reaction was stopped by the addition of 1.5 ml of ferric chloride reagent and the absorbance was measured at 535 nm in a Pye Unicam SP-500 spectrophotometer. Protein was estimated according to the method of Lowry et al. using bovine serum albumin as the standard (11). One unit of enzyme activity is defined as the amount of enzyme required to produce 1 pmol of y-glutamyl hydroxamate per minute at pH 7.2 and 37°C.
588
REGULATION
OF MUNG BEAN GLUTAMINE
SYNTHETASE
589
RESULTS
Inhibition of Mung Bean Glutamine Synthetase Activity by Amino Acids and Nucleotides
The inhibition of mung bean glutamine synthetase activity by alanine, glycine, and histidine is shown in Fig. 1. The plot of the percentage activity remaining against the inhibitor concentration was hyperbolic and the residual activity reached a finite value indicating partial inhibition. This was further contlrmed by the double-reciprocal plot of l/fractional inhibition versus l/[inhib-
0
I
I
20
40
INHIBITOR Ibl
0
0.08
0.16
0
0.01
(mM)
FIG. 2. Effect of AMP and ADP on mung bean glutamine synthetase activity. The enzyme assays were performed in the same manner as described in Fig. 1, except that the enzyme was preincubated with different concentrations of the adenine nueleotides indicated in the figure. Inset: Plot of l/i vs l/I where i denotes the fractional inhibition calculated as described in Fig. 1 and I denotes the concentration of AMP (0) or ADP (0).
0.02
100 z 5: F :: = E a 2
-0d
50
1 0
I
1
125
250
INHIBITOR
I
(mM)
FIG. 1. Inhibition of mung bean glutamine synthetase activity by amino acids. The reaction mixture contained 80 mM imidazole hydrochloride buffer (pH 7.21, 10 mM MgCl,, 10 mM b-mercaptoethanol, 100 mM glutamate, 10 mM ATP, 10 mM NH,OH, and enzyme which had been preincubated for 5 min at 37°C with different concentrations of the amino acids indicated in the figure. The velocity was expressed as pmol of y-glutamylhydroxamate formed per min per mg protein. The enzyme activity in the absence of the added amino acid was normalized to 100. (0) Glycine; (0) alanine; (A) histidine. Inset (a): Plot of l/i vs l/I where i represents the fractional inhibition calculated according to the equation i = (V, - VJV,, V, is the enzyme activity in the absence of the inhibitor, and Vi represents the activity in the presence of the inhibitor. I denotes the concentration of alanine (0) or glycine (0). Inset (b): Plot of l/i vs l/I where I denotes the concentration of histidine (A).
itor] (inset, Fig. 1). The ordinate intercept value of greater than unity is characteristic of partial inhibition (12). The effects of ADP and AMP on the enzyme activity are shown in Fig. 2. AMP and ADP inhibited the enzyme activity completely as evidenced by the ordinate intercept of unity in the double-reciprocal plot of l/fractional inhibition versus l/[inhibitor] (inset Fig. 2). Guanosine nucleotides were without effect (data not given). The effect of the inhibitors on the velocity of the reaction at saturating and subsaturating concentrations of the three substrates was studied in order to gain .an insight into the interaction of these ligands with the substrate binding site. As evident from Table I, at a subsaturating concentration of glutamate, the inhibition caused by glycine and histidine was larger than that at a saturating concentration suggesting that these ligands may be affecting the binding of glutamate. A comparison of the inhibitory effects by these amino acids at saturating and subsaturating levels of ATP and hydroxylamine revealed that the inhibition was not altered
590
SEETHALAKSHMI
AND RAO
TABLE I EFFECT OF SUBSTRATECONCENTRATIONON THE EXTENT OF THE INHIBITION OF MUNG BEAN GLLITAMINE SYNTHETASEACTIVITY BY AMINO ACIDS AND NUCLEOTIDES~ Fractional inhibition x 100 Sub&rating concentration Inhibitor (mM)
Saturating concentration of substrates
Glutamate
(2 mM)
ATP (0.8 mM)
NH,OH (0.6 mM)
Alanine (50) Glycine (50) Histidine (150) AMP (10) ADP (10)
40 50 25 30 25
43 66 78 45 39
35 48 20 68 43
42 47 26 30 30
a The enzyme was preincubated with the inhibitors for 5 min at 37°C and the reaction was started by the addition of substrates. In column 2, glutamate (100 mM), ATP (10 mM), NH,OH (10 mM) were used. When nonsaturating concentration of one substrate (indicated in the table) was used, the other two substrates were at the saturating levels.
GLYCINE(mM)
l/GLUTAMATE lb)
2
ImMl-’ (c I
ALANINE CmM)
HISTIDINE
by a variation in the concentrations of these substrates. This suggested that alanine, glycine, and histidine may not be interfering with the binding of ATP and hydroxylamine. ADP and AMP were more potent inhibitors at subsaturating ATP concentration than at saturating concentration implying that they were interacting at the ATP binding site. The extent of inhibition by these nucleotides was not altered by a change in the concentration of either glutamate or hydroxylamine. Kinetics
of Inhibition
The noncompetitive inhibition by alanine, mixed-type inhibition by glycine, and competitive inhibition by histidine are depicted in Fig. 3 which represents the Lineweaver0
1 l/GLUTAMATE
2 , mMr’
0 0.25 0 5 l/GLUTAMATE imMi'
FIG. 3. Kinetics of inhibition of mung bean glutamine synthetaae activity by amino acids. (a) Mixed-type inhibition by glycine. The reaction mixture contained 80 mM imidazole hydrochloride buffer (pH 7.2), 10 mM MgCl,, 10 mM pmercaptoethanol, 10 mM ATP, 10 mM NH,OH, and enzyme which had been preincubated for 5 min at 37”C, with the concentrations of glycine shown in the figure. Glutamate concentration was varied in the range l-50 mM at the two different concentrations of glycine indicated in the figure. Double-reciprocal plot is given in the figure.
(0) Minus glycine; (0) 30 mM glycine; (A) 50 mM glycine. (b) Noncompetitive inhibition by alanine. The enzyme assays were carried out in the same manner as described in Fig. 3a with glutamate concentration varied in the range 0.5-50 mM at the two indicated concentrations of alanine. (0) Minus alanine; (0) 30 mM alanine; (A) 50 mre alanine. (c) Competitive inhibition by histidine. The reaction mixtures were identical to those described in Fig. 3a, except that glutamate concentration was varied in the range l-50 mM at the indicated concentrations of histidine. (0) Minus histidine; (0) 150 mM histidine; (A) 1’75mhf histidine.
REGULATION
OF MUNG BEAN GLUTAMINE
0
2.5 l/ATP
5
0
I mM 1-l
591
SYNTHETASE
1.5
3
VATP
( mM I-’
FIG. 4. Competitive inhibition of mung bean glutamine synthetase activity by AMP and ADP. (a) Double-reciprocal plot of velocity vs ATP concentration. The reaction mixture contained 80 mMimidazole hydrochloride buffer (pH 7.2), 10 mEdp-mercaptoethanol, 10 mMATP, 10 mMNHzOH and enzyme preincubated with the concentrations of AMP indicated in the figure. ATP concentration was varied from 0.4-5 mM at different fixed concentrations of AMP. (0) Minus AMP; (0) 7 mM AMP; (A) 10 mM AMP. (b) Double-reciprocal plot of the velocity of mung bean glutamine synthetase reaction when ATP concentration was varied in the presence of ADP. The reaction mixtures were similar to those described in Fig. 4a, except that ATP concentration was varied at different fixed concentrations of ADP indicated in the figure. (0) Minus ADP, (0) 10 mM ADP; (A) 20 mM ADP.
Burk plots when glutamate concentration was varied at different fixed concentrations of the inhibitors. The values of (Y = 2.4 and KHis = 100 mM were calculated from Fig. 3c using the expressions:
1 1 + [His]/aKHi, - K - GlUapp. = - &dl + VWKd and
1 + IHisllKm l.+ [His]/aKHi,
1 ’
1 *
TABLE II
CUMULATIVEINHIBITIONOFMUNGBEANGLUTAMINESYNTHETASE ACTIVITYBY EFFECTORS"
Inhibitors Ala Ala Gly Ala Ala Gly Gly His His
+ + + + + + + + +
Gly His His AMP ADP AMP ADP AMP ADP
Observed inhibition (8)
Calculated value (%) Cumulative
Additive
Antagonistic
62 53 59 67 71 74 67 59 61
64 52 57 65 67 69 71 60 62
79 61 69 82 85 89 92 71 74
43 36 43 46 49 46 49 46 49
a The enzyme was preincubated with the inhibitors for 5 min at 37°C. The reaction was started by the addition of saturating concentrations of the substrates. The velocity was determined by the estimation of y-glutamylhydroxamate formed in the reaction mixture. Inhibition in the presence of a single inhibitor was: Ala (50) 36%, Gly (50) 438, His (150) 25%, AMP (10) 462, ADP (20) 49%. The numbers in the parentheses indicate the concentrations of the inhibitors. From the extent of inhibition produced by each inhibitor, the following formulae were used to predict the percentage inhibition when both the inhibitors were present. Additive = Ii + X,; Cumulative = I, + (lOO-1,)X,/100; Antagonistic = less than Xi; where Xi and Ii represent the percentage inhibition produced by the inhibitors, X and I, respectively. Xi is greater than Ii.
SEETHALAKSHMI
AND RAO
Multiple Inhibition
TABLE III
Studies
KINETICCONSTANTSFORTHE INTERACTIONOF
In order to determine the mutual exclusivity or nonexclusivity of the interaction of these inhibitors, multiple inhibition analysis was carried out. Inhibitor Multiple inhibition analyses for the amino acids are shown in Figs. 5 and 6. A linear Ala 21 (NC) 1 0.53 ‘JY 22.5 (MT) 3.014 0.137 intersecting pattern of lines was obtained in the Dixon plot for glycine in the presence His 100 (C) 2.4 1 AMP 3 (Cl of different fixed concentrations of alanine ADP 8 (0 and histidine. A similar multiple inhibition analysis for the AMP-ADP pair gave a a Factor by which K, for glutamate is altered in the parallel set of lines in the Dixon plot presence of glycine and histidine. (Fig. 7). b Factor by which k, is altered in the presence of In order to determine whether the inhibialanine and glycine. tion by these modifiers was cumulative, c NC, noncompetitive; MT, mixed type; C, competiantagonistic, or additive, the enzyme active. tivity in the presence of two inhibitors was determined. From the extent of inhibition The values of p = 0.53 and K,,, (21 mM> produced by each inhibitor, calculations were calculated from Fig. 3b using the were made to predict the percentage inhibition when both the inhibitors were present equations: depending on whether the effects were additive, cumulative, or antagonistic (14). As shown in Table II, cumulative inhibition was observed when the amino acid inhibitor 1 + [AlalIKAla 1 + PIAla]/KALa * AMINOACIDSAND NUCLEOTIDESWITHTHE MUNG BEANGLUTAMINESYNTHETASE
1
The values of (Y = 3.014, /3 = 0.137, and K GlY = 22.5 mM were calculated from Fig. 3a using the following equations: 1 1 a-1 at intersection = - v va-p’
[ 1 1-P [ 1 I.
1 1 at intersection = K Glu K GIU a-p
-=1 V Gly
’
1 - Glyll&a,
[ 1 + P[Glyll&a,
Competitive inhibition by AMP and ADP is shown in Fig. 4 which depicts the Lineweaver-Burk plots when ATP concentration was varied at different fixed concentrations of the inhibitors. The values of Ki for AMP and ADP were calculated from the slope and x-axis intercepts of Fig. 4 (13). The Ki values for the amino acids and nucleotides, and the values of (Yand p are given in Table III.
ALANINE
0
25 50 GLYCINE ImM)
FIG. 5. Inhibition of mung bean glutamine synthetase activity by glycine in the presence of alanine. The enzyme assays were identical to those described in other figures. The enzyme was preincubated with alanine for 5 min at 37°C followed by a second preincubation with the different concentrations of glycine. The reaction was started by the addition of the saturating concentrations of the substrates. The velocity was determined by estimating y-glutamylhydroxamate in the reaction mixture. The figure represents the Dixon plot of l/v vs the concentration of glycine at different fixed concentrations of alanine. Inset: The reciprocal of the velocity computed assuming equilibria shown in Fig. 11 and the constants determined in the study are plotted against concentration of glycine. (0) Minus alanine; (0) 30 mM &nine; (A) 50 mMalanine.
REGULATION
OF MUNG BEAN GLUTAMINE
HISTIDINElmM)
50
0
GLYCINE
(mM1
FIG. 6. Multiple inhibition analysis for the inhibition by glycine in the presence of histidine. The enzyme was preincubated with the indicated concentrations of histidine for 5 min at 37”C, followed by a second preincubation with different concentrations of glycine. The reaction was started by the addition of saturating concentrations of the substrates and the velocity was determined by estimating y-glutamylhydroxamate formed in the reaction mixture. The figure represents the Dixon plot of l/v vs the concentration of glycine at different fixed concentrations of histidine. Inset: The reciprocal of the velocity calculated for nonexclusive binding shown in Fig. 12, using the constants given in Table III are plotted against the concentration of glycine. (0) Minus histidine; (0) 150 mM histidine; (A) 1’75mM histidine.
was present with another amino acid or a nucleotide.
593
SYNTHETASE
specific susceptibility to the different inhibitors. (c) The existence of a single enzyme possessing multiple catalytic sites that differ from each other in their ability to be affected by the inhibitors. In both these situations, the inhibitory effects are additive. (d) The presence of separate and specific site for each inhibitor on the enzyme. From among the possibilities listed above, (a), (b), and (c) could be ruled out in the case of mung bean glutamine synthetase for the following reasons: (i) a single protein band was observed on polyacrylamide gel electrophoresis of the enzyme suggesting the absence of isoenzymes (8); (ii) additive inhibition was not observed when a combination of inhibitors was used (Table II) suggesting the absence of isoenzymes and multiple catalytic sites on the enzyme; (iii) cumulative inhibition of enzyme activity was noticed in the presence of two inhibitors (Table II). Although the calculated values for cumulative inhibition agree with those observed, the method of assay is not sensitive enough to reach the unequivocal conclusion that a single nonspecific allosteric site is absent. The partial inhibition observed with mung bean glutamine synthetase could be fitted into the model of Stadtman et al. (20) and Segel (13).
DISCUSSION
The regulation of enzyme activity by partial inhibition by end products was reported for several enzymes (15-19). A number of hypotheses were proposed to explain partial inhibition (13, 15, 18). According to Woolfolk and Stadtman (15), the partial inhibitory effects could arise due to one of the following situations. (a) The existence of an enzyme with a single nonspecific allosteric site. Binding to this site by any one of the inhibitors caused a conformational change resulting in a catalytically less active form of the enzyme. In this case, the inhibition in the presence of saturating concentrations of several inhibitors is equal to that obtained in the presence of a single inhibitor. (b) The presence of a heterogeneous population of closely related isoenzymes that differ from each other only in their
0.1 ADP
t I 0
I 10
5 AMP
(mM)
(mM)
FIG. 7. Inhibition of mung bean glutamine synthetase activity by AMP in the presence of ADP. The figure depicts the Dixon plot of l/v vs the concentration of AMP at different fixed concentrations of ADP at saturating concentrations of glutamate (100 mM), ATP (10 mru), NH,OH (10 mM). (0) Minus ADP; (0) 10 mM ADP; (A) 20 mM ADP.
594
SEETHALAKSHMI
In the model of Stadtman et al. (ZO), each inhibitor possessed two distinct and mutually exclusive binding sites. When the inhibitor was bound to one of the sites, the “inhibitory site,” it induced a conformational change in the enzyme that either prevented the substrate from binding or decreased the catalytic efficiency even though the substrate was bound to the enzyme. When the inhibitor was bound to the “noninhibitory site,” binding to the “inhibitory site” was prevented without any effect on the catalytic efficiency and on the binding of substrate. The partial competitive inhibition by histidine could be explained by the following equilibria (Fig. 8). The partial noncompetitive and mixedtype inhibition by alanine and glycine could be explained by the following equilibria (Fig. 9). The inhibition cannot be overcome by high substrate concentration due to the formation of inactive E *I 1Glu complex. Partial noncompetitive inhibition by alanine might be due to the reduction in the concentration of the productive enzyme complexes, leading to a decrease in the value of V. The partial inhibition by glycine was due to the formation of Gly . E complex followed by Gly +E. Glu and products. Mixed-type inhibition could be due to the decreased affinity of glutamate for E *Gly complex and the reduction in the concentration of productive enzyme-substrate complexes.
AND RAO IE+GIu
=zt
II
I t E +Glu
IEGlu
-
E+P
I t E Glu -
E+P
II
\=
t I 1t EI + Glu e
t I It EI Glu
FIG. 9. Equilibria for partial noncompetitive and mixed-type inhibition of mung bean glutamine synthetase activity. I, alanine or glycine. I .E., inhibitor bound to the noninhibitory site. E. I., inhibitor bound to the inhibitory site. E, enzyme in the presence of saturating concentrations of ATP and hydroxylamine.
Although the data on partial inhibition could be adequately explained by these equilibria, an alternate explanation based on the model of Segel(13) could not be ruled out and is described below. The inhibitors bind to their single specific sites to yield enzyme-inhibitor and enzymesubstrate inhibitor complexes. The enzymesubstrate inhibitor complex could yield products with equal or less facility than the enzyme-substrate complex. The inhibitors exert their action either by increasing the K, or decreasing the V of the enzyme (Fig. 10). In the above equilibria, (Yrepresents the factor by which KGluchanged when I occupied the enzyme. /3 is the factor by which the rate constant of the uninhibited His. E + Glu e His,E,Glu--+E + P reaction was altered in the presence of I. Ki and aKi are the constants for the dissociation of E. I and E. Glu *I complexes, His His respectively. t t The partial competitive inhibition by histiE +Glu dine is explained by the value of cx = 2.4 and e E.GIu -E+P KHis = 100 mM. In this case pk, = k, as both E *Glu and E. Glu . His complexes are His equally efficient in producing the products. The partial noncompetitive inhibition by it alanine could be due to a decrease in the E. His rate constant by a factor, p = 0.53 and by FIG. 8. Equilibria for partial competitive inhibition the absence of a change in the K, value of mung bean glutamine synthetase activity by histidine. E ‘His, histidine bound to the inhibitory site. His. E, for glutamate at different concentrations histidine bound to the noninhibitory site. E, enzyme in of alanine (CX= 1). The partial mixed type the presence of saturating concentrations of ATP and of inhibition by glycine could occur due to a change in k, and K, for glutamate hydroxylamine.
REGULATION KGlu E
+
Glu
~
OF MUNG
BEAN
GLUTAMINE
SYNTHETASE
595
kP
L
E.Glu
-E+P
I QKi E.1
+Glu
e
E.I.GIu
pkp_E+P
a KGlu
FIG. 10. Equilibria for partial inhibition of the enzyme activity. E, enzyme in the presence of saturating concentrations of ATP and hydroxylamine. I, histidine, alanine, or glycine.
t
E+P
FIG. 12. Equilibria among enzyme forms in the by factors, /3 = 0.137 and Q = 3.014, presence of the competitive inhibitor, histidine (I), and respectively. the mixed-type inhibitor, glycine (X). S represents the The equilibria (Fig. 11) representing the substrate, glutamate, and E represents the enzyme in nonexclusivity of the binding sites for alanine the presence of saturating concentrations of ADP and and glycine can be used to calculate the con- hydroxylamine. Ki and K, are the constants for the centration of the enzyme-substrate and dissociation of EI and EX complexes respectively. enzyme-substrate-inhibitor complexes. a,K, and a,K, are the constants for the dissociation The velocity of the reaction in the presence of ES1 and ESX complexes respectively. a,, a2, and of alanine and glycine can be computed from aloZ are the factors by which KGlu changes when I, X, and XI occupy the enzyme, pl, p2, and p3 are the values of Ki, (Y,and /3 determined in this the constants by which the rate of the uninhibited study (Table III). A plot of the calculated reaction is altered in the presence of the inhibitors. velocity against the concentration of glycine is shown in inset to Fig. 5. The linear intersecting plot observed experimentally (Fig. 5) to support the assumption of nonexclusive and computed as described above seems binding of these inhibitors to mung bean glutamine synthetase. A similar analysis with histidine and glycine using equilibria (Fig. 12) drawn assuming nonexclusivity in the binding of the inhibitors and taking into consideration the type of inhibition by these compounds gave a linear Dixon plot (inset, Fig. 6). It is pertinent to point out that a nonlinear Dixon plot is predicted for nonexclusive
9I
K,
EtP
FIG. 11. Equilibria among enzyme species in the presence of the noncompetitive inhibitor, alinine (I), and the mixed type inhibitor, glycine (X). S represents the substrate, glutamate, and E represents the enzyme in the presence of saturating concentrations of ATP and hydroxylamine. a is the factor by which K,,, changes when X occupies the enzyme. K, and K, are the constants for the dissociation of EI and EX respectively. cxKx is the constant for the dissociation of ESX, and ESIX complexes. &, p2 and fi3 are the factors by which the rate of the uninhibited reaction is reduced in the presence of the inhibitors.
E. AMP
II
K AMP
AMP t E + ATP +
~E.ATP-E+P Keel
ADP
It
KADP
E. ADP
FIG. 13. Equilibria for the interaction of AMP and ADP with mung bean glutamine synthetase. E, enzyme in the presence of saturating concentration of glutamate and hydroxylamine.
596
SEETHALAKSHMI
partial inhibitors (13). The apparent discrepancy between the predicted pattern and the observed pattern may possibly be due to the use of inhibitors at concentrations close to their Ki values and the decreased rate constants for the dissociation of the E. S. I complexes. The mutual exclusivity of the binding for AMP and ADP could be explained by equilibria shown below (Fig. 13). The equation for Dixon plot when the concentration of AMP was varied at different fixed concentrations of ADP is given below: 1
-=
K ATP
2) [ATPIVK,,,
[AMP1
1
AND RAO
plant roots is regulated by the end products of glutamine metabolism. The nucleotides AMP, ADP, GTP, and IDP cause substantial inhibition of the enzyme activity (6). The homogeneous enzyme isolated from soybean root nodules is feedback regulated by GTP, ADP, AMP, histidine, and glycine (5). Our results as well as the observations discussed above indicate that the amino acids and nucleotides regulate the activity of glutamine synthetase from plant sources. The possible existence of separate binding sites on the mung bean glutamine synthetase for the effecters needs to be confirmed by differential inactivation of the sites combined with kinetic studies and by binding and isotope exchange studies.
*
The absence of [ADP] in the slope term of the above equation explained the parallel set of lines obtained when AMP concentration was varied in the presence of different fixed concentrations of ADP. Cumulative inhibition observed with alanine, glycine, and histidine lent some support to the contention that these amino acids possessed separate and nonexclusive binding sites. Cumulative inhibition was also observed when the amino acids interacted with the enzyme in the presence of AMP or ADP, indicating that the binding sites for these nucleotides were different from the amino acid binding sites. The observation that high concentrations of the effecters are required for partial inhibition is not unique to mung bean glutamine synthetase as Stadtman et al. (20) have reported that relatively high concentrations of partial inhibitors are needed to cause 50% inhibition of Escherichja coli glutamine synthetase activity. Pea leaf glutamine synthetase is significantly inhibited by ADP and AMP in the Mgzf-dependent biosynthetic assay. Among the amino acids, histidine and ornithine are the most inhibitory, but significant inhibition
ACKNOWLEDGMENTS The authors thank Professor C. S. Vaidyanathan for his advice and helpful suggestions during the course of this investigation. One of the authors (S. S.) thanks the Council of Scientific and Industrial Research for a Fellowship. REFERENCES 1. STADTMAN,E. R. (1973)in The Enzymes of Glutamine Metabolism (Prusiner, S. and Stadtman, E. R., eds.), Vol. 3, Academic Press, New York. 2. O’NEAL, D. T., AND JOY, K. W. (1973)Arch. Biothem. Biophys. 159, 113-122. 3. O’NEAL, D. T., AND JOY, K. W. (1975) Plant Physiol. 54, 968-974. 4. KINGDON, H. S. (1974) Arch. Biochem.
5.
6. 7. 8. 9.
is observed only in the presence of Mn2+.
Alanine, glycine, and serine also cause slight 10. inhibition of Mn2+-dependent enzyme activity. Cumulative inhibition of enzyme activity is observed in the presence of these amino acids (2, 3). The enzyme from rice
Biophys.
163, 429-431. MCPARLAND, R. H., GUEVARA, J. G., BECKER, R. R., AND EVANS, H. J. (1976) Biochem. J. 153, 597-606. KANAMORI,T., AND MATSUMOTO,H. (1972)Arch. Biochem. Biophys. 152, 464-412. SEETHALAKSHMI,S., AND APPAJI RAO, N. (1979) J. Biosci. 13-25. SEETHALAKSHMI,S., VAIDYANATHAN, C. S., AND APPAJI RAO, N. (1977) Ind. J. Biochem. Biophys. 14, 112-117. SEETHALAKSHMI,S. (1978) Studies on Glutamine Synthetase from Phaseolus aweus (Mung Bean) Seedlings: Purification, Kinetic and Regulatory Properties of the Enzyme, Ph.D. Thesis, Indian Institute of Science, Bangalore, India. ROWE, W. B., RONZIO,R. A., WELLNER, A. P., AND MEISTER, A. (1970) in Methods in Enzymology (Tabor, H., and Tabor, C. A., eds.), Vol. 17, Pt. A, pp. 990-910, Academic Press, New York.
REGULATION
OF MUNG
BEAN
11. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Eliol. Chem. 193, 265-275. 12. WEBB, J. L. (1964) in Enzymes and Metabolic Inhibitors, Vol. 1, p. 49, Academic Press, New York. 13. SEGEL, I. H. (1975) in Enzyme Kinetics, pp. 161, 465, Wiley, New York. 14. WEDLER, F. C., AND HOFFMAN, F. M. (1974) Biochemistry 13, 3215-3220. 15. WOOLFOLK, C. A., AND STADTMAN, E. R. (1967) Arch. Biochem. Biophys. 118, 736-755. 16. WANG, J. H., Tu, J. I., AND Lo, F. M. (1970) J. Biol. Chem. 245, 3115-3121.
GLUTAMINE
SYNTHETASE
597
17. GOLD, M. H., FARRAND, R. J., LIVONI, J. P., AND SEGEL, I. H. (1974) Arch. Biochem. Biophys. 161, 515-527. 18. RHEE, S. G., VILLAFRANCA, J. J., CHOCK, P. B., AND STADTMAN, E. R. (1977) Biockm. Biophys. Res. Commun. 78, 244-250. 19. RHEE, S. G., VILLAFRANCA, J. J., CHOCK, P. B., AND STADTMAN, E. R. (1978) in Frontiers of Biological Energetics (Dutton, P. L., Leigh, J., and Scarpa, A., eds.), pp. 725-733, Academic Press, New York. 20. STADTMAN, E. R., SHAPIRO, B. M., KINGDON, H. S., WOOLFOLK, C. A., AND HUBBARD, J. S. (1968) Advan. Enzyme Regul. 6, 257-289.
Regulation
of the Activity of Mung Bean (Phaseolus aureus) Synthetase by Amino Acids and Nucleotides S. SEETHALAKSHMI
AND
Department of Biochemistry, Indian Institute
Glutamine
N. APPAJI RAO
of Science, Bangalore-
012, India
Received November 15, 1978; revised March 26, 1979 The activity of glutamine synthetase isolated from the germinated seedlings of Phaseolus aureus was regulated by feedback inhibition by alanine, glycine, histidine, AMP, and ADP. When glutamate was the varied substrate, alanine, histidine, and glycine were partial noncompetitive, competitive, and mixed-type inhibitors, respectively. The type of inhibition by these amino acids was confirmed by fractional inhibition analysis. The adenine nucleotides, AMP and ADP, completely inhibited the enzyme activity and were competitive with respect to ATP. Multiple inhibition analyses revealed the presence of separate and nonexclusive binding sites for the amino acids and mutually exclusive sites for adenine nucleotides. Cumulative inhibition was observed with these end products.
Glutamine synthetase (L-glutamate:ammonia ligase, EC 6.3.1.2) occupies a key position in a highly branched metabolic pathway and its activity is under rigorous cellular control. The mammalian and bacterial glutamine synthetases are subject to feedback inhibition by the end products of glutamine metabolism (1). It is known that glutamine serves diverse functions in different cells and hence a study of the regulatory mechanism of this enzyme isolated from different sources is of great significance. Although the enzyme from plant sources has been examined in some detail (2-6), the kinetic features of the regulation of this enzyme activity have not been thoroughly investigated. A study of the regulatory properties of this enzyme isolated from plant sources is of special interest in view of a large number of nitrogenous end products of the secondary metabolism in plants. We have recently reported the purification of glutamine synthetase from mung bean (Phaseohs aweus) seedlings and the alteration of the enzyme-antibody reaction on the addition of the substrates (7). The y-glutamyl transferase reaction catalyzed by this enzyme proceeds by a ping-pong mechanism 0003-9861/79/100588-10$02.00/O copyright 0 1979by Academic Press, Inc. All rights of reproduction in any form reserved.
(8). The present study describes the regulation of the enzyme activity by feedback inhibitors.
MATERIALS
AND METHODS
Reagents including ATP, ADP, AMP, sodium L-glutamate, hydroxylamine hydrochloride, and amino acids were purchased from Sigma Chemical Company, St. Louis, Missouri. All other reagents were of the analytical reagent grade. Mung bean seeds were purchased from the local market. Enzyme assay. Glutamine synthetase was purified to homogeneity and characterized as reported earlier (7, 9). Glutamine synthetase activity was determined by estimating y-glutamylhydroxamate formed in the reaction (10). The reaction mixture (1.0 ml) contained 80 mM imidazole hydrochloride buffer (pH 7.2), 10 mM MgCl*, 10 mM P-mercaptoethanol, 100 mM glutamate, 10mM NHZOH, 10mM ATP, and enzyme. After incubation at 37°C for 15 min, the reaction was stopped by the addition of 1.5 ml of ferric chloride reagent and the absorbance was measured at 535 nm in a Pye Unicam SP-500 spectrophotometer. Protein was estimated according to the method of Lowry et al. using bovine serum albumin as the standard (11). One unit of enzyme activity is defined as the amount of enzyme required to produce 1 pmol of y-glutamyl hydroxamate per minute at pH 7.2 and 37°C.
588
REGULATION
OF MUNG BEAN GLUTAMINE
SYNTHETASE
589
RESULTS
Inhibition of Mung Bean Glutamine Synthetase Activity by Amino Acids and Nucleotides
The inhibition of mung bean glutamine synthetase activity by alanine, glycine, and histidine is shown in Fig. 1. The plot of the percentage activity remaining against the inhibitor concentration was hyperbolic and the residual activity reached a finite value indicating partial inhibition. This was further contlrmed by the double-reciprocal plot of l/fractional inhibition versus l/[inhib-
0
I
I
20
40
INHIBITOR Ibl
0
0.08
0.16
0
0.01
(mM)
FIG. 2. Effect of AMP and ADP on mung bean glutamine synthetase activity. The enzyme assays were performed in the same manner as described in Fig. 1, except that the enzyme was preincubated with different concentrations of the adenine nueleotides indicated in the figure. Inset: Plot of l/i vs l/I where i denotes the fractional inhibition calculated as described in Fig. 1 and I denotes the concentration of AMP (0) or ADP (0).
0.02
100 z 5: F :: = E a 2
-0d
50
1 0
I
1
125
250
INHIBITOR
I
(mM)
FIG. 1. Inhibition of mung bean glutamine synthetase activity by amino acids. The reaction mixture contained 80 mM imidazole hydrochloride buffer (pH 7.21, 10 mM MgCl,, 10 mM b-mercaptoethanol, 100 mM glutamate, 10 mM ATP, 10 mM NH,OH, and enzyme which had been preincubated for 5 min at 37°C with different concentrations of the amino acids indicated in the figure. The velocity was expressed as pmol of y-glutamylhydroxamate formed per min per mg protein. The enzyme activity in the absence of the added amino acid was normalized to 100. (0) Glycine; (0) alanine; (A) histidine. Inset (a): Plot of l/i vs l/I where i represents the fractional inhibition calculated according to the equation i = (V, - VJV,, V, is the enzyme activity in the absence of the inhibitor, and Vi represents the activity in the presence of the inhibitor. I denotes the concentration of alanine (0) or glycine (0). Inset (b): Plot of l/i vs l/I where I denotes the concentration of histidine (A).
itor] (inset, Fig. 1). The ordinate intercept value of greater than unity is characteristic of partial inhibition (12). The effects of ADP and AMP on the enzyme activity are shown in Fig. 2. AMP and ADP inhibited the enzyme activity completely as evidenced by the ordinate intercept of unity in the double-reciprocal plot of l/fractional inhibition versus l/[inhibitor] (inset Fig. 2). Guanosine nucleotides were without effect (data not given). The effect of the inhibitors on the velocity of the reaction at saturating and subsaturating concentrations of the three substrates was studied in order to gain .an insight into the interaction of these ligands with the substrate binding site. As evident from Table I, at a subsaturating concentration of glutamate, the inhibition caused by glycine and histidine was larger than that at a saturating concentration suggesting that these ligands may be affecting the binding of glutamate. A comparison of the inhibitory effects by these amino acids at saturating and subsaturating levels of ATP and hydroxylamine revealed that the inhibition was not altered
590
SEETHALAKSHMI
AND RAO
TABLE I EFFECT OF SUBSTRATECONCENTRATIONON THE EXTENT OF THE INHIBITION OF MUNG BEAN GLLITAMINE SYNTHETASEACTIVITY BY AMINO ACIDS AND NUCLEOTIDES~ Fractional inhibition x 100 Sub&rating concentration Inhibitor (mM)
Saturating concentration of substrates
Glutamate
(2 mM)
ATP (0.8 mM)
NH,OH (0.6 mM)
Alanine (50) Glycine (50) Histidine (150) AMP (10) ADP (10)
40 50 25 30 25
43 66 78 45 39
35 48 20 68 43
42 47 26 30 30
a The enzyme was preincubated with the inhibitors for 5 min at 37°C and the reaction was started by the addition of substrates. In column 2, glutamate (100 mM), ATP (10 mM), NH,OH (10 mM) were used. When nonsaturating concentration of one substrate (indicated in the table) was used, the other two substrates were at the saturating levels.
GLYCINE(mM)
l/GLUTAMATE lb)
2
ImMl-’ (c I
ALANINE CmM)
HISTIDINE
by a variation in the concentrations of these substrates. This suggested that alanine, glycine, and histidine may not be interfering with the binding of ATP and hydroxylamine. ADP and AMP were more potent inhibitors at subsaturating ATP concentration than at saturating concentration implying that they were interacting at the ATP binding site. The extent of inhibition by these nucleotides was not altered by a change in the concentration of either glutamate or hydroxylamine. Kinetics
of Inhibition
The noncompetitive inhibition by alanine, mixed-type inhibition by glycine, and competitive inhibition by histidine are depicted in Fig. 3 which represents the Lineweaver0
1 l/GLUTAMATE
2 , mMr’
0 0.25 0 5 l/GLUTAMATE imMi'
FIG. 3. Kinetics of inhibition of mung bean glutamine synthetaae activity by amino acids. (a) Mixed-type inhibition by glycine. The reaction mixture contained 80 mM imidazole hydrochloride buffer (pH 7.2), 10 mM MgCl,, 10 mM pmercaptoethanol, 10 mM ATP, 10 mM NH,OH, and enzyme which had been preincubated for 5 min at 37”C, with the concentrations of glycine shown in the figure. Glutamate concentration was varied in the range l-50 mM at the two different concentrations of glycine indicated in the figure. Double-reciprocal plot is given in the figure.
(0) Minus glycine; (0) 30 mM glycine; (A) 50 mM glycine. (b) Noncompetitive inhibition by alanine. The enzyme assays were carried out in the same manner as described in Fig. 3a with glutamate concentration varied in the range 0.5-50 mM at the two indicated concentrations of alanine. (0) Minus alanine; (0) 30 mM alanine; (A) 50 mre alanine. (c) Competitive inhibition by histidine. The reaction mixtures were identical to those described in Fig. 3a, except that glutamate concentration was varied in the range l-50 mM at the indicated concentrations of histidine. (0) Minus histidine; (0) 150 mM histidine; (A) 1’75mhf histidine.
REGULATION
OF MUNG BEAN GLUTAMINE
0
2.5 l/ATP
5
0
I mM 1-l
591
SYNTHETASE
1.5
3
VATP
( mM I-’
FIG. 4. Competitive inhibition of mung bean glutamine synthetase activity by AMP and ADP. (a) Double-reciprocal plot of velocity vs ATP concentration. The reaction mixture contained 80 mMimidazole hydrochloride buffer (pH 7.2), 10 mEdp-mercaptoethanol, 10 mMATP, 10 mMNHzOH and enzyme preincubated with the concentrations of AMP indicated in the figure. ATP concentration was varied from 0.4-5 mM at different fixed concentrations of AMP. (0) Minus AMP; (0) 7 mM AMP; (A) 10 mM AMP. (b) Double-reciprocal plot of the velocity of mung bean glutamine synthetase reaction when ATP concentration was varied in the presence of ADP. The reaction mixtures were similar to those described in Fig. 4a, except that ATP concentration was varied at different fixed concentrations of ADP indicated in the figure. (0) Minus ADP, (0) 10 mM ADP; (A) 20 mM ADP.
Burk plots when glutamate concentration was varied at different fixed concentrations of the inhibitors. The values of (Y = 2.4 and KHis = 100 mM were calculated from Fig. 3c using the expressions:
1 1 + [His]/aKHi, - K - GlUapp. = - &dl + VWKd and
1 + IHisllKm l.+ [His]/aKHi,
1 ’
1 *
TABLE II
CUMULATIVEINHIBITIONOFMUNGBEANGLUTAMINESYNTHETASE ACTIVITYBY EFFECTORS"
Inhibitors Ala Ala Gly Ala Ala Gly Gly His His
+ + + + + + + + +
Gly His His AMP ADP AMP ADP AMP ADP
Observed inhibition (8)
Calculated value (%) Cumulative
Additive
Antagonistic
62 53 59 67 71 74 67 59 61
64 52 57 65 67 69 71 60 62
79 61 69 82 85 89 92 71 74
43 36 43 46 49 46 49 46 49
a The enzyme was preincubated with the inhibitors for 5 min at 37°C. The reaction was started by the addition of saturating concentrations of the substrates. The velocity was determined by the estimation of y-glutamylhydroxamate formed in the reaction mixture. Inhibition in the presence of a single inhibitor was: Ala (50) 36%, Gly (50) 438, His (150) 25%, AMP (10) 462, ADP (20) 49%. The numbers in the parentheses indicate the concentrations of the inhibitors. From the extent of inhibition produced by each inhibitor, the following formulae were used to predict the percentage inhibition when both the inhibitors were present. Additive = Ii + X,; Cumulative = I, + (lOO-1,)X,/100; Antagonistic = less than Xi; where Xi and Ii represent the percentage inhibition produced by the inhibitors, X and I, respectively. Xi is greater than Ii.
SEETHALAKSHMI
AND RAO
Multiple Inhibition
TABLE III
Studies
KINETICCONSTANTSFORTHE INTERACTIONOF
In order to determine the mutual exclusivity or nonexclusivity of the interaction of these inhibitors, multiple inhibition analysis was carried out. Inhibitor Multiple inhibition analyses for the amino acids are shown in Figs. 5 and 6. A linear Ala 21 (NC) 1 0.53 ‘JY 22.5 (MT) 3.014 0.137 intersecting pattern of lines was obtained in the Dixon plot for glycine in the presence His 100 (C) 2.4 1 AMP 3 (Cl of different fixed concentrations of alanine ADP 8 (0 and histidine. A similar multiple inhibition analysis for the AMP-ADP pair gave a a Factor by which K, for glutamate is altered in the parallel set of lines in the Dixon plot presence of glycine and histidine. (Fig. 7). b Factor by which k, is altered in the presence of In order to determine whether the inhibialanine and glycine. tion by these modifiers was cumulative, c NC, noncompetitive; MT, mixed type; C, competiantagonistic, or additive, the enzyme active. tivity in the presence of two inhibitors was determined. From the extent of inhibition The values of p = 0.53 and K,,, (21 mM> produced by each inhibitor, calculations were calculated from Fig. 3b using the were made to predict the percentage inhibition when both the inhibitors were present equations: depending on whether the effects were additive, cumulative, or antagonistic (14). As shown in Table II, cumulative inhibition was observed when the amino acid inhibitor 1 + [AlalIKAla 1 + PIAla]/KALa * AMINOACIDSAND NUCLEOTIDESWITHTHE MUNG BEANGLUTAMINESYNTHETASE
1
The values of (Y = 3.014, /3 = 0.137, and K GlY = 22.5 mM were calculated from Fig. 3a using the following equations: 1 1 a-1 at intersection = - v va-p’
[ 1 1-P [ 1 I.
1 1 at intersection = K Glu K GIU a-p
-=1 V Gly
’
1 - Glyll&a,
[ 1 + P[Glyll&a,
Competitive inhibition by AMP and ADP is shown in Fig. 4 which depicts the Lineweaver-Burk plots when ATP concentration was varied at different fixed concentrations of the inhibitors. The values of Ki for AMP and ADP were calculated from the slope and x-axis intercepts of Fig. 4 (13). The Ki values for the amino acids and nucleotides, and the values of (Yand p are given in Table III.
ALANINE
0
25 50 GLYCINE ImM)
FIG. 5. Inhibition of mung bean glutamine synthetase activity by glycine in the presence of alanine. The enzyme assays were identical to those described in other figures. The enzyme was preincubated with alanine for 5 min at 37°C followed by a second preincubation with the different concentrations of glycine. The reaction was started by the addition of the saturating concentrations of the substrates. The velocity was determined by estimating y-glutamylhydroxamate in the reaction mixture. The figure represents the Dixon plot of l/v vs the concentration of glycine at different fixed concentrations of alanine. Inset: The reciprocal of the velocity computed assuming equilibria shown in Fig. 11 and the constants determined in the study are plotted against concentration of glycine. (0) Minus alanine; (0) 30 mM &nine; (A) 50 mMalanine.
REGULATION
OF MUNG BEAN GLUTAMINE
HISTIDINElmM)
50
0
GLYCINE
(mM1
FIG. 6. Multiple inhibition analysis for the inhibition by glycine in the presence of histidine. The enzyme was preincubated with the indicated concentrations of histidine for 5 min at 37”C, followed by a second preincubation with different concentrations of glycine. The reaction was started by the addition of saturating concentrations of the substrates and the velocity was determined by estimating y-glutamylhydroxamate formed in the reaction mixture. The figure represents the Dixon plot of l/v vs the concentration of glycine at different fixed concentrations of histidine. Inset: The reciprocal of the velocity calculated for nonexclusive binding shown in Fig. 12, using the constants given in Table III are plotted against the concentration of glycine. (0) Minus histidine; (0) 150 mM histidine; (A) 1’75mM histidine.
was present with another amino acid or a nucleotide.
593
SYNTHETASE
specific susceptibility to the different inhibitors. (c) The existence of a single enzyme possessing multiple catalytic sites that differ from each other in their ability to be affected by the inhibitors. In both these situations, the inhibitory effects are additive. (d) The presence of separate and specific site for each inhibitor on the enzyme. From among the possibilities listed above, (a), (b), and (c) could be ruled out in the case of mung bean glutamine synthetase for the following reasons: (i) a single protein band was observed on polyacrylamide gel electrophoresis of the enzyme suggesting the absence of isoenzymes (8); (ii) additive inhibition was not observed when a combination of inhibitors was used (Table II) suggesting the absence of isoenzymes and multiple catalytic sites on the enzyme; (iii) cumulative inhibition of enzyme activity was noticed in the presence of two inhibitors (Table II). Although the calculated values for cumulative inhibition agree with those observed, the method of assay is not sensitive enough to reach the unequivocal conclusion that a single nonspecific allosteric site is absent. The partial inhibition observed with mung bean glutamine synthetase could be fitted into the model of Stadtman et al. (20) and Segel (13).
DISCUSSION
The regulation of enzyme activity by partial inhibition by end products was reported for several enzymes (15-19). A number of hypotheses were proposed to explain partial inhibition (13, 15, 18). According to Woolfolk and Stadtman (15), the partial inhibitory effects could arise due to one of the following situations. (a) The existence of an enzyme with a single nonspecific allosteric site. Binding to this site by any one of the inhibitors caused a conformational change resulting in a catalytically less active form of the enzyme. In this case, the inhibition in the presence of saturating concentrations of several inhibitors is equal to that obtained in the presence of a single inhibitor. (b) The presence of a heterogeneous population of closely related isoenzymes that differ from each other only in their
0.1 ADP
t I 0
I 10
5 AMP
(mM)
(mM)
FIG. 7. Inhibition of mung bean glutamine synthetase activity by AMP in the presence of ADP. The figure depicts the Dixon plot of l/v vs the concentration of AMP at different fixed concentrations of ADP at saturating concentrations of glutamate (100 mM), ATP (10 mru), NH,OH (10 mM). (0) Minus ADP; (0) 10 mM ADP; (A) 20 mM ADP.
594
SEETHALAKSHMI
In the model of Stadtman et al. (ZO), each inhibitor possessed two distinct and mutually exclusive binding sites. When the inhibitor was bound to one of the sites, the “inhibitory site,” it induced a conformational change in the enzyme that either prevented the substrate from binding or decreased the catalytic efficiency even though the substrate was bound to the enzyme. When the inhibitor was bound to the “noninhibitory site,” binding to the “inhibitory site” was prevented without any effect on the catalytic efficiency and on the binding of substrate. The partial competitive inhibition by histidine could be explained by the following equilibria (Fig. 8). The partial noncompetitive and mixedtype inhibition by alanine and glycine could be explained by the following equilibria (Fig. 9). The inhibition cannot be overcome by high substrate concentration due to the formation of inactive E *I 1Glu complex. Partial noncompetitive inhibition by alanine might be due to the reduction in the concentration of the productive enzyme complexes, leading to a decrease in the value of V. The partial inhibition by glycine was due to the formation of Gly . E complex followed by Gly +E. Glu and products. Mixed-type inhibition could be due to the decreased affinity of glutamate for E *Gly complex and the reduction in the concentration of productive enzyme-substrate complexes.
AND RAO IE+GIu
=zt
II
I t E +Glu
IEGlu
-
E+P
I t E Glu -
E+P
II
\=
t I 1t EI + Glu e
t I It EI Glu
FIG. 9. Equilibria for partial noncompetitive and mixed-type inhibition of mung bean glutamine synthetase activity. I, alanine or glycine. I .E., inhibitor bound to the noninhibitory site. E. I., inhibitor bound to the inhibitory site. E, enzyme in the presence of saturating concentrations of ATP and hydroxylamine.
Although the data on partial inhibition could be adequately explained by these equilibria, an alternate explanation based on the model of Segel(13) could not be ruled out and is described below. The inhibitors bind to their single specific sites to yield enzyme-inhibitor and enzymesubstrate inhibitor complexes. The enzymesubstrate inhibitor complex could yield products with equal or less facility than the enzyme-substrate complex. The inhibitors exert their action either by increasing the K, or decreasing the V of the enzyme (Fig. 10). In the above equilibria, (Yrepresents the factor by which KGluchanged when I occupied the enzyme. /3 is the factor by which the rate constant of the uninhibited His. E + Glu e His,E,Glu--+E + P reaction was altered in the presence of I. Ki and aKi are the constants for the dissociation of E. I and E. Glu *I complexes, His His respectively. t t The partial competitive inhibition by histiE +Glu dine is explained by the value of cx = 2.4 and e E.GIu -E+P KHis = 100 mM. In this case pk, = k, as both E *Glu and E. Glu . His complexes are His equally efficient in producing the products. The partial noncompetitive inhibition by it alanine could be due to a decrease in the E. His rate constant by a factor, p = 0.53 and by FIG. 8. Equilibria for partial competitive inhibition the absence of a change in the K, value of mung bean glutamine synthetase activity by histidine. E ‘His, histidine bound to the inhibitory site. His. E, for glutamate at different concentrations histidine bound to the noninhibitory site. E, enzyme in of alanine (CX= 1). The partial mixed type the presence of saturating concentrations of ATP and of inhibition by glycine could occur due to a change in k, and K, for glutamate hydroxylamine.
REGULATION KGlu E
+
Glu
~
OF MUNG
BEAN
GLUTAMINE
SYNTHETASE
595
kP
L
E.Glu
-E+P
I QKi E.1
+Glu
e
E.I.GIu
pkp_E+P
a KGlu
FIG. 10. Equilibria for partial inhibition of the enzyme activity. E, enzyme in the presence of saturating concentrations of ATP and hydroxylamine. I, histidine, alanine, or glycine.
t
E+P
FIG. 12. Equilibria among enzyme forms in the by factors, /3 = 0.137 and Q = 3.014, presence of the competitive inhibitor, histidine (I), and respectively. the mixed-type inhibitor, glycine (X). S represents the The equilibria (Fig. 11) representing the substrate, glutamate, and E represents the enzyme in nonexclusivity of the binding sites for alanine the presence of saturating concentrations of ADP and and glycine can be used to calculate the con- hydroxylamine. Ki and K, are the constants for the centration of the enzyme-substrate and dissociation of EI and EX complexes respectively. enzyme-substrate-inhibitor complexes. a,K, and a,K, are the constants for the dissociation The velocity of the reaction in the presence of ES1 and ESX complexes respectively. a,, a2, and of alanine and glycine can be computed from aloZ are the factors by which KGlu changes when I, X, and XI occupy the enzyme, pl, p2, and p3 are the values of Ki, (Y,and /3 determined in this the constants by which the rate of the uninhibited study (Table III). A plot of the calculated reaction is altered in the presence of the inhibitors. velocity against the concentration of glycine is shown in inset to Fig. 5. The linear intersecting plot observed experimentally (Fig. 5) to support the assumption of nonexclusive and computed as described above seems binding of these inhibitors to mung bean glutamine synthetase. A similar analysis with histidine and glycine using equilibria (Fig. 12) drawn assuming nonexclusivity in the binding of the inhibitors and taking into consideration the type of inhibition by these compounds gave a linear Dixon plot (inset, Fig. 6). It is pertinent to point out that a nonlinear Dixon plot is predicted for nonexclusive
9I
K,
EtP
FIG. 11. Equilibria among enzyme species in the presence of the noncompetitive inhibitor, alinine (I), and the mixed type inhibitor, glycine (X). S represents the substrate, glutamate, and E represents the enzyme in the presence of saturating concentrations of ATP and hydroxylamine. a is the factor by which K,,, changes when X occupies the enzyme. K, and K, are the constants for the dissociation of EI and EX respectively. cxKx is the constant for the dissociation of ESX, and ESIX complexes. &, p2 and fi3 are the factors by which the rate of the uninhibited reaction is reduced in the presence of the inhibitors.
E. AMP
II
K AMP
AMP t E + ATP +
~E.ATP-E+P Keel
ADP
It
KADP
E. ADP
FIG. 13. Equilibria for the interaction of AMP and ADP with mung bean glutamine synthetase. E, enzyme in the presence of saturating concentration of glutamate and hydroxylamine.
596
SEETHALAKSHMI
partial inhibitors (13). The apparent discrepancy between the predicted pattern and the observed pattern may possibly be due to the use of inhibitors at concentrations close to their Ki values and the decreased rate constants for the dissociation of the E. S. I complexes. The mutual exclusivity of the binding for AMP and ADP could be explained by equilibria shown below (Fig. 13). The equation for Dixon plot when the concentration of AMP was varied at different fixed concentrations of ADP is given below: 1
-=
K ATP
2) [ATPIVK,,,
[AMP1
1
AND RAO
plant roots is regulated by the end products of glutamine metabolism. The nucleotides AMP, ADP, GTP, and IDP cause substantial inhibition of the enzyme activity (6). The homogeneous enzyme isolated from soybean root nodules is feedback regulated by GTP, ADP, AMP, histidine, and glycine (5). Our results as well as the observations discussed above indicate that the amino acids and nucleotides regulate the activity of glutamine synthetase from plant sources. The possible existence of separate binding sites on the mung bean glutamine synthetase for the effecters needs to be confirmed by differential inactivation of the sites combined with kinetic studies and by binding and isotope exchange studies.
*
The absence of [ADP] in the slope term of the above equation explained the parallel set of lines obtained when AMP concentration was varied in the presence of different fixed concentrations of ADP. Cumulative inhibition observed with alanine, glycine, and histidine lent some support to the contention that these amino acids possessed separate and nonexclusive binding sites. Cumulative inhibition was also observed when the amino acids interacted with the enzyme in the presence of AMP or ADP, indicating that the binding sites for these nucleotides were different from the amino acid binding sites. The observation that high concentrations of the effecters are required for partial inhibition is not unique to mung bean glutamine synthetase as Stadtman et al. (20) have reported that relatively high concentrations of partial inhibitors are needed to cause 50% inhibition of Escherichja coli glutamine synthetase activity. Pea leaf glutamine synthetase is significantly inhibited by ADP and AMP in the Mgzf-dependent biosynthetic assay. Among the amino acids, histidine and ornithine are the most inhibitory, but significant inhibition
ACKNOWLEDGMENTS The authors thank Professor C. S. Vaidyanathan for his advice and helpful suggestions during the course of this investigation. One of the authors (S. S.) thanks the Council of Scientific and Industrial Research for a Fellowship. REFERENCES 1. STADTMAN,E. R. (1973)in The Enzymes of Glutamine Metabolism (Prusiner, S. and Stadtman, E. R., eds.), Vol. 3, Academic Press, New York. 2. O’NEAL, D. T., AND JOY, K. W. (1973)Arch. Biothem. Biophys. 159, 113-122. 3. O’NEAL, D. T., AND JOY, K. W. (1975) Plant Physiol. 54, 968-974. 4. KINGDON, H. S. (1974) Arch. Biochem.
5.
6. 7. 8. 9.
is observed only in the presence of Mn2+.
Alanine, glycine, and serine also cause slight 10. inhibition of Mn2+-dependent enzyme activity. Cumulative inhibition of enzyme activity is observed in the presence of these amino acids (2, 3). The enzyme from rice
Biophys.
163, 429-431. MCPARLAND, R. H., GUEVARA, J. G., BECKER, R. R., AND EVANS, H. J. (1976) Biochem. J. 153, 597-606. KANAMORI,T., AND MATSUMOTO,H. (1972)Arch. Biochem. Biophys. 152, 464-412. SEETHALAKSHMI,S., AND APPAJI RAO, N. (1979) J. Biosci. 13-25. SEETHALAKSHMI,S., VAIDYANATHAN, C. S., AND APPAJI RAO, N. (1977) Ind. J. Biochem. Biophys. 14, 112-117. SEETHALAKSHMI,S. (1978) Studies on Glutamine Synthetase from Phaseolus aweus (Mung Bean) Seedlings: Purification, Kinetic and Regulatory Properties of the Enzyme, Ph.D. Thesis, Indian Institute of Science, Bangalore, India. ROWE, W. B., RONZIO,R. A., WELLNER, A. P., AND MEISTER, A. (1970) in Methods in Enzymology (Tabor, H., and Tabor, C. A., eds.), Vol. 17, Pt. A, pp. 990-910, Academic Press, New York.
REGULATION
OF MUNG
BEAN
11. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Eliol. Chem. 193, 265-275. 12. WEBB, J. L. (1964) in Enzymes and Metabolic Inhibitors, Vol. 1, p. 49, Academic Press, New York. 13. SEGEL, I. H. (1975) in Enzyme Kinetics, pp. 161, 465, Wiley, New York. 14. WEDLER, F. C., AND HOFFMAN, F. M. (1974) Biochemistry 13, 3215-3220. 15. WOOLFOLK, C. A., AND STADTMAN, E. R. (1967) Arch. Biochem. Biophys. 118, 736-755. 16. WANG, J. H., Tu, J. I., AND Lo, F. M. (1970) J. Biol. Chem. 245, 3115-3121.
GLUTAMINE
SYNTHETASE
597
17. GOLD, M. H., FARRAND, R. J., LIVONI, J. P., AND SEGEL, I. H. (1974) Arch. Biochem. Biophys. 161, 515-527. 18. RHEE, S. G., VILLAFRANCA, J. J., CHOCK, P. B., AND STADTMAN, E. R. (1977) Biockm. Biophys. Res. Commun. 78, 244-250. 19. RHEE, S. G., VILLAFRANCA, J. J., CHOCK, P. B., AND STADTMAN, E. R. (1978) in Frontiers of Biological Energetics (Dutton, P. L., Leigh, J., and Scarpa, A., eds.), pp. 725-733, Academic Press, New York. 20. STADTMAN, E. R., SHAPIRO, B. M., KINGDON, H. S., WOOLFOLK, C. A., AND HUBBARD, J. S. (1968) Advan. Enzyme Regul. 6, 257-289.
