Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase, ATPase reactions

BIOCHIMIE, I97,6, 58, 35-~9.

Mechanistic studies of glutamine synthetase from Escherichia colt. An integrated mechanism for biosynthesis, transferase, ATPase reactions (*). S. G. RHEE (**), P. B.

CHOCK,a n d E. R. STADTMAN ~.

Laboratory of B i o c h e m i s t r y , Section on E n z y m e s , National Heart and L u n g Institute, National Institutes o[ Health, Bethesda, Maryland 20014. Summary. - - The m e c h a n i s m of biosynthetic, transferase, ATPase, and t r a n s p h o s p h o r v l a t i o n reactions catalyzed by u n a d e n y l y l a t c d g l u t a m i n e s y n t h e t a s e f r o m E. colt was studied. Activation eomplex(es) involved in the b i o s y n t h e t i c r e a c t i o n are produced in the presence of e i t h e r Mg2÷ or Mn2+ ; however, w i t h the Mn2+-enzyme i n h i b i t i o n by the produet, ADP, is so great t h a t the overall f o r w a r d b i o s y n t h e t i c r e a c t i o n c a n n o t be detected w i t h the kno'wn assay methods. Binding studies show t h a t s u b s t r a t e s (except for NH3 a n d NHeOH which are not reported here) can b i n d to the enzyme in a r a n d o m m a n n e r a n d t h a t b i n d i n g of the ATP-glutamate, ADP-P~ or ADP-arsenate pairs is strongly synergistic. I n h i b i t i o n a n d b i n d i n g studies show t h a t the same b i n d i n g site is utilized for g l u t a m a t e a n d g]utamine in b i o s y n t h e t i c a n d t r a n s f c r a s e reactions, respectively, a n d t h a t a c o m m o n nucleotide b i n d i n g site is used for all reactions studied. Studies of the reverse biosynthetic reaction and results of fluorescent t i t r a t i o n e x p e r i m e n t s suggest t h a t b o t h a r s e n a t e a n d o r t h o p h o s p h a t e b i n d at a site w h i c h overlaps the ~-phosphate site of nueleoside t r i p h o s p h a t e . In the reverse b i o s y n t h e t i c a n d t r a n s f e r a s e reactions, ATP serves as a s u b s t r a t e for the Mn2+-enzyme b u t not f o r the Mg2+-enzyme. The ATP supported t r a n s f e r a s e activity of Mn2+-enzyme is p r o b a b l y facilitated by the generation of ADP t h r o u g h ATP hydrolysis. W h e n AMP was the only nneleotide s u b s t r a t e added, it was converted to ATP w i t h c o n c o m i t a n t f o r m a t i o n of two e q u i v a l e n t s of glutamate, u n d e r the reverse b i o s y n t h e t i c r e a c t i o n conditions, a n d no ADP was detected. The r e v e r s i b i l i t y of 180 t r a n s f e r between o r t h o p h o s p h a t e a n d ~-acyl group of g l u t a m a t e was confirmed. ATPase activity of Mg2÷ a n d Mn2+ u n a d e n y l y l a t e d enzymes is a b o u t the same. Both enzyme f o r m s catalyze t r a n s p h o s p h o r y ] a t i o n reactions between various p u r i n e nueleoside triphosphates a n d nueleoside d i p h o s p h a t e s u n d e r b i o s y n t h e t i c reaction conditions. The data are consistent w i t h the h y p o t h e s i s t h a t a single active center is utilized for all reactions studied. Two stepwise m e c a n i s m s t h a t could explain the results are discussed.

M e 2+

INTRODUCTIOn. G l u t a m i n e synt, h e t a s e is a k e y e n z y m e i n n i t r o g e n m e t a b o l i s m [1]. T h e e n z y m e f r o m E. colt is kno~ccn t o c a t a l y z e r e a c t i o n s (1-5) i n w h i c h t h e a b b r e v i a t i o n Me 2+, Pi, N1 a n d No r e p r e s e n t d i v a lent metal ions, orthophosphate, nucleoside 1 and nucleosi~de 2, r e s p e c t i v e l y . (See r e c e n t r e v i e w s b y Sta, d t m a n an,d G i n s b u r g [2J a n d M,eister [3]). Me 2÷ L-Glutamate + ATP + NH a ~ L - ~ l u t a m i n e + AD,P + Pi

(1)

]~[e2+ L-Glutamine ÷ NH2OH < ADP, Pi o r A r s e n a t e , t - g l u t a m y l h y d r o x a m a t e -4- NH3

(2)

(*) This p a p e r is P a r t II of a series on m e c h a n i s t i c studies of t h i s enzyme. (**) Recipient of a N a t i o n a l H e a r t a n d Lung Institute Postdoctoral Fello~vship. O To w h o m all correspondence should be addressed.

L - G l u t a m a t e -b A T P ~ p y r r o l i d o n e c a r b o x y l a t e A- AD'P + Pi

(3)

.Me2+ L-~lutamine + H20 ~" ADP, A r s e n a t e L - g l u t a m a t e + N.H 3

(4)

Me2+, Pi o r A r s e n a t e N I T P + N2DP ..(_ ~Glutamate, Glntamine N2TP + NIDP

(5)

The enzymic activities which catalyze the various r e a c t i o n s a r e r e f e r r e d to as f o l l o w s : r e a c t i o n (1), b i o s y n t h e t i c a c t i v i t y ; r e a c t i o n (2), t r a n s f e r a s e ; r e a c t i o n (3), A T P a s e ; r e a c t i o n (4), a r s e n a t e dep e n d e n t g l u t a m i n a s e ; a n d r e a c t i o n (5), t r a n s p h o s phorylase. The physiological significance of react i o n s (2), (3), (4), a n d (5) h a s n o t b e e n d e t e r m i n e d . To date, there have been numerous studies and s p e c u l a t i o n s o n t h e m e c h a n i s m of r e a c t i o n (1) i n

36

S. G. R h e e , P. B. C h o c k a n d E. R. S t a d t m a n .

E. colt [4-7] and in a n i m a l s [;3, 8], on the m e c h a n i s m of r e a c t i o n (2) in E. colt E4, 9] and in anim a l s ~3, 10], on the m e c h a n i s m s of r e a c t i o n (3) in E. colt [11], a n d on the m e c h a n i s m of r e a c t i o n (4) in E. colt [2J an~d in peas [12]. A tentative m e c h a n i s t i c s c h e m e w h i c h in,cludes r e a c t i o n s (1) to (4) has been p r o p o s e d for glutamine s y n t h e t a s e from m a m m a l s [3, 8]. H o w e v e r , t h e r e is c o n t r o versy as to w h e t h e r such a m e c h a n i s m can e x p l a i n the data o b t a i n e d w i t h the E. colt enzyme [5, 6, 7, 13, 14]. In a d d i t i o n , it w a s u n c e r t a i n w h e t h e r the same c a t a l y t i c site is i n v o l v e d for all these r e a c t i o n s since Mn 2+ is k n o w n to s u p p o r t o n l y t r a n s f e r a s e a c t i v i t y w h i l e Mg 2+ s u p p o r t s b o t h b i o s y n t h e t i c a n d t r a n s f e r a s e activities of the una d e n y l y l a t e d enzyme.

In this p a p e r , w e r e p o r t results of f ~ r t h e r studies on r e a c t i o n s (1) to (5) as c a t a l y z e d b y u n a d e n y l y l a t e d a n d in some cases a d e n y l y l a t e d glutam i n e synthetase f r o m E. colt in the p r e s e n c e pC e i t h e r Mg e÷ or Mn2+. The e x p e r i m e n t a l d a t a suggest that the enzyme utilizes the same c a t a l y t i c site for r e a c t i o n s (1) to (5), a n d an i n t e g r a t e d m e c h a n i s m is p r o p o s e d to a c c o u n t for these r e a c tions. W i t h the p r o p o s e d m e c h a n i s m , one c a n recon.cfle most e x p e r i m e n t a l data in the literature. MATERI&LS AND M~THO~)S. (a) Materials : Both the u n a d e n y l y l a t e d a n d a d e n y l y l a t e d forms of glutamin.e s y n t h e t a s e w e r e isolated f r o m E. colt using the p r o c e d u r e develo p e d b y W o ~ l f o l k et al. [4~. U n a d e n y l y l a t e d enzyme (El%) (the s u b s c r i p t in,dicates the a v e r a g e n u m b e r o~f a d e n y l y l a t e d subunits p e r do,decamer) was isolated f r o m E. colt t h a t h a d been g r o w n in a m e d i u m c o n t a i n i n g 20 ram NH4C1 and 0.67 M glycerol. F o r the adeny']ylated e n z y m e (E~y), E. colt w a s g r o w n in a m e d i u m c o n t a i n i n g 35 raM gl~atamate an.d 0.6.7 M glycerol and, i m m e d i a t e l y p r i o r to harvesting, NH4C1 was a d d e d to 40 mM final c o n c e n t r a t i o n . U n d e r these c o n d i t i o n s , the enzyme is c o n v e r t e d v e r y r a p i d l y to the a d e n y l y l ated form [15J. The state ~f a d e n y l y l a t i o n of glut a m i n e synthetase p r e p a r a t i o n s was d e t e r m i n e d both by the s p e c t r o p h o t o m e t r i c m e t h o d a n d b y the ~-glutamyltrans,ferase assay [16]. The s p e c i f i c a c t i v i t y of the p u r i f i e d enzyme Yeas d e t e r m i n e d b y a m o d i f i e d p r o c e d u r e d e v e l o p e d b y G i n s b u r g et al [5~ a n d the results o b t a i n e d agree well w i t h p u b l i s h e d values for the p u r i f i e d enzyme. L - [ l ~ C ] g l u t a m i c a c i d (200 m'Ci/mmole), [~2.p] i n o r g a n i c phos~phate a n d 5 ' - a d e n y l y l i m i d o d i p h o s p h a t e (AMP-P-N-P) w e r e p u r c h a s e d from I n t e r n a t i o n a l Chemical Nuclear. [v-a2PJATP (2-1.0 C i / BIOCHIMIE, 1976, 58, n ° 1-2.

mmo'le) an.d I,-[14C]gl.utamine ( > 200 m g / m o l e ) w e r e o b t a i n e d f r o m New E n g l a n d N u c l e a r Corp. [ 1 8 0 ] i n o r g a n i c p h o s p h a t e ( d i s o d i u m salt; 9.4.4 p e r cent 180,) was p u r c h a s s e d f r o m Bio-Rad L a b o r a tories. ATP, ADP, AMP, L-glutamine, L-g]utamic act,d, GTP, GDP, IDP, UD:P, C,DP, a n d c~-methylD-manosi.de w e r e o b t a i n e d from Sigma Chemical Go. P o l y g r a m CJ~L 300 P E I ( P o l y e t h y l e n e i m i n e i m p r e g n a t e d ) c h r o m a t o g r a p h y sheets w e r e p u r c h a s e d from Brin.kman I n s t r u m e n t s , Inc. Snake venom p h o s p h o d i e s t e r a s e w a s o b t a i n e d f r o m W o r thingto.n Biochemica.1 C o r p o r a t i o n . Concanavalin-A c o v a l e n t l y b o u n d to s e p h a r o s e 4B was obtained from P h a r m a c i a F i n e C,hemicals AB. The c o m m e r c i a l p r e p a r a t i o n s o~f ATP and AMP-P-N-P (about 910~p e r cent p u r e ) w e r e p u r i f i e d b y c h r o m a t o g r a p h y on D~EAE~Sephadex A-25 p r i o r to use. E l u t i o n of the p u r e nucleotides was a c h i e v e d w i t h a KC1 g r a d i e n t from 50 mM to 200 mM in 20 mM Tris-HCl (pH 8.3). W h e n h i g h p u r i t y was r e q u i r e d , g l u t a m i n e a n d glutamate, w h e t h e r l a b e l e d or u n l a b e l e d , w e r e p u r i f i e d b y a d s o r p t i o n to a n d elution from Dowex-1 (.C.1) columns b y s'light modifi,catio~ns of the p r o c e d u r e of P r u s i n e r a n d Milner [17]. Completely u n a d e n y l y l a t e d glutamine synthetase, E y , w a s o b t a i n e d b y t r e a t m e n t of the EE5 enzyme w i t h snake v e n o m p h o s p h o d i e s t e r a s e . The rea.etion m i x t u r e , 0.2 ml, c o n t a i n i n g 30 mM T r i s (pH 8.0,), 18 m g o~ E ~ a n d 0.2 m g of v e n o m phosp h o d i e s t e r a s e , w h i c h h a d been h e a t e d at 37 ° for 3 h o u r s in 1.0 p e r cent acetic a c i d solution to i n a c t i v a t e c o n t a m i n a t i n g 5 ' - n u d e o t i d a s e E18], was i n c u b a t e d at 37 ° for 30 minutes. A p a r a l l e l e x p e r i m e n t s h o w e d that u n d e r simi~lar con, ditions, Eyy- was d e a d e n y l y l a t e d to EE~ w i t h o u t significant loss of a c t i v i t y ( < 10 p e r cent). To r e m o v e the venom p h o s p h o d i e s t e r a s e [19] the r e a c t i o n m i x t u r e was then p l a c e d on a c o n c a n a v a l i n - A S e p h a r o s e column (0.9 × 11 cm) vzhich h a d been first w a s h e d w i t h 0.1 M a - m e t h y l - D - m a n n o s i d e at p H 6.0 a n d then w i t h 0.2 M p o t a s s i u m acetate, p H 6.0. Elution of the l o a d e d column w i t h 0.2 M p o t a s s i u m acetate buffer at p H 6.0 r e m o v e d glut a m i n e s y n t h e t a s e (6 to 20 ml eluate fractions) and v e n o m p h o s p h o d i e s t e r a s e was i m m o b i l i z e d on the column. The eluted enzyme was di,alyzed first against 5,0 rrrM K-HEP.ES buffer (pH 7.0) containing 1 mM EpDTA and 0.1 M KC1, then against the same buffer from w h i c h EDTA was onfitted. Tke state of a d e n y l y l a t i o n of the r e s u l t i n g p r e p a r a t i o n w a s O as d e t e r m i n e d b y both ~,-glutamyltransferase and s p e c t r o p h o t o m e t r i c assay methods. (b) Methods : L-glutaruine, L-glutamate, and p y r r o l i d o n e c a r b o x y l a t e w e r e d e t e r m i n e d by a

Glutamine synthelase -- Mechanistic studies. m o d i f i e d p r o c e d u r e of P v u s i n e r a n d Mitner [17] a n d / o r the method p r e v i o u s l y described by K r i s h n a s ~ , a m y et al [8]. I n e x p e r i m e n t s w i t h [14C]glutamine the r e a c t i o n was q u e n c h e d by the a d d i t i o n of 1 ml of 3,0 mM u n l a b e l e d glutamine in 10 mM imadozole-H,C1 (pH 7.09 to a 50 to 100 ~xl reaction m i x t u r e s at 0 °. The diluted sample was i m m e d i a t e l y placed on a Do~ex-1 (C1) c o l u m n (0.5 × 5.5 cm) to separate the products, labeled glutamate and pyrroli,done carboxylate, from residual [~4C]glutamine. E l u t i o n of the c o l u m n with the 10 mM imidazole-HC1 (pH 7.0)-3.0 mM glutam i n e solution r e m o v e d the [~4C] glutamine quantitatively in the first 4 ml of e~uate w h i c h was collected i n two s c i n t i l l a t i o n vials. The [14C]glutamate that r e m a i n e d on the c o l u m n u n d e r these c o n d i t i o n s was eluted out q u a n t i t a t i v e l y with 4 ml of 0.1 M HC1 a n d collected in two s c i n t i l l a t i o n vials. Since p y r r o l i d o n e carboxylate also was eluted in the glutamate fractions, it was separated from glutamate by p a p e r c h r o m a t o g r a p h y in a solvent system of ethanol-acetic acid-water (4-1-1). The a m o u n t of [~4,C] i n each sample was determ i n e d by c o u n t i n g in a Beckman LS-250 l i q u i d s c i n t i l l a t i o n counter. Nucleotides and [32p]orthophosphate were separated by the method of Cashel et al [20] b y c h r o m a t o g r a p h y on PEI thin layer sheets developed i n phosphate buffer (pH 3.4). Tile concent r a t i o n of the buffer used (0.5 to 1.5 M) was v a r i e d d e p e n d i n g on whi.ch nucleotides were to be separated. The areas c o r r e s p o n d i n g to a u t h e n t i c nucleotide m a r k e r s were h~cated u s i n g a UV lamp and were cut out for counting. W h e n the experim e n t r e q u i r e d no authentic nucleotide to be coc h r o m a t o g r a p h e d w i t h the sample, radioautograp h y was used to locate the position of the labeled nucleotide on the thin layer sheet. [32p] orthophosphate p r o d u c e d from [v-szP] ATP d u r i n g the reaction was d e t e r m i n e d by a m o d i f i e d m e t h o d of Goldmark and L i n n [21] in w h i c h charcoal was used to remove the nucleotide. The enzym i c r e a c t i o n was stopped b y r a p i d a d d i t i o n to the sample solution (50-10,0 ~I) 1 ml of charcoal s u s p e n s i o n p r e p a r e d by m i x i n g 3.0 g of Norit charcoal, 15:0 ml of 1 M orthophosphate at pH 6.7, 150 ml of 1 M HE1, a n d 400 ml of water. After a few minutes, the s u s p e n s i o n was filtered t h r o u g h glass wool directly into a s c i n t i l l a t i o n vial. One ml of acidic phosphate solution (0.3,8 M) was used to w a s h the charcoal a n d the glass wool. The wash was collected i n a second s c i n t i l l a t i o n vial. I n controlled studies, 74 p e r cent o,f the [z2p]orthophosphate w.as recovered i n the first vial a n d 25 per cent i n the second vial.

BIOCHIMIE, 1976, 58, n ° 1-2.

37

The [lS0]glutamate o b t a i n e d from the reverse b i o s y n t h e t i c reaction was first tri-fluoroacetylated a n d then esterified w i t h d i a z o m e t h a n e to form a volatile derivative [22], w h i c h was analyzed with a LKB Type 9000 g a s - l i q u i d c h r o m a t o g r a p h y - m a s s spectrometer. F l u o r e s c e n c e m e a s u r e m e n t s were ma,de u s i n g a H i t a c h i - P e r k i n - E l m e r MPF-2A i n s t r u m e n t equipped with a Hewlett-Packard 7004B X-Y recorder. Constant t e m p e r a t u r e was m a i n t a i n e d u s i n g thermostated cell holders a n d c o n s t a n t t e m p e r a t u r e c i r c u l a t i n g baths. A Cary 17 s p e c t r o p h o t o m e t e r e q u i p p e d w i t h thermostated cells was used for a b s o r p t i o n spectra m e a s u r e m e n t s a n d for lhe bios y n t h e t i c coupled-enzyme assays. Special care was taken to remove trace a m o u n t s of a m i n o n i a in the enzyme [8]. Redistilled deionized w a t e r was used. The a m o u n t of NH a p r e s e n t in the system was negligible as i n d i c a t e d by the fact that on,ly a trace a m o u n t of [~4C]glutamine was formed w h e n enzyme was a d d e d to flaG]glutamate, ATP, a n d Mg2+. RESULTS.

Divalent Cation Effects. Mn 2+ supports the bios y n t h e t i c activity of a d e n y l y l a t e d but not una d e n y l y l a t e d glutamine s y n t h e t a s e ; h o w e v e r it can s u p p o r t the transferase activity of both adenylylated and u n a d e n y l y l a t e d enzyme forms [2325]. To investigate the possibility that separate catalytic sites are i n v o l v e d for reactions (1) a n d (2), the reverse b i o s y n t h e t i c r e a c t i o n was c a r r i e d out in the p r e s e n c e of eilher Mg2+ or Mn "2-+. Completely u n a d e n y l y l a t e d enzyme, E ~ , (see Methods) TABLE I.

R e v e r s e B i o s y n t h e t i c A c t i v i t y of the Unadenylylated E n z y m e in the P r e s e n c e of Mge+ or Mn ~+ at D i f f e r e n t ADP and P~ Concentrations (a). t ADP] mM

[mPi] mM

5.0 05 0.1 O. 02 0.005

5.0 0.5 0.1 O. 02 0.005 j

activity, (b) ~M/min

Mg~+ supported activity, (b) ~tM/miu

0.58 0.57 0.39 0.18 0.057

2.8 0.75 0.14 0.013 O. 0012

MIJ~+ supported

I

MB2+

/Mg~+ 0.21 0.76 28 14 48

(a) The reaction ~vas followed at 37 ° by the formation of [~t-32p]ATP. The reaction mixtures contained ADP, 32p I as indicated, 46 mM K-HEPES (pH 7.0), 92 mM KC1, 3 nM Eo (subunit concentration), 10 mM L-glutamine and 0.4 mM Mn2+ or 20 mM Mg2+. (b) The activity was an average of two experimental points obtained at 7 and 17 rain after the addition of enzyme.

S. G. R h e e , P. B. C h o c k a n d E. R. S t a d t m a n .

38

was used for these e x p e r i m e n t s to elilninate the possibi'lity that any o b s e r v e d activity could be due to the p r e s e n c e of t r a c e amounts of the aden y l y l a t e d subunit. The results s h o w that both Mg 2* a n d Mn 2÷ s u p p o r t e d activity is a f u n c t i o n o,f the c o n c e n t r a t i o n s of ADP and P] (table I). W h e n the c o n c e n t r a t i o n s of ~DIP and P~ are the same, the Mg2*-enzyme is m o r e active at h i g h AD,P and Pi c o n c e n t r a t i o n s , w h e r e a s the Mn2*-enzyme is m o r e active at l o w ADP and Pi c o n c e n t r a t i o n s . I n o r d e r to e x p l a i n the v a r i a t i o n in a.ctivity sho~vn in table I, f l u o r o m e t r i c titrations w e r e c a r r i e d out at pH 7.6, 25 °, in 5,0 mM K-HJEPES buffer and 0.1 M I~C1. The dissociation constants o b t a i n e d f o r Pi f r o m MgEy6-ADP-P i and Mn,EI.o-ADP-P ] are 5 mM and 0.0.8 raM, r e s p e c t i vely a n d the c o r r e s p o n d i n g dissociation constants for ADP in the p r e s e n c e of Pi av.e 25 ~tM a n d 20 nM, r e s p e c t i v e l y .

attributed to the fact that Mg2÷ and Mn 2+ stabilize different conformationa'l s,tates of the enzyme [26, 27J. It is significant, h o w e v e r , that w h e r e a s binding of the analog of ATP, 5 ' - a d e n y i y l i m i d o dip h o s p h a t e (AMP-P-N-P), to both MgEy6 and M n E ~ p r o d u c e s the same fluorescence e n h a n c e ments as are o b t a i n e d w i t h AT,P, no a d d i t i o n a l e n h a n c e m e n t of f l u o r e s c e n c e is o b t a i n e d w i t h the subsequ.ent a d d i t i o n of L-glutamate. This suggests that w i t h both Mn2+ arid Mg 2+ a c t i v a t e d e n z y m e forms, a c o m p l e x is p r o d u c e d b e t w e e n ATP and glutamate (possibly ~,-glutamyl-phosphate transition state i n t e r m e d i a t e ) nvhile such a c o m p l e x cannot be f o r m e d w i t h AM,P-P-N-P and glutamate.

t-

/

20! /

j

(

x

S i m i l a r changes in the i n t r i n s i c t r y p t o p h a n f l u o r e s c e n c e of u n a d e n y l y l a t e d e n z y m e are oh-

/'

L0 ~"

K, = 30 mM f

'

/ /

0 Glut

//

/

!V

//

02

(J

/

£"

,

~,;;,/

//

.,"

/

/ 2' / ,r , - ,

.

Glut

,,z,

/

.

/

4O

/

°,

>-

)-

/ ' / / / /'ll

/

/

80

_z 60

/

30 610 ~ GLUTAMATEqmM) /

,

/

/ •

/-"

/~

/

/

./

/-~

.A

/

~ . ./ . - --

. -

ATP

LL

<,

- .- ,7

LU Q:

. - j " . - , :,y

0

MgE~

SUBSTRATE ADDITION

Fro. 1. - - Relative fluorescent intensity enhanced by A TP addition follolwed by glutamate addition for MgET.~ and MnET~. The reactions were carried out in 50 mM K-HEPES (pH 7.0) balffer containin,g 0.1 M KC1 at 25 ° . Saturated level of ATP and glutamate "were added. Excitation wavelength was 300 mM and emission was measured at 336 mM. rained by stepwise addi'fions of ATP and glutam a t e to M g E y 5 and M n E i . 0 ~figure 1.). This suggests that the same i n t e r m e d i a t e s are f o r m e d in the p r e s e n c e of both d i v a l e n t cations. The fact that g r e a t e r e n h a n c e m e n t of fluores.cence is obt a i n e d w i t h MnE1z~ than w i t h MgE1. o m a y be

BIOCHIMIE, 1976, 58, n ° 1-2.

200

I (M_~) Glutamme

AnE 1-~

o

100

Fic,. 2. - - Competitive inhibition exhibited by glutamate in the ~.,-glulamyl-transfer reaction at 37 °. The reaction mixtures contained the indicated concentration of glutamine, 50 mM K-HEPES (pH 7.6) 0.1 M KC1, 20 mM Mg9+, 1 mM ADP, 20 mM inorganic phosphate, 40 mM NH2OH, 18 nM E~.6 (suhunit concentration) and glutamate at O mM f~), 5 mM (D), 15 mM (O), 30 mM (A), 45 mM (m) and 60 mM (O) final concentrations.

COMPARISON OF REACTIONS.

BIOSYNTHETIC

AND

TRANSFERASE

(a) Glutamate and Glutamine Binding Site : M p H 7,6 w i t h MgEy6 , L-glutamate is a c o m p e t i t i v e i n h i b i t o r w i t h r e s p e c t to L-glutamine in the v-glutamyl t r a n s f e r reaction. F i g u r e 2 shows the corn-

G l u t a m i n e stjnthetase - - Mechanistic studies.

petitive p a t t e r n observed w h e n the L-glutamine c o n c e n t r a t i o n is varied from 4 to 80 mM at constant L-glutamate c o n c e n t r a t i o n s of 0, 5, 15, 3'0, 45 and 60 raM. The K m obtained for L-glutamine is 11 raM. F r o m the s e c o n d a r y 9lot of the slopes of the lines in figure 2 vs L-glutamate concentration, the K i for L-glutamate was estimated to be 30 mM. T i t r a t i o n of i n t r i n s i c t r y p t o p h a n fluorescence e n h a n c e m e n t associai.ed w i t h the b i n d i n g of L-glutamate to ELo MgATP, a complex req,uired for b i o s y n t h e t i c reaction, showed that the L-glutamate dissociation constants increase w i t h i n c r e a sing c o n c e n t r a t i o n of added L-glutamine (table II). With the assumption that L-glutamine a n d L-glutamate are m u t u a l l y exclusive w i t h respect to enzyme b i n d i n g , a dissociation c o n s t a n t for L-glula-

39

level of arsenate is 25 vM at pH 7.2, 25 ° [28]. There might be slight synergism in the b i n d i n g of ADP and arsenate. Figure 3 clearly demonstrates that AMP-P-N-P is a competitive i n h i b i t o r of ADP in the v-glutamyltransferase reaction with a K i value of 80 ,:xM. This value agrees well with a value of 70 ~M for the dissociation constant o b t a i n e d by

/ ×

S 03

//

/

14`

,,-~ /t,

K I = 0 08 m M

/" /

02

0.4

A M P P N P {mM)

,"

~."

[Glutamine] 0 mM

12 mM 24 raM

Kais~ 3

//-

0,4 1 V

/

/

...

/' /./ / i, A// // / // ." Q,/ / / /~ /" ~/ / / / J-

/

/' .-

/ //// / /

mM

6 raM 8.5 mM

(a) Data obtained from fluorescence titration 'with 1.3 i~xM E]~ (subunit concentration), 10 mM Mg2+, 0.9 mM ATP, 100 mM KC1, and 50 mM K-HEPES at 25 °.

m i n e is calculated to be 12 mM at pH 7.2. Fluorescence titration in the p r e s e n c e of a s a t u r a t i n g a m o u n t of ADP (0.9 raM), 20 mM Mg2+, 2.6 ~M E~U~ (subunit c o n c e n t r a t i o n ) , 50 mM K-HE.PES, and 0.1 M KC1, at pH 7.6, gives a disso,ciation c o n s t a n t of 24 mM for L-glutamate. This value is in relatively good agreement with the Ki for L-glutamate obtained from figure 2, and the dissociation constant, 20 raM, estimated by T i m m o n s et al C7] based on a scheme i n v o l v i n g r a n d o m a d d i t i o n of ATP a n d L-glutamate to the MgE-~u~ , (b) A T P and A D P B i n d i n g Site : T i m m o n s et al [7] showed that AMP-P-N-P h i n d s to the ATP site in the b i o s y n t h e t i c reaction. Therefore, to avoid c o m p l i c a t i o n s that w o u l d he caused by the presence of trace a m o u n t s o.f AD,P i n ATP p r e p a r a tions, purified AMP-P-N-P was substituted for ATP in the studies to d e t e r m i n e the i n h i b i t i o n p a t t e r n w i t h respect to A:DP d u r i n g the transferase reaction. Figure 3 shows the L i n e w e a v e r - B u r k ' s plot obtained for the transferase r e a c t i o n at pH 7.0 w h e n a constant a m o u n t of AMP-P-N-P is present. The K~ for ADP in the p r e s e n c e of a s a t u r a t i n g BIOCHIMIE, 1976, 58, n ° 1-2.

.,

//

0,6

/

, /

TABLE II. E q u i l i b r i u m Constants [or the Dissociation o f Glutamate [rom E F.o MgATP-Glu in the Presence and Absence of L-Glutamine at p H 7.2 (a).

/

0.2

0.5

10

1.5

20

2.5

(M"~ x 10-~

Fro. 3. - - - Competitive inhibition exhibited by AMPP-N-P in the v-glutamyl-transferase reaction. The reactions 'were carried out at 37 °, pH 7.0 in a mixture containing 40 mM K-HEPES, 80 mM KCI, 24 mM Mg2+, 24 mM arsenate, 40 mM NHoOH, 40 mM glutamine, 30.8 nM E ~ (subunit concentration) and AMP-P-N-P at 0 mM (A), 0.06 mM (H), 0.14 mM (O), 0.24 mM (A) and 0.4 mM (I) final concentrations.

direct fluorometric titration o,f MgE1. o w i t h AMPP~N-P at pH 7.0, 20 °. It is n o t e w o r t h y that the affinity of MgE1. o for AMP-P-N-P is a p p r o x i m a t e l y three times greater t h a n for ATP. (c) Orthophosphate and Arsenate Site : Figure 4 shows the rate of [14C] glutamate formation by the reverse b i o s y n t h e t i c r e a c t i o n w h e n [14C] glutamine, Pi or arsenate and AD,P or GDP are substrates. In parallel e x p e r i m e n l s w i t h [32p] orthophosphate as substrate, the a m o u n t of either [-/-a~P]ATP o r [y_32p]GTP formed was equivalent to the a m o u n t of glutamate formed, in a c c o r d a n c e w i t h the s t o i c h i o m e t r y expected by reversal of reaction (1). The c u r v i l i n e a r r e l a t i o n s h i p between glutamate f o r m a t i o n and time observed in the e x p e r i m e n t s w i t h Pt as substrate is p r o b a b l y due

S. G. R h e e , P. B. C h o c k a n d E. R. S t a d t m a n .

40

to p r o d u c t i n h i b i t i o n , since the e n z y m e has relatively h i g h affinity for n u c l e o s i d e t r i p h o s p h a t e s . No i n h i b i t i o n o c c u r s w i t h arsenate since the ADPOAs0~- = f o r m e d u n d e r g o e s r a p i d s p o n t a n e o u s hydrolysis.

i 1200 ~

,,,

"

~

800

400

!

0

20

40 -60 MINUTES

80

100

me.d (2,4 ~M) is about the sanre as the a m o u n t of AD'P (28 '~M) expe.cie,d f r o m ATP hydrolysi's in the p r e s e n c e of Mn 2+, a c c o r d i n g to the data of Tetas and Lo:wenstein [303. W i t h c o m m e r c i a l ATP as substrate anid s'aturating levels of arsenate, glutarain.e, and NH2OH, t r a n s f e r a s e activity was observed w i t h b o t h MgE1. o and MnEy. o. In the case of M g E ~ the activity i n c r e a s e s as a f u n c t i o n of ATP c o n c e n t r a t i o n until it r e a c h e s a plateau at about 5.0 p e r cent of the Vm~x o b s e r v e d for ADP. T h e c o n c e n t r a t i o n of ATP r e q u i r e d to r e a c h the plateau is 0.8 raM. H o w e v e r , w i t h f r e s h l y p u r i f i e d ATP and glutamine as substrates, only MnE~:~ and not MgEiT6 exhibits t r a n s f e r a s e activity. This activity is ATP c o n c e n t r a t i o n d e p e n d e n t until it r e a c h e s about 60 p e r cent obf the Vm,,x value obtained w i t h A DP as substrate. The c o n c e n t r a t i o n of &DP r e q u i r e d to r e a c h the plateau is 2 I~M.

(e) AMP is a Substrate in the Reverse Biosynthetic and Trans[erase Reactions. &MP is an inhi, b i t o r of the f o r w a r d biosynthetic r e a c t i o n [31~ an~d will r e p l a c e A~DP as a c a t a l y t i c sub strate in t h e t r a n s f e r a s e r e a c t i o n in the p r e s e n c e of Mn 2+ or Cd 2+ but not in the pre-

FIG. 4. Formation of glutamate by the reverse biosynthetic reaction. The reactions Were carried out at 37 °, pH 7.6, in a mixture containing 50 mM K-HEPES, 0.1 M KC1, 40 nM E ~ (subunit concentration), 50 mM Mg2+ and 8 mM ADP plus 10 mM P~ (O) or 8 mM GDP plus 10 mM P~ (E]) or 8 mM ADP plus 10 mM arsenate (A). The rate was followed by [14C] glutamate, formation. -

-

120

/

8O

(d) A T P does not S u p p o r t the Reverse and Transferase Reactions : W e l l n e r and Meister [29] r e p o r t e d that ~-glutamyl t r a n s f e r a s e activity of purified sheep b r a i n glutamine synthetase is activated by l o w c o n c e n t r a t i o n s of either ATP or AD,P. Our data s h o w that n e i t h e r the r e v e r s e bios y n t h e t i c nor the -~-glutamyt t r a n s f e r r e a c t i o n is catalyzed by the E. colt e n z y m e w h e n f r e s h l y purifield ATP and g l u t a m i n e are p r o v i d e d as substrates. If AT:P c o u l d s u p p o r t the r e v e r s e r e a c t i o n , a d e n o s i n e t e t r a p h o s p h a t e should be f o r m e d ; h o w e v e r , in the studies w i t h both Mg E ~ and MnEL6, a d e n o s i n e t e t r a p h o s p h a t e could not be detected in the r e a c t i o n m i x t u r e s i n i t i a l l y containing 5.0 mM glutamine, 2.4 m~M [32p] o r t h o p h o s phat.e and 5 mM A T P at p H 7.0. H o w e v e r , w i t h 9.4 ~M M n E ~ , 1.0 p e r cent of the a d d e d [32p]p~ w a s i n c o r p o r a t e d into ATP after i n c u b a t i o n of the r e a c t i o n m i x t u r e for 25 m i n at 37 ° . This [32p] ATP is undoubte~dly d e r i v e d fro.m trace a m o u n t s of ADP p r o d u c e d by slow hy~drolysis of ATP dur i n g the i n c u b a t i o n . The a m o u n t of [a2p]ATP for-

BIOCHIMIE, 1976, 58, n ° 1-2.

rf

100

::L 6O 40 20

0

20

40

60 80 100 120 MINUTES Fro. 5. - - AMP supported reverse biosynthetic reaction. The reaction 'was carried out at 37 °, pH 7.0 in a mixture containing 46 mM K-HEPES, 92 mM KC1, 0.5 mM Mn2+, 10 mM glutamine, 5 mM AMP, 10 mM orthophosphate and 3 ~tM E ~ (subunit concentration). The rate was follo~ved by the formation of [14C]glutamate and [32P]ATP.

sence o.f Mg2+ ( S m y r n i o t i s and Stadtman, unpub l i s h e d data cited in [2]). In a d d i t i o n , W e d l e r and B o y e r [32] h a v e s h o w n that AMP, like GDP, completely inhibits the Pi ~ > " ATIP e x c h a n g e but

G l u t a m i n e s y n t h e t a s e - - M e c h a n i s t i c studies. depresses only slightly the glutamate ~)glut a m i n e e x c h a n g e , c a t a l y z e d b y M n E T ~ . To e x p l a i n these observations, they suggested that nucleotide d i s s o c i a t i o n f r o m t h e e n z y m e is b l o c k e d b y AMP. F i g u r e 5 s h o w s t h a t AMP also s u p p o r t s t h e r e v e r s e b i o s y n t h e t i c r e a c t i o n in t h e p r e s e n c e of MnEi76 an,d t h e l a b e l e d substrates, [14C] g l u t a m i n e o r tamp] o r t h o p h o s p h a t e . At a n y g i v e n t i m e , t h e a m o u n t of [B, :,-s'~P]ATP f o r m e d is e q u a l to a b o u t 40 p e r c e n t of t h e [14C] g l u t a m a t e p r o d u c e d (figure 5). T h e s m a l l d e v i a t i o n f r o m an e x p e c t e d s l o i c h i o m e t r y o,f 1 : 2 for t h e A T P : g l u t a m a t e r a t i o , is w i t h i n e x p e r i m e n t a l e r r o r s . [B-a2P]ADP w a s not d e t e c t e d a m o n g t h e r e a c t i o n p r o d u c t s . U n d e r t h e s a m e c o n d i t i o n s u s e d in s t u d i e s of the reverse biosynthetic reaction, except that 25 mM NH2OH w a s also p r e s e n t , t r a n s f e r a s e a c t i v i t y w a s o b s e r v e d w i t h MnE1z ~ b u t n o t w i t h MgEi-_6. H u n t et al [9], s h o w e d t h a t AMP a n d arsenate support the transfer reaction catalyzed by b o t h Mn ~ a n d Cd 2+ s u p p o r t e d e n z y m e s . H o w e v e r , w e foun,d t h a t i n t h e absen,ce of CA-, Cd~+-enzyme cannot catalyze the reverse biosynthetic reaction, whereas t h e Mn-~+-enzyme can. I n t e r e s t i n g l y , the i n i t i a l r a t e f o r the t r a n s f e r r e a c t i o n is a b o u t

41

60 t i m e s f a s t e r t h a n t h e r e v e r s e r e a c t i o n r a t e f o r the Mn2+-enzyme s y s t e m . T h e s i g n i f i c a n c e of these results w i l l be d i s c u s s e d later. It is notew o r t h y t h a t whe~:eas A M P c a n r e p l a c e A D P in the r e v e r s e b i o s y n t h e t i c r e a c t i o n (figure 5), A D P is u n a b l e to r e p l a c e A T P as a s u b s t r a t e in the forw a r d re,action ; n e i t h e r AM~P n o r Pi is p r o d u c e d in t h e p r e s e n c e of A D P , g l u t a m a t e a n d a m m o n i a . (f) Oxygen Trans[er and Relative Activity : O x y g e n - 1 8 l a b e l l e d g l u t a m a t e h a s b e e n u s e d to s h o w t h a t t h e o x y g e n of the v - c a r b o x y l g r o u p w a s t r a n s f e r r e d to o r t h o p h o s p h a t e in t h e c o u r s e of b i o s y n t h e t i c r e a c t i o n c a t a l y z e d by the p e a e n z y m e [33, 34]. It is b e l i e v e d t h a t a s i m i l a r o x y g e n ]

Mg++l ~/fMn++ I / / Mg+'l~

600 -

ET~

TABI~ III.

Relative Activity [or Reverse Biosynthetic and Transferase Reactions (a).

[ati;ncarboxylateL z -

Relative activity Group

Effectors present

pH

Reverse biosynthetic

Transterase

A(b)

ADP, Mg~+, Arsenate 7.6 A D P , Mg~+, Pi 7.6

3.6 8.1

100 50

B(e)

ADP, Pi, Mn "2+

8 32

100 92

0.4

23

A D P , Pi, Mg~+ C (d)

AMP, Pi, Mn ~+

7.0

(a) The relative activity at 37 ° is normalized with the assumption that 100 is (1) the activity of the transferase reaction with ADP, Mg2+, arsenate in the pH 7.6 system, and (2) the activity of the transferase reaction with ADP, P~, MnU÷ in the pH 7.0 system. (b) Reactions were carried out in a mixture containing 50 mM K-HEPES, 0.1 M KC1, 20 mM Gln, 2.8 mM (8 mM for reverse biosynthetic reaction) ADP, 20 mM Mg2+, 0.15 luM E y ~ (subnnit concentration), 10 mM arsenate, and additional 50 mM NH~OH for the transfer reactions. (c) Reactions 'were carried out in a mixture containing 40 mM IK-HEPES, 92 mM KC1, 10 mM Gln, 5 mM ADP, 10 mM P~, 0.1 t~M El. 0 (subunit concentration). [Me `'+] used is 0.5 mM for Mn2+ and 50 mM for Mg2+. For the transfer reaction 25 mM NH2OH was added. (d) The reactions were carried out with 46 mM K-HEPES, 92 mM KC1, 0.5 mM Mn2+, 10 mM Gln, 5 mM AMP, 10 mM P,, 3 i~zME ~ (subunit concentration) and additional 50 mM NH2OH for the transfer reaction.

BIOCHIMIE, 1976, 58, n ° 1-2.

J,F

0

!

iorma.o.

50

i00 MINUTES

150

Fro. 6. - - Rates of pyrrolidone carboxflate and orthophosphate formation. The reactions were carried

out at 37 °, pH 7.0, in a mixture containing 45 mM K-HEPES, 90 mM KC1, 4.3 mM ATP, 7.1 mM glutamate, 7.4 aM E Lo (subunit concentration) and either 20 mM Mg2+ or 3 mM Mn2 ÷. The rate of the reaction ~vas followed by the formation of 32Pi and [14C]pyrrolidone carboxylate.

e x c h a n g e is also c a t a l y z e d b y t h e E. colt glutam i n e s y n t h e t a s e . In s t u d i e s of t h e r e v e r s e b i o s y n t h e t i c r e a c t i o n , [asO] o r t h o p h o s p h a t e (94.4 p e r c e n t 1so) w a s i n c u b a t e d w i t h u n a d e n y l y l a t e d enz y m e , g l u t a m i n e , a n d A T P in t h e p r e s e n c e of Mg 2+ at 37 °, p H 7.6 f o r I h o u r . T h e glut,amate f o r m e d w a s s u b j e c t e d to m a s s s p e c t r a l a n a l y s i s as d e s c r i b e d u n d e r M e t h o d s . T h e r e s u l t s s h o w e d t h a t 1so f r o m Pi w a s t r a n s f e r r e d to t h e g l u t a m a t e . By anal o g y it is b e l i e v e d t h a t t h e a r s e n a t e o x y g e n is also t r a n s f e r r e d to glu~tamate w h e n a r s e n a t e , i n s t e a d of p h o s p h a t e , is u s e d as n u c l e o p h i l e . Some relative activities for the transferase and r e v e r s e b i o s y n t h e t i c r e a c t i o n s are t a b u l a t e d in table I I I to s h o w (1) r e l a t i v e e f f e c t i v e n e s s of arse-

42

S. G. R h e e , P. B. C h o c k a n d E . R . S t a d t m a n .

nate and Pi as substrate for the MgEi~ at pH 7.6, 37 ° ; (2) relative catalytic activities of MgE ~6 a n d Mn,Ekuo at pH 7.0, 37 ° ; a n d (3) the effectiveness o~f AMP as substrate i n the t r a n s f e r and reverse b i o s y n t h e t i c reactions. A T P a s e Reaction : The glutamine synthetases from sheep b r a i n [8] a n d from E. colt [11] catalyze rea.ction (3), i n w h i c h L-glutamate is converted to pyrroli, done carboxylate w i t h concomiten,t h y d r o l y s i s of ATP. The rate of this r e a c t i o n is about 100 times slower t h a n the b i o s y n t h e t i c reaction. I n the p r e s e n c e of a m m o n i a no pyrroli,done carboxyla.te can be ,detected. Quantitative analysis of this reaction has been c a r r i e d out w i t h both Mg2÷ a n d Mn 2+ s u p p o r t e d enzymes. The results (figure 6) show that both M g E y z aud MnE~6 catalyze the g l u t a m a t e - d e p e n d e n t ATPase r e a c t i o n almos,t equally well ; this is in contrast to the data of W e i s b r o d a n d Meister [11] w h i c h show that the ATPase activity of the Mn 2÷ s u p p o r t e d u n - d e n y l y l -

I n contrast to results w i t h u n a d e n y l y l a t e d enzyme, the data i n figure 7 show that Mn ~+ is m u c h more effective t h a n Mg -°+i n s u p p o r t i n g cat,alysis of r e a c t i o n (3) by the a d e n y l y l a t e d enzyme. I n fact, it is likely that fully a d e n y l y l a t e d enzyme is comp,letely inactive in the p r e s e n c e of Mg2÷, since the low activity observed w i t h MgE~-i- can, w i t h i n e x p e r i m e n t a l error, be a c c o u n t e d for b y the unad e n y l y l a t e d s u b u n i t s presents i n the enzyme used.

20 -

Mg

'T E

7

E

15 o

E

E.

/

Eii

400

/ /

-

10-

6.0

7.0

8.0

P~

200

-

-

~

i00

0

_

FIc,. 8. - - ATPase activity expressed as a fnnction of pH. Radioassays were carried out in 45 mM K-HEPES, 90 mM KC], 2 mM [~t-32P]ATP, 27 mM glutamate, 8 tLM E ~ (subunit concentration) and either 20 mM My2÷ or 3 mM Mn2+ at 37 °.

-

15

30

45

MINUTES

Fro. 7. - - Pyrrolidone carboxylate formation at pH 7.0 and 37 °. The reaction mixture contained 45 mM K HEPES, 90 mM KC1, 28 mM glutamate, 2 mM ATP, 9.4 ~,M E 1i-(subunit concentration) and either 20 mM My2+ or 3 mM Mn2+. The reactions ~vere followed by [14C]p~crrolidone earboxylate formation..

ated enzyme is only 15 to 3,6 per cent of the activity exhibited by the Mg2÷ s u p p o r t e d enzyme. Figure 6 shows also that pyrroli~done carboxylate f o r m a t i o n is a c c o m p a n i e d by the p r o d u c t i o n of I)i i n a c c o r d a n c e w i t h r e a c t i o n (3). The fact that somewhat more Pi is formed t h a n p y r r o l i d o n e carboxylate is p r o b a b l y the result of net h y d r o ]ysis o,f a small a m o u n t of glutamine derived fronl trace a m o u n t s of NH~ ÷ in the reagents u~ed, BIOCHIMIE~ 1976~ 58, ~1° ~-~,

F i g u r e 8 shows the p H depen, dence of the glutam a t e , d e p e n d e n t ATPase activity of E ~ in the presence of Mg2+ a n d Mn 2+, at 37 ° . The activity of MgE1u6 decreases from pH 6.0 to pH 6.8, a n d is almost c o n s t a n t from pH 6.8 to p,H 8.0, xvhereas M n E ~ activity increases gradually from pH 6.0 to 8.,0. This p H profile is quite different from that of the b i o s y n t h e t i c activity of M g E ~ w h i c h exhibits a maximu.m at pH 7..6 [5] a n d is different, also, from that of the t r a n s f e r activity w h i c h exhibits m a x i m a at pH 7.5 a n d at 7.3 for MnEzz6 and M g E ~ , respectively. T r a n s p h o s p h o r y l a t i o n : Data i n figures 4 and 9 show that in order of their reactivity, the p u r i n e nuedeoside diphosphates (&DP > GDP > IDP) can serve as p h o s p h o r y l group acceptors i n the reverse b i o s y n t h e t i c reaction. However, only negligible a m o u n t s of the c o r r e s p o n d i n g nucleoside triphosphates are p r o d u e e d with either UDP, CDP, 5'-deoxy-ADP or 5'-deoxy-GDP. In view of

Glulamine

synthetase

the r e l a t i v e l y n o n s p e c i f i c r e q u i r e m e n t for a pur i n e nucleoside d i p h o s p h a t e , it a p p e a r e d likely that c o u p l i n g of the f o r w a r d a n d r e v e r s e biosynthetic r e a c t i o n s could lead to t r a n s p h o s p h o r y l a tion b e t w e e n v a r i o u s n u c l e o s i d e t r i p h o s p h a t e s and diphosphates. This is veri,fied by the data in figure 10 s h o w i n g that w h e n [v-32p]ATP was incubated w i t h an equal a m o u n t of GD,P in the presence of glutamate, glutamine and either MgEv.~ (figure 10A) or M n E ~ .o (figure 10B), the c o n c e n t r a tion of ATP decreased, and a s t o i c h i o m e t r i c a m o u n t of [7-.~-°P]GTP was forme,d. F i g u r e IO'C s u m m a r i z e s results of a r e c i p r o c a l e x p e r i m e n t w i t h M g E ~ s h o w i n g that the ,t-phosphoryl group

-- Mechanistic

43

studies.

result of the b i o s y n t h e t i c r e a c t i o n . Of the t h r e e systems p r e s e n t e d in figure 10, t r a n s p h o s p h o r y l a tion fr(Hn ATP to GTP in the p r e s e n c e of Mg -~+ is t h e most effective t r a n s p h o r y l a i i o n system. T h e

\-~

A

40

_ / / ' ~

~

80

~

GTP

~

ATP ~ 4o GTP + ATP

1.0

g

ATP

r~ I---

z

80

~-~ ~---~

O.--

0.5

GTP

I

I

I

]

01 ~ ~ ~ - - - ~ 25

ITP + ATP =E

ATP 0.5

ITP I

0

20

40 MINUTES

I

60

I 8O

100

_

FIG. 9. - - Rate of nucleoside triphosphate (NTP) f o r m a t i o n at .37° in the presence of two d i f f e r e n t nucleoside diphosphates. The data were obtained bv radioassay of [~-32P]NTP. Reactions were carried ot{t in a mixture containing 40 mM K-HEPES (pH 7.6), 80 mM KC1, 20 mM Mg2+, 19 mM glutamine, 9.4 mM 32P1, 11 uM E ~ (subunit concentration) plus (A) 4.8 mM ADP and 4.5 mM GDP, (B) 4.8 mM ADP and 4.8 mM IDP.

of a d d e d [y-32P]GTP is t r a n s f e r r e d to ADP. In all of these e x p e r i m e n t s , 6.5 ~M of EL--A (subunit concen~tration) was used, w h i c h is about hal,f the conc e n t r a t i o n p r e s e n t in E. c o l t g r o w n u n d e r derep r e s s i o n conditions. At this h i g h enzyme c o n c e n t r a t i o n and in the p r e s e n c e of glutamine and glutamate, about 10 p e r cent of the initial nucleoside t r i p h o s p h a t e was d e c o m p o s e d to yield Pi as a BIOCHIM1E, 1976, 58, n ° 1-2.

Pi

FIo. 10. - - Rate of t r a n s p h o s p h o r y l a t i o n as measured by radioassag at 37 °. Reaction mixtures contained 42 mM K-HEPES (pH 7.0), 84 mM KC1, 9.1 mM glutamine, 3 mM glutamate, 6.5 :aM E ~ (subunit concentration) and (A) 20 mM Mg2+, 4.2 mM [,t-32pJATP, 4.2 mM GDP, (B) 3 mM Mn2+, 4.2 mM [~-32P]ATP, 4.2 mM GDP, (C) 20 mM Mg2+, 4 mM [~.-32P]GTP and 4.6 mM ADP.

1.0

a,. I-

z

50 75 MINUTES

r e l a t i v e l y slow t r a n s f e r o:f p h o s p h o r y l groups f r o m ATP to GDP c a t a l y z e d in the p r e s e n c e of Mn 2+ is r e a d i l y e x p l a i n e d by the fact that dissociation of ADP f r o m the i n t e r m e d i a t e M n E I ~ ADP c o m p l e x is v e r y slow.

DISCUSSION. Table I shows that both Mg~ and Mn 2+ s u p p o r ted u n a d e n y l y l a t e d enzymes are c a p a b l e of catal y z i n g the r e v e r s e b i o s y n t h e t i c reaction. The Mg 2÷e n z y m e is m o r e active at h i g h ADP an,d Pi c o n c e n trations ; whereas, the Mn2+-enzyme is m o r e active at l o w ADP and Pi c o n c e n t r a t i o n s . The specific a c t i v i t y of the Mg2+-enzyme is 5 times or m o r e g r e a t e r than the Mn-°+-euzyme. The difference in c o n c e n t r a t i o n d e p e n d e n c e reflects differences in b i n d i n g constants for ADP and Pi b e t w e e n the

44

S. G. Rhee, P. B. C h o c k a n d E. R. S t a d t m a n .

Mg2+ a n d Mn 2+ s u p p o r t e d enzyme. The a p p a r e n t dissociation constants for P~ o b t a i n e d at pH 7.6 a n d 25 ° from MgEy~- ADP-P i a n d MnE~z6 - ADP-P i are 5 mM a n d 0.0,8 raM, respectively, a n d the corr e s p o n d i n g dissociation constants for ADP i n the p r e s e n c e of Pi a r e 2,5 t~M a n d 2.0 riM, respectively. F u r t h e r m o r e , a d d i t i o n o.f ATP a n d L-glutamate p r o v o k e d a s i m i l a r type of fluorescence e n h a n c e m e n t for both MgZ+ a n d Mn 2+ enzymes (figure 1). Moreover, the e n h a n c e m e n t obtained i n the presence 0¢ both ATP a n d glutamate is greater than the sum o.f e n h a n c e m e n t s o b t a i n e d with each ind e p e n d e n t l y since in the absence of ATiP, L-glutamate did nol p r o v o k e an e n h a n c e m e n t of the protein fluorescence intensity. This sugge~sts that w h e n both substrates are b o u n d to the enzyme, they stabilize a u n i q u e c o n f o r m a t i o n a r i s i n g from either an in,terac,tion b e t w e e n the separate binding sites or from the b i n d i n g of an i n t e r m e d i a t e (possibly glutan~yl phosphate) p r o d u c e d by direct i n t e r a c t i o n b e t w e e n the two substrates on the enzyme. The latter possibility seems most likely since the e n h a n c e m e n t cff fluorescence p r o d u c e d by the bin,ding of A~P-P-N-P (an analog of ATP) is almost the same as that p r o d u c e d by ATI~ alone ; however, in contrast to results w i t h ATP, the fluorescence associated w i t h the b i n d i n g of AMP-P-N-P is not augmented by tlle s u b s e q u e n t a d d i t i o n of glutamate. Since M n E ~ a n d MgE~6 catalyze the reverse b i o s y n t h e t i c reaction a n d both enzyme forms react with ATP a n d gl.utamate "to form a n ATPglutamate complex, as judge,d by the observed fluorescence changes, it seems reasonable that both forms of enzyme coul,d catalyze the f o r w a r d b i o s y n t h e t i c reaction. Nevertheless, efforts to dem o n s t r a t e catalysis of the f o r w a r d reaction by u n a d e n y l y l a t e d enzyme i n the presen~ce of Mn2+ (i.e. b y MnE~z6) have failed. This failure is probably e x p l a i n e d b y the very high affinities of MnEy:6 for ADP (K a = 20' n:M) a n d Pi (Kd = 0.08 raM). These high affinities for ADP a n d Pi w o u l d present no special p r o b l e m i n the reverse biosynthetic r e a c t i o n btrt in the f o r w a r d r e a c t i o n they wou'ld cause such severe p r o d u c t i n h i b i t i o n that a significant r e a c t i o n ~vould not be detected by the c o n v e n t i o n a l methods. I n the s p e c t r o p h o t o m e t r i c assay metho,d [35] A~D'Pf o r m a t i o n is coupled w i t h the c o n v e r s i o n of phosph, o e n o l p y r u v a l e to lactate in the presence of p y r u v a t e kinase and lactate dehydrogenase. The K~ for A,DP i n the p y r u v a t e kinase system at pH 7.0, 25 ° i n the p r e s e n c e of Mn z+ is 0.2 mM [36, 37]. This is several orders of m a g n i t u d e greater t h a n the a p p a r e n t Ka o.f MnEy-.6 for AD,P (2.0 nM at pH 7..6). Moreover, i n the b i o s y n t h e t i c assay m e t h o d i n ~vhich P~ forma-

BIOCHIMIE, 1976, 58, n ° 1-2.

tion is m e a s u r e d [16] the lower limit of Pi measur e m e n i is 0.25 mM whi, ch is well above the K d of M n E ~ for Pi (0.08 mM at pH 7.6). In contrast MgEu.5 can catalyze the f o r w a r d reaction because this enzyme form has relatively low affinities for A~D,P and Pi a n d is, therefore, not subject to strong produ.ct i n h i b i t i o n . W i t h respect to substrate b i n d i n g , data o b t a i n e d from fluorescence ti,trations r e p o r t e d here, a n d in a pre.vious p a p e r [7] and the fact that a m m o n i a can b i n d to the enzyme p r i o r to ATP (Rhee, u n p u blished data 1975) indi.cate that substrates can b i n d to the enzyme in a ranldom m a n n e r . The high substrate specificity m a y be due i n part, if not entirely, to s y n e r g i s i i c effects asso, ciated with substrate b i n d i n g . This s y n e r g i s m is p a r t i c u l a r l y pronoun,ee,d in the case of L-glutamate a n d ATP binding [28] and ADP a n d Pi or arsenate b i n d i n g [9, 2,8]. Synergistic b i n d i n g r e q u i r e s either direct substrate-substrate i n t e r a c t i o n a n d / o r substrate i n d u c e d p r o t e i n c o n f o r m a t i o n a l change(s). Data o b t a i n e d from k i n e t i c studies on substrate b i n d i n g show b i n d i n g o,f ATP, ADP, glutamate, a n d Pi induces enzyme con~formational change(s) (Rhee, u n p u b l i s h e d data 1975). The hypothesis that the saine active c e n t e r is used for the catalysis of reactions (1) to (5) is supp o r t e d by the following c o n s i d e r a t i o n s : (1) Glutamine and glutamate b i n d to the same site on the enzyme. This follows from the data in figure 2 s h o w i n g that L-glutamate is a competitive i n h i b i t o r for the L-glutamine site o~ the transferase reaction in w h i c h the K i for L-glutamate is 3'0 mM at p H 7.6, 37 ° a n d the K m for L-glutamine is 11 raM, and also from the observation that under the b i o s y n t h e t i c or ATPase reaction conditions, L-glu~amine competes with L-glutamate a n d exhibits a dissociation c o n s t a n t of 12 mM at pH 7.2 (table II). Therefore the L-glutamine binding site for the tran.sferase reaction is likely the same site w h i c h b i n d s L-glutamate d u r i n g the bios y n t h e t i c or ATPase reaction. I n addition, the dissociation c o n s t a n t of 2,4 mM o b t a i n e d for the binding of L-glutamate w i t h MgEK6, w h e n saturated w i t h ADP at pH 7.6, 2,5 ° , is i n good agreement w i t h the K i value o b t a i n e d from figure 2 a n d the K d (20 raM) for glutamate estimated b y T i m m o n s et at. [7] based on a r a n d o m a d d i t i o n scheme of ATP a n d glutamate to the enzyme. The fact that these three cons,t,ants are i n relatively good agreement, suggests that the same glutamate b i n d i n g site is i n v o l v e d in the b i o s y n t h e t i c a n d ATPase reactions a n d in the i n h i b i t i o n of the transferase reaction. All o.f ,these observations i n d i c a t e that glutamate a n d glutamine b i n d to the same site.

Glutamine synthetase -- Mechanistic studies.

(2) ATP and ADP b i n d to the same site. F i g u r e 3 shows that AM,P-P-N-P, an ATP analog w h i c h provokes the same p r o l e i n fluorescence e n h a n c e m e n t as does ATP E7], is a competitive i n h i b i t o r for ADP in the MgEi76 catalyzod transferase reaction. The K i (pH 7.0, 37 ° ) calculated from the data in figure 3 (80 ~M) is in good agreement with a value of 70 ~M for the disso.ciation constan~ o b t a i n e d from direct AM~P-P-N-P titration at pH 7.0, 25 °. These results suggest that there is a c o m m o n binding site for ADP, ATP a n d AMP-P-N-P. (3) Ovthophosphate a n d arsenate b i n d at the same sRe. Arsenate is an allernative substrate of P~ in the reverse b i o s y n t h e t i c reaction (,figure 4). W i t h P~ as su,bstra~e, p r o d u c t i n h i b i t i o n is observed since the e n z y m e has relatively high affinity for nucleoside triphosphate. However, no i n h i b i tion is observed w i t h arsenate since the p r e s u m e d product, P~DP-OAsO 3 undergoes r a p i d s p o n t a n e o u s hydrolysis. This leads to an essentially irreversible r e a c t i o n in w h i c h glu~amine is c o n v e r t e d to glutamate and NH~ +. This capacity of arsenate to f u n c t i o n in a catalytic fashion i n the reverse biosynthetic reaction was noted by L e v i n t o w and Meister [12] i n s.tudi,es on the glutamine synthetase from peas, a n d these investigators referred to the reaction as the arsenolysis o'f glulamine. P~ also will substitute for arsenate i n the 7-glutamyl t r a n s f e r reaction (Huang .an,d P u r i c h , u n p u b l i s h e d data 19,73). The fact that P~ a n d arsenate are interchangeable substrates in bo.th the reverse b i o s y n thetic a n d the t r a n s f e r reactions is con,sistent w i t h the view that the sa~me P~ b i n d i n g site is utilized for both reactions. (4) The ~,-phosphate of nueleoside t r i p h o s p h a t e bind,s to the Pi site. A'PP ~¢as reported to substitute for AI)P i n the t r a n s f e r reaction catalyzed by the sheep b r a i n enzyme [20] ; however, this is not true for either the Mg2+ or the Mn -0+ activated enzyme from E. colt. I n addition, ATP also fails to s u p p o r t the reverse b i o s y n t h e t i c r e a c t i o n since a d e n o s i n e tetraphosphate (the expected product of the reverse b i o s y n t h e t i c reaction) is not formed. However, a small a m o u n t of [a2P]Pi (24 wM) was f o u n d incorvporated into ATP when Mn~Ei~ was i n c u b a t e d w i t h r e a c t i o n m i x t u r e s cont a i n i n g freshly p u r i f i e d (ADP-free) ATP p r e p a r a tions. This a m o u n t of [~2P]ATP formed was just about equal to the a m o u n t oi A ~ P (2,8 r~M) expected to be formed by slow n o n e n z y m i c h y d r o l y s i s of ATP in the p r e s e n c e o,f Mn ~+ [30]. The observation that MnE 1.o catalyzes i n c o r p o r a t i o n of [32p] Pi into ATP whereas MgEy5 does not is e x p l a i n e d b y the fact that Mn 2+ is a better catalyst for ATP h y d r o l y s i s (31 ~M per 25 min) than is Mge+ (7 !~M

B[OCHIM1E, 1 9 7 6 ,

58, n ° 1 - 2 .

45

per 25 min) [30], a n d by the fact that i n the presence of Pi or arsenate the M n E i : 5 has a higher affinity for ADP (K D = 20 n[M) t h a n does M g E ~ (K n = 25 ~xM). Thus the a m o u n t of AD,P formed by n o n e n z y m i c h y d r o l y s i s of ATP is sufficient to s u p p o r t a significant r e a c t i o n w i t h M n E ~ but not with MgEv.o. The d e p e n d e n c e of A T P - s u p p o r t e d transferase activity on the f o r m a t i o n ot ADP by ATP h y d r o l y s i s is s u p p o r t e d further by the observation th,at although AMP-P-N-P p r o b a b l y b i n d s to the ATP site, as judge,d by the extent of fluorescence e n h a n c e m e n t , it c a n n o t serve as a substrate in the transferase r e a c t i o n catalyzed by eilher M g E ~ or MnEvA. The i n a b i l i t y of ATP to s u p p o r t either the reverse reaction or the transferase r e a c t i o n is con,sistent w i t h a plausible r e a c t i o n m e c h a n i s m in w h i c h the Pi a n d the v-phosphoryl group o,f ATP compete with one a n o t h e r for b i n d i n g at the same site on the enzyme i n the b i o s y n t h e t i c and transferase (or reverse biosynthetic) reactions, respectively. Such competition is also i n d i c a t e d by fluorescence t i t r a t i o n studies s h o w i n g that Pi i n h i b i t s the b i n d i n g of ATP to MnEv.~ a n d by k i n e t i c studies s h o w i n g that ATP i n h i b i t i o n of the transferase reaction catalyzed by MgEvA is competitive w i t h respect to the substrate arsenate (Park, u n p u b l i s h e d da~a 19:74). (5) AMP b i n d s to the ADP site d u r i n g the catalysis of the reverse b i o s y n t h e t i c a n d t r a n s f e r reactions. Figure 5 shows that AMP supports the reverse b i o s y n t h e t i c reaction i n the p r e s e n c e of MnEy-.c, The A T P : g l u t a m a t e ratio is, w i t h i n exper i m e n t a l error, 1:2. The fact that no [32p]ADP was detected a m o n g the reaction p r o d u c t s suggests that the ADP pro,duced i n the first reaction of glutamine w i t h AMP is p h o s p h o r y t a t e d to ATP in the second reaction w i t h glutamine, before it can dissociate from the enzyme. This implies that release of ADP from the MnEy.0.0 is a slow process. Res.ults of stopped-flow fast reaction k i n e t i c studies (Rhee, u n p u b l i s h e d data, 1975) show that this is the case. Moreover, the affinity of MnEy5 for ADP is about 2.5 X 103 times greater than AMP, as judged by the K m values derived from steadystate kinetic studies of v-glutamyl transferase reaction (Smyrniotis a n d St~dtman, u n p u b l i s h e d date 1973). A dissociation constant (0.125 raM) d e t e r m i n e d by direct b i n d i n g m e a s u r e m e n t s rL38] is a,bout 10 times lower t h a n the observed K i or Km and is not significantly different for MnEyo, MgEyo or relaxed enzyme. The a p p a r e n t d i s c r e p a n c y b e t w e e n k i n e t i c a n d direct b i n d i n g m e a s u r e m e n t s is likely due to the fact that the b i n d i n g m e a s u r e m e n t s were made in the absence

46

S. G. Rhee, P. B. Chock and E. R. S t a d t m a n .

of o t h e r substrates. O~ther studies indi,cate that i n t e r a c t i o n s b e t w e e n b o u d s u b s t r a t e s a n d / o r betw e e n s u b s t r a t e b i n d i n g sites does affect the bind i n g affini,ties for different suhstra.tes [7, 28]. In a d d i t i o n , Hunt et aI. [9] s h o w e d that AMP and a r s e n a t e 'support the t r a n s f e r r e a c t i o n c a t a l y z e d b y M,nEys. The fact that AMP can r e p l a c e ADP in the r e v e r s e b i o s y n t h e t i c a n d trans~fer r e a c t i o n s suggests t h a t AMP p r o b a b l y b i n d s to the ADP site. F i g u r e 6 s h o w s that b o t h M g E ~ .o and MnEx. o catalyze the glutamate dependen,t ATPase re,action, almost equally well, a n d the pyrroli,done carb o x y l a t e f o r m a t i o n is ac.co~mpani~d by the p r o d u c tion of n e a r l y s t o i c h i o m e t r i c a m o u n t of P~, in a c c o r d a n c e w i t h r e a c t i o n (3). This is in c o n t r a s t to the d a t a of W e i s b r o d and Meister [11] s h o w i n g that Mn 2÷ s u p p o r t e d u n a d e n y l y l a t e d enzyme exhibited only 15 to 3;6 p e r cent of the Mg2+ s u p p o r t e d glutamate d e p e n d e n t ATPase activity. The fact that both Mg-~÷ and Mn e+ s u p p o r t the ATPase activity is consistent w i t h the d,ata w h i c h s h o w both d i v a l e n t metal ions s u p p o r t the f o r m a t i o n of an a c t i v a t i o n i n t e r m e d i a t e be~veen ATI~ a n d glutamate as j.udged b y the fluorescent s p e c t r a l changes a n d b y the stu, dies of r e v e r s e b i o s y n t h e t i c react i o n in the p r e s e n c e o,f Mg 2+ a n d Mn 2÷. It is believed that the activ.ation i n t e r m e d i a t e is likely to be :-gluta~nyl p h o s p h a t e w h i c h is a s s u m e d to be the p r e c u r s o r o,f p y r r o l i d o n e c a r b o x y ] a t e formation. The rates o b s e r v e d (~igure 6) at p H 7.0 s h o w that Mg2+-enzyme is slightly m o r e active t h a n Mn 2+enzynae. The r e l a t i v e a c t i v i t y b e t w e e n Mg2+- and Mn2÷-enzyme is p H d e p e n d e n t (figure 8) ; a n d the p H p r o f i l e in F i g u r e 8 is different f r o m those e x h i b i t e d by the c o r r e s p o n d i n g r e a c t i o n s catatyzed b y the sheep b r a i n e n z y m e in the p r e s e n c e o,f ~Mg2+ [8]. This suggests that the rate l i m i t i n g step is d e p e n d e n t on the c o n f o r m a t i o n a l state of the enzyme. The rate l i m i t i n g step p r o b a b l y is the c y c l i z a t i o n p r o c e s s t a k i n g p l a c e on the enzyme or the release o.f v-glutamyl p h o s p h a t e from t.he enzyme [3]. This rate shoul,d be so slow such that the release c~f AI)P from the MnE
BIOCHIMIE, 1976, 58, n ° 1-2.

tic reaction, in w h i c h case GTP is the p r o d u c t . This p r o v i d e s a di,fferent i n t e r p r e t a t i o n of e a r l i e r studies b y W e d l e r an, d B o y e r [32], sho'wing that @DP supresses Pi ~ " ATP exchange, ~vithout affecting.the glutamate ~ glutamine exchange. To e x p l a i n this result, t h e y p r o p o s e d a <> nmdel, b a s e d on the a s s u m p t i o n tha,t GDP i n t e r f e r e s s e l e c t i v e l y w i t h the d i s s o c i a t i o n of ATP from the enzyme. H o w e v e r , since GDP can r e p l a c e AD,P in the r e v e r s e r e a c t i o n (figure 4), the ~failure to observe Pi ~ ATP e x c h a n g e in the p r e s e n c e of GDP could be due to d i r e c t comp e t i t i o n o,f GDP w i t h ADP as a p h o s p h a t e a c c e p tot. This i n t e r p r e t a t i o n is s u p p o r t e d b y the data in figure 10, s h o w i n g that the enzyme catalyzes t r a n s f e r of the v - p h o s p h o r y t group of ATP to GD,P. The results suggest that, w h e r e a s GDP w i l l s u p p r e s s the P~ ~ - - ~ - ATP exchange, it w o u l d not supp,ress the e x c h a n g e of Pi into total nucleoside t r i p h o s p h a t e s , i.e. the P~ :< ~ ATP + GTP exchange. I n a d d i t i o n to GDP, IDP can serve as p h o s p h o ryl group a c c e p t o r in the r e v e r s e b i o s y n t h e t i c rea, ction as s h o w n in figure 9. The fact that the enzyme exhibits r e l a t i v e l y n o n s p e c i f i c r e q u i r e m e n t s for p u r i n e n u c l e o t i d e suggests that c o u p l i n g of the f o r w a r d a n d r e v e r s e b i o s y n t h e t i c r e a c t i o n s s h o u l d lead to t r a n s p h o s p h o r y l . a t i o n b e t w e e n w a r i o u s n u c l e o s i d e t r i p h o s p h a t e s and d i p h o s p h a t e s as d e s c r i b e d in r e a c t i o n (5). F i g u r e 10 s ho~vs so,me d a t a on t r a n , s p h o s p h o r y l a t i o n reactions. Of the r e a c t i o n s presente,d, the MgEy~ is the most effective and MnE1. o is the least effective catalyst for the t r a n s p h o s p h o r y l a t i o n f r o m ATP to GDP. The slow rate e x h i b i t e d for MnEy~ is r e a d i l y e x p l a i n e d b y the fact that d i s s o c i a t i o n of A ~ P from MnE~z~-ADP-P i c o m p l e x is a v e r y slow process. This is consistent w i t h the p r o p o s e d m e c h a n i s m w h i c h is si,mply the c o u p l i n g of the f o r w a r d and r e v e r s e b i o s y n t h e t i c r e a c t i o n . F u r t h e r m o r e , .the orde,r o¢ relative r e a c t i v i t y is exhib i t e d by the nu, cleoside d i p h o s p h a t e s a,s p h o s p h o ryl group a c c e p t o r s in the r e v e r s e b i o s y n t h e t i c and t r a n , s p h o s p h o r y l a t i o n reactions. Even t h o u g h the o b s e r v e d t r a n s p h o s p h o r y l a t i o n r a t e is relatively slow, it can be i m p o r t a n t p h y s i o l o g i c a l l y since glutamine s y n t h e t a s e c o n c e n t r a t i o n in the n i t r o g e n s u p p r e s s e d cell is v e r y high (,~ 14 .aM). Based on the overall dat,a, w h i c h also i n d i c a t e that the same active site is b e i n g utilize,d by the e n z y m e in t,he c a t a l y s i s of r.e.action (1) to (5), a s t e p w i s e m e c h a n i s t i c s c h e m e ~s p r e s e n t e d h e r e (figure 1,1) to accoun,t for all r e a c t i o n s s t u d i e d . In a d d i t i o n , an a c t i v a t i o n e n e r g y d i a g r a m , designed to e x p l a i n the relative rate of b i o s y n t h e t i c

Glntamine synthetase -- Mechanistic studies.

a n d t r a n s f e r a s e r e a c t i o n s (table HI) b a s e d on the p r o p o s e d m e c h a n i s m s is given in figure 12. This scheme, to a large exten,t, agrees w e l l w i t h that p r o p o s e d b y Meister a n d his c o - w o r k e r s for the m a m m a l i a n glutamine s y n t h e t a s e [3]. In the p r o p o s e d scheme, there are two p o s s i b l e m e c h a n i s m s ,

47

action b e t w e e n an oxygen atom f r o m P~ w i t h the 8-carbon of glutamine to i n i t i a t e the r e v e r s e biosyntheEtic r e a c t i o n , followed b y a m o r e e n e r g e t i c step in w h i c h oxygen is t r a n s f e r r e d f r o m Pi to glutamine and ATP is f o r m e d from P~ a n d ADP. This is s u b s t a n t i a t e d by the o b s e r v e d t r a n s f e r of

Glut + ATP

0 N

Pyrrohdone Carboxylate

0

0

O

!_AO.l

"

--0 • R 0

+ PI + ADP

NH3

R

~GIuNOH i Pi+ ADP~

0

0

L,'"-0

--ADP? 0

LH2N--!'''O--i'''AD ~l

FIG. 1 L Proposed mechanistic scheme for biosynthetic, transferase, ATPase, and transphosphorglation reactions. The parenthesis indicates

enzyme bound.

Gin + PI + ADP

w h i c h are consistent w i t h all da~a available. One i n v o l v i n g the f o r m a t i o n of v-glu,tamyl p h o s p h a t e (or arsenate) as a r e q u i r e d i n t e r m e d i a t e in the b i o s y n t h e t i c r e a c t i o n an,d the other i n v o l v i n g p a r tial b o n d b r e k i n g a n d f o r m i n g t r a n s i t i o n state i n t e r m e d i a t e s . Both m e c h a n i s m s r e q u i r e the i n t e r -

>(.9 n,LU Z UJ Z 0

S

s

< > t-0 <

Products

REACTION COORDINATE

FiG. 12. - - Relative activation energy profile ~ i t h respect to activation complexes indicated in figure 11. BIOCHIMIE, 1976, 58, n ° 1-2.

180 ,from Pi to form 180-glutamate. The f o r m a t i o n of enzyme b o u n d a c t i v a t i o n c o m p l e x I (figure 11) is also s u p p o r t e d b y o x y g e n t r a n s f e r e x p e r i m e n t s s h o w i n g that the 180 from 7 - c a r b o x y l group of glutamate is t r a n s f e r r e d to Pi [33, ?,4]. Mechanism one involves the f o r m a t i o n of enzyme b o u n d 7-glutamyl p h o s p h a t e (V) (or arsenate) from a c t i v a t i o n co;mplex I, or from activation c o m p l e x H, w i t h the a s s u m p t i o n t h a t II cannot r e a c t w i t h NH 3 to f o r m glutamine, ADP a n d Pv y-Glutamyl p h o s p h a t e (V) w i l l react r e a d i l y w i t h NH 3 or NH20H to f o r m g l u t a m i n e o r 7-glutamyl hydroxamate respectively, or decompose to pyrroli,done carboxyla~e an,d Pi in the a b s e n c e of NH 3 or NH2OH. In the r e v e r s e b i o s y n t h e t i c a n d t r a n s f e r reactions, this m e c h a n i s m calls for a d i r e c t i n t e r a c t i o n b e t w e e n glutamine a n d Pi (or arsenate) to form 7-glutamyl p h o s p h a l e (or arsenate) w i t h o u t the requiremen.t of d i r e c t ADP-P i i n t e r a c t i o n . H o w e v e r , the b i n d i n g e n e r g y of ADP is utilized for i n d u c i n g (or stabilizing) the p r o t e i n c o n f o r m a t i o n n e e d e d to p r o v i d e the r e q u i r e d tert i a r y s t r u c t u r e and some of the free e n e r g y for the v-glutamyl p h o s p h a t e (arsenate) f o r m a t i o n [39, 40]. If d i r e c t ADP-Pi i n t e r a c t i o n is r e q u i r e d for the r e v e r s e b i o s y n t h e t i c r e a c t i o n , then enzyme

48

S. G. Rhee, P. B. Chock and E. R. S t a d t m a n .

b o u n d aativation complex III should be formed first, followed by I I - ~ - ' V -- ~ I. The f o r w a r d biosynthetic r e a c t i o n will then proceed 'from I-->-V-~-II->-III. This p a t h w a y requires the AD,P-Pi b o n d to be b r o k e n , formed a n d broken, a n d suggests that NH 3 will not react w i t h II such that V is the o n l y rea~ctive i n t e r m e d i a t e . Mechanism two involves no f o r m a t i o n of "¢-glutamyl phosphate w h e n NH 3 a n d NH20H are present. NH 3 reacts w i t h a c t i v a t i o n colrrplex II w h i c h is a relatively unstable complex c o m p a r e d to v-glutamyl phosphate. W h e n .NHa a n d NH2OH are absent, this u n s t a b l e complex II will p r o c e e d to form 7-glutamyl phosphate, w h i c h will f u r t h e r decompose slowly to p y r r o R d o n e carboxylate a n d Pv W i t h this m e c h a n i s m , b i o s y n t h e t i c r e a c t i o n

~

will p r o c e e d from glutamate, ATP ~ _ I H ->" III ~ glutamine, ADP, a n d Pi ; while the transfer reaction p a t h w a y is g l u i ' a m i n e - > I I I - ~ - I I - ~ - I V - > - 7 - g l u t a m y l h y d r o x a m a t e . Direct interaction of &DP-P i is r e q u i r e d for both reverse bios y n t h e t i c and t r a n s f e r re.actions. Such an interaction is consistent wi~.h the fact that A,D,P a n d P~ are b o u n d to the ATP site. The fa.ct that t r a n s f e r r e a c t i o n is faster t h a n the reverse b i o s y n i h e t i c reacAion ; a n d arsenate is a more effective substrate t h a n Pi for the transferase reaction, whereas the opposite is true for the reverse b i o s y n t h e t i c reac~tion (table l i D , suggems that only a relatively low degree of interaction b e t w e e n arsenate or Pi with glutamine is r e q u i r e d to activate the a~nide c a r b o n for nucleop h i l i c attack by NrH20,H. This indicates that a relatively low actival[on energy is r e q u i r e d for the formation of an activation complex for the t r a n s f e r reaction, e.g. from p r o d u c t s to ¥ or to II (figure 12). Therelfore, the rate l i m i t i n g step for the t r a n s f e r rea.ction w o u l d be the i n t e r a c t i o n betw e e n the 8-carbon o,f glutamine a n d the oxygen of either Pi or arsenate. Since arsenMe is a better nucleophi'le t h a n Pi [41], the r e a c t i o n goes better w i t h arsenate t h a n w i l h Pi. However, i n the reverse biosynbhetic eeaction, the pa*hway wJ~ll i n c l u d e the f o r m a t i o n of the activation complex of the t r a n s f e r reaction, followed b y a more energetic process lo form I (V to I or II to I i n figure 12), i n w h i c h b o n d s b e t w e e n oxygen of Pi a n d 8-carbon of glutamine, a n d b e t w e e n Pi a n d AD'P are largely formed. Sin,ce ATP, d e r i v e d from Pi a n d ADP, is m o r e stable t h a n the c o r r e s p o n d i n g arsenate derivative, (AI)P-arsenate) Pi should be a better substrate t h a n arsenate for the reverse b i o s y n t h e t i c reaction. It is believed that the m e c h a n i s m s p r e s e n t e d in figure 11 will hold for E. colt enzyme w h e n diva-

BIOCHIMIE, 1976, 58, n ° 1-2.

lent metal ion other than Mg2+ or Mn2÷ is used as activator. Bu,t, o n e expects to observe differences i n relative ac~ivi~ty a n d pH profile, w h e n p r o t e i n c o n f o r m a t i o n a l states are different due to stabilization by di,fferent metal ions. Table HI, group B, shows that at pH 7.0, w i t h ADP a n d Pi as substrafes, MnEv.~ is more effeotive than M g E : ~ in catalyzing the t r a n s f e r reaction. This is c o n s i s t e n t w i t h observations of H u n t et al. [9]. Hoavever, i n catalyzing the reverse b i o s y n t h e t i c reaction, M g E ~ .o is more aeAive tha,n MnEI7~. These differences are believed to he caused by the fact Mg2+ a n d Mn2+ stabilize different c o n f o r m a t i o n a ] states of the en.zyme [26, 27]. W h e n AMP is a substrate, MnET.0 system exhibits relatively good transferase activity (23 per cent of ADP system) a n d poor reverse b i o s y n t h e tic activity. The ratio b e t w e e n transferase activity a n d reverse b i o s y n t h e t i c activity is 6,0 f o r the AMP system a n d 12 for the ADP system (table HI). By assuming the a d e n o s i n e moiety b i n d s at the same site, the observed ratios can be explained by m e c h a n i s m s one a n d two, since the distances b e t w e e n AMP or ADP and Pi or arsenate, a n d the 8-carbon of glutamine are c r u c i a l for the reverse b i o s y n t h e t i c r e a c t i o n a n d less so for the t r a n s f e r reaction. I n s u m m a r y , we have s h o w n that glutamate a n d glutamine b i n d to the same site, ADP a n d ATP share one b i n d i n g s i t e ; a n d in p r i n c i p l e both Mg2+ and Mn 2÷ can s u p p o r t the f o r m a t i o n of activation complex w h e n u n ~ d e n y l y l a t e d enzyme, ATP, and glutamate are present. This implies thai the same ac~tive center is utilized for the catalysis of biosynthetic, transferase, ATPase, t r a n s p h o s phoryl.ation a n d arsenate d e p e n d e n t glutaminase reactions by the u n a d e n y l y l a t e d glutamine synthetase f r o m E. colt. A m e c h a n i s t i c scheme w h i c h is designed to e x p l a i n the data observed a n d those p r e s e n t in the literature is proposed, Of the two m e c h a n i s m s discussed here, both are sequential in n a t u r e w i t h respect to lhe f o r m a t i o n of activation co,mplexes, even lhough substrates b i n d to the enzyme i n a ran,dora m a n n e r . One requires the f o r m a t i o n of 7-glutamyl phosphate as rea'ctive i n t e r m e d i a t e , the other involves activated t r a n s i tion i n t e r m e d i a t e s a n d 7-glutamyl p h o s p h a t e is formed only i n the absen,ce of N,Hz or N H20H.

Aeknawledgment. We thank Dr. D. L. Luterman for determining s o m e of the binding constants presented. R~su~r~.

m~canismes des diverses activit~s (biosynth~tique, transf~rante, ATPasique et transphosphorylante) Les

Glutamine synthelase -- Mechanistic studies. c a t a l y s ~ e s p a r la g l u t a m i n e s y n t h ~ t a s e n o n a d 6 n y l ~ e de E. colt o u t ~16 6tudi6s. L e s c o m p l e x e s d ' a e t i v a t i o n i m p l i q u ~ s darts la r 6 a c t i o n b i o s y n t h 6 t i q u e se p r o d u i s e n t s o i t e n p r 6 s e n c e de Mg ÷+ o u de M n ÷÷ ; t o u t e f o i s , avec l ' e n z y m e - M n ÷+ l ' i n h i b i t i o n p a r le p r o d u i t (ADP) e s t si g r a n d e q u e la r ~ a c t i o n b i o s y n t h 6 t i q u e g l o b a l e ne p e u t p a s 6tre d6tect6e. Des 6 t n d e s de l i a i s o n s m o n t r e n t q u e les s u b s t r a t s ( s a u l NH~ et NH._,OH n o n ~ t u d i ~ s ici) p e u v e n t se l i e r h l ' e n z y m e a u h a s a r d et q u e la l i a i s o n de l ' A T P - g l u t a m a t e , A D P - P i et de l ' A D P - a r s ( ~ n i a t e e s t f o r t e m e n t s y n e r g i q u e . Le m ~ i n e site de f i x a t i o n est n t i l i s 6 p o u r le g l u t a m a t e et la g l u t a m i n e d a n s les rt~actions b i o s y n t h 6 t i q u e et t r a n s f ~ r a s e , et u n site de l i a i s o n c o m m u n p o u r les n u c l ~ o t i d e s e s t u t i l i s 6 p o u r t o u l e s les r 6 a c t i o n s 6tudi~es. Des 6 t u d e s de l a r ~ a c t i o n b i o s y n t h 6 t i q u e r 6 v e r s e et lcs r S s u l t a t s d ' e x p 6 r i e n c e s f l u o r i m 6 t r i q u e s s u g g 6 r e n t q u e l ' a r s 6 n i a t e et l ' o r t h o p h o s p h a t e se l i e n t h u n site c h c v a u c h a n t le s i t e y - p h o s p h a t e d e s n u c l ~ o t i d e s trYp h o s p h a t e s . D a n s les r 6 a e t i o n s b i o s y n t h t ~ t i q u e s r 6 v e r s e et de t r a n s f 6 r a s e , I ' A T P e s t u n s u b s t r a t p o u r l ' e n z y m e M n ++ m a t s p a s p o u r l ' e n z y m e - M g +÷. L ' a c t i v i t 6 t r a n s f 6 r a s e de l ' e n z y m e - M n ÷÷ e s t p r o b a b l e m e n t f a e i l i i 6 e p a r la f o r m a t i o n d ' A D P e o n s 6 c u t i v e h l ' h y d r o l y s e de I ' A T P . Q u a n d I'AMP est le s e u l n u e l 6 o t i d e a j o u t 6 , il e s t c o n v e r t i e n A T P avee f o r m a t i o n e o n e o m i t a n t e de d e u x 5 q u i v a l e n t s de g l u t a m a t e , d a n s les c o n d i t i o n s de ]a r ~ a e t i o n r~verse, et o n n e d~teete p a s d ' A D P . O n c o n f i r m e la r 6 v e r s i b i l i t 6 d u t r a n s f e r t d'~80, e n t r e l ' o r t h o p h o s p h a t e et le g r o u p e 7 - a c y l e d u g l u t a m a t e . L ' a c t i v i t ~ A T P a s e d e s e n z y m e s n o n a d ~ n y l 6 s e s t h p e u p r 6 s ]a m ~ m e a v e c Mg ++ et M n ÷÷. D a n s les d e u x cas, d e s r 6 a c t i o n s de t r a n s p h o s p h o r y l a t i o n entre divers purine n u c l S o s i d e t r i p h o s p h a t e s et n u c l 6 o s i d e d i p h o s p h a t e s s o n t c a t a l y s 6 e s d a n s les c o n d i t i o n s de la r 6 a e t i o n b i o s y n t h ~ t i q u e . L e s r 6 s u l t a t s s o n t e n a c c o r d avec l'hypoth6se selon laquelle un seul centre actif est u t i l i s 6 p o u r r o u t e s l e s r 6 a c t i o n s 6tudi~es. D e u x mt~canismes s~quentiels expliquant les r ~ s u l t a t s s o n t discut~s. REFERENCES. 1. S t a d t m a n , E. R. (1973) i n <>, S. P r u s i n e r a n d E. R. S t a d t m a n , Eds., N e w York, N.Y., A c a d e m i c P r e s s , p. 1-6. 2. S t a d t m a n , E. R. ~ G i n s b u r g , A. (1974) i n ¢ The E n z y m e s >>, 3rd. ed., Vol. 10, P. D. Boyer, Ed., Ne'w York, N.Y., A c a d e m i c P r e s s , p. 755-807. 3. M e i s t e r , A. (1974) i n <> 3rd ed., Vol. 10, P. D. B o y e r , Ed., N e w York, N.Y., A c a d e m i c P r e s s , p. 699-754. 4. W e o l f o l k , C. A., S h a p i r o , B. M. a S t a d t m a n , E. R. (1966) Arch. Biochem. Biophys., 116, 177-192. 5. G i n s b u r g , A., Yeh, J., H e n n i g , S. B. ~ D e n t o n , M. D. (1970) Biochemistry, 9, 633-648. 6. W e d l e r , F. C. a B o y e r , P. D. (1972) J. Biol. Chem.. 247, 984-992. 7. T i m m o n s , R. B., R h e e , S. G., L u t e r m a n , D. L. Chock, P. B. (1974) Biochemistry, 13, 4479-4485. 8. K r i s h n a s w a m y , P. R., P a m i l j a n s , V. ~ Meister, A. (1962) J. Biol. Chem., 237, 2932-2940.

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