BIOCHIMIE,
1979, 61, 663-669.
Spatial proximity of a tyrosyl and a lysyl residue in the active site region of yeast 3-phosphoglycerate kinase. Claude ROUSTAN , Abdellatif FATTOUM and Louise-Anne PRA~DEL.
Centre de R e c h e r c h e s de B i o c h i m i e M a c r o m o l ~ c u l a i r e du CNRS, B.P. 5051, 34033 M o n t p e l l i e r C e d e x , F r a n c e .
Rdsum~.
En conclusion, ce r6sidu 1ysyl est situ6 & proximit6 du q r o u p e m e n t t y r o s y l essentiel darts la r6qion du centre actif.
L'emploi de r6actifs c h i m i q u e s sp6cifiques darts l a modification d e la 3-PGK de l e v u r e a v a i t r6v616 l ' i m p o r t a n c e de certains r6sidus d ' a c i d e s a m i n e s tels q u e des 1ysines, arqinines, u n e tyrosine et un a c i d e q l u t a m i q u e a u sein du centre actif de cet e n z y m e . De plus d e s r6sidus t y r o s y l et g l u t a m y l a v a i e n t 6t6 localis6s d a n s la structure p r i m a i r e . La raise en 6 v i d e n c e d ' u n e proximit6 s p a t i a l e entre u n e Iysine d ' u n e p a r t et la tyrosine essentieUe d ' a u t r e p a r t a n6cessit6 l ' e m p l o i d ' u n r6actif dit d e transfert, le 7-chloro-4-nitrobenzof u r a z a n e . Ce r6actif modifie u n seul r6sidu tyrosine, e n t r a i n a n t l'inactivation totale de Fenz y m e s u i v a n t u n e r6action de p s e u d o p r e m i e r ordre. Cette modification n ' e n t r a l n e a u c u n c h a n q e m e n t si~/nificatif du contenu en h61ice a de la prot6ine. Une protection totale contre l'inactivation et la modification d e la t y r o s i n e a 6t6 o b t e n u e en p r 6 s e n c e d e s substrats. De plus I'AMP, le t r i p o l y p h o s p h a t e et le p y r o p h o s p h a t e exercent 6 q a l e m e n t un effet protecteur. En outre, & p H alcalin, u n d 6 p l a c e m e n t du spectre caract6ristique du d6riv6 tyrosine-O-nitrob e n z o f u r a z a n e est o b s e r v & I1 c o r r e s p o n d a u transfert du r6actff de l ' o x y q 6 n e de la tyrosine & l'azote d ' u n e lysine. Le d6riv6 /V-nitrobenzof u r a z a n e form6 est lui a u s s i inactif.
Summary. The effect of 7-chloro-4-nitrobenzofurazan on y e a s t 3 - p h o s p h o q l y c e r a t e k i n a s e c a u s e s a modification of one t y r o s y l r e s i d u e c o n c o m i t a n t l y with a total loss of activity of the e n z y m e . The modification is not a c c o m p a n i e d b y a n y siqnificant c o n f o r m a t i o n a l c h a n q e . A total protection a q a i n s t inactivation is o b s e r v e d with the substrates ; furthermore, AMP, t r i p o l y p h o s p h a t e a n d p y r o p h o s p h a t e afford a n effective protection. At pH 9, a shift in the a b s o r b a n c e spec~ trum of the tyrosine O - n i t r o b e n z o f u r a z a n deriv a t i v e of 3 - p h o s p h o q l y c e r a t e k i n a s e is observed. It c a n b e r e l a t e d to the transfer of the reaqent from tyrosine to lysine. The N-nitrobenzof u r a z a n d e r i v a t i v e is also c o m p l e t e l y inactive. It is c o n c l u d e d that a Iysine residue is located close to the essential t y r o s y l residue.
Key words : Tyr-Lys proximity/Active site/Yeast 3-phosphoglycerate kinase.
Introduction. A d d r e s s to w h i c h correspondence s h o u l d be s e n t : Centre de Recherches de B i o c h i m i e Macromol~culaire, CNRS, B.P. 5051, 34033 M o n t p e l l i e r Cedex, France. Abbreviations : -- PGK : 3-phosphoglycerate kinase. - - NBD-CI : 7-chloro-~-nitrobenzofurazan. - - PCMB p - c h l o r o m e r c u r i b e n z o i c acid. Code n u m b e r o[ e n z y m e s : 3-phosphoglycerate k i n a s e EC 2.7.2.3. Glyceraldehyde-3-phosphate dehtldrogenase EC 1.2.1.12.
Yeast 3-phosphoglycerate kinase (EC 2.7.2.3) is characterized in the glycolytic system by its monomeric form. The folding of its single polypeptide chain has been established from X-ray studies. However, in the absence of sufficient data on the p r i m a r y structure, the crystallographic picture [1] of the 3-phos,phoglycerate kinase molecule cannot include the description of the active
664
C. Rouslan and coll.
center. Furthermore, whereas the nucleotide bind i n g r e g i o n has b e e n a l r e a d y s p e c i f i c a l l y c h a r a c t e r i z e d , t h e 3 - p h o s p h o g l y c e r a t e b i n d i n g site is alw a y s u n c e r t a i n Ell.
(109 mCi/mM) was from C.E.A. (France) ; p-chloromereuribenzoie acid was obtained from NBC. ATP (sodium salt), ADP (sodium salt), AMP (sodium salt), adenosine, NADH (sodium salt) and 3-phosphoglyceratc were from Boehringer.
A s t u d y of the s t r u c t u r e of t h e a c t i v e site of yeast 3-phosphoglycerate kinase has been investig a t e d b y c h e m i c a l m o d i f i c a t i o n s of s e v e r a l a m i n o a c i d s s u c h as c y s t e i n e E2], ]ysine [3j, t y r o s i n e [3, 4, 5, 6 1 , a r g i n i n e [7, 81 a n d g l u t a m i c a c i d [9t. T h e l o c a l i s a t i o n in t h e p o l y p e p t i d e c h a i n of cyst e i n e [101, g l u t a m i c a c i d [113, t y r o s i n e [12] h a s been achieved.
Protein concentration. 3-phosphoglyeerate kinase concentration was determined from absorbance at 280 nm using "a l1 cm % = 5 [18] ; the concentration of modified protein samples was measured either by UV absorbanee or by the mierobiuret method. A moleeular weight of 42000 [191 was used in the calculations.
However, the spatial relationship between these r e s i d u e s in r e g i o n of t h e a c t i v e c e n t e r is still to be d e t e r m i n e d . T h e i r close p r o x i m i t y c o u l d l e a d to i n t e r a c t i o n s w h i c h w o u l d a l t e r t h e i r i n t r i n s i c p r o p e r t i e s i.e., o n e t y r o s i n e p o s s e s s e s an a b n o r m a l p K of i o n i s a t i o n [5]. I n o r d e r to d e t e r m i n e w h e t h e r t w o c r i t i c a l r e s i d u e s a r e a d j a c e n t in t h e t e r t i a r y s t r u c t u r e , t h e use of a m o n o f u n c t i o n a ] r e a g e n t w h i c h c a n u n d e r g o an i n t r a m o . l e c u l a r t r a n s f e r f r o m one r e s i d u e to a n o t h e r is of p a r t i c u l a r i n t e r e s t [13, 141. It is t h e case of 7-chloro-4n i t r o b e n z o f u r a z a n (NBD-C1) w h i c h c a n r e a c t read i l y e i t h e r w i t h t y r o s y l h y d r o x y l or a m i n o g r o u p s of l y s i n e a n d w h i c h h a s b e e n sho~vn to u n d e r g o an i n t r a m o l e c u l a r t r a n s f e r at p H 9 f r o m a t y r o s i n e to a l y s i n e in m i t o c h o n d r i a l Aq~Pase [131.
Physical measurements. Absorption speetra were done on a Cary Model 15 Speetrophotometer. Optieal rotatory dispersion measurements were performed on a Flea type Speetropol I Speetropolarimeter in 0.05 M Tris HC1 buffer pH 7.3 or 9 at room temperature using a 1 em light path cell. The protein eoneentration was 0.1-0.2 m g / m l . The 14C counting was carried out using instagel (Packard) as seientillator in Interteehnique liquid scintillation Spectrometer Model SL 30.
In this communication we present data which i d e n t i f y o n e r e a c t i v e e s s e n t i a l t y r o s i n e r e s i d u e in 3-phosphoglycerate kinase and which show the s p a t i a l p r o x i m i t y of t h i s r e s i d u e w i t h a l y s i n e at t h e a c t i v e site of t h e e n z y m e .
Chemical modi[ication. All operations, unless otherwise stated, were carried out at 30°13 and in 0.05 M Tris HC1 at pH 7.3 or 8.9. Before any modification, the - - S H group of the enzyme was masked with PCMB with an enzyme reagent molar ratio of 1:1. Then, in a typical experiment at pH 7.3, reactions with NBD-CI were initiated by addition of 75 Ixl of 80 mM reagent (in ethanol) to 1 ml of enzyme solution (71 I~M). Aliquots of the mixture were taken at different times and tested for residual enzymic activity. The reactions were stopped by precipitation with (NI4~)~SO4 (600 g/liter) and freed of the excess of reagent by exhaustive dialysis or Sephadex G-25 filtration. The extent of the reagent incorporation is determined by radioactivity measurements. In this ease, the enzyme is incubated with [14C] NBD-C1 as described above. After removing of excess of reagent, the sample was submitted to the radioaetivity counting. To identify and quantify the NBD-amino acid derivatives, the same sample is speetrophotometrieally analyzed. The NBD derivatives can be characterized by an absorption m a x i m a at 475 n m and 350 nm for amino group, 385 nm for the phenolic group. The amount of NBDlysine and NBD-tyrosine can be determined from the E M475 m n = 2fi 0 0 0 and £M3sa nm ~--- 11 600 respectively [13, 20]. The absorbanee of the lysine derivative (partieularly below 385 nm) affects the 385 nm peak. So, this contribution (e N-NBD ---- 3000) [21] has been M38~ nm taken into account for the t y r o s y l determination.
Materials and Methods.
Results.
3-phosphoglycerate kinase f r o m baker's yeast was prepared according to Scopes [16]. A specific activity of about 1300 U/nag at 30°C and pH 7.3 was determined spectrophotometrieally [17] ; glyeeraldehyde 3-phosphate dehydrogenase f r o m yeast (80 units/rag at 25°C) was obtained from Boehringer. 7-ehloro-4-nitrobenzofurazan (7-chloro-4-nitrobenzo-2-oxa-l,3-diazole or NBD-CI) was Merck reagent. The [14C] (U) NBD-C1
Inactivation o[ the enzyme.
T h e N B D d e r i v a t i v e s h a v e i n t e n s e s p e c i f i c abs o r p t i o n s in the v i s i b l e r e g i o n of t h e s p e c t r u m a n d so c a n be u s e d e f f i c i e n t l y at one ~o p e r f o r m k i n e t i c s t u d i e s a n d at the s a m e t i m e to i d e n t i f y the modified amino acid residues. Unfortunately, o n e of the d i s a d v a n t a g e s of N B D - d e r i v a t i v e s is t h e i r l a b i l i t y i n t h e c o n d i t i o n s r e q u i r e d for a m i n o a c i d d e t e r m i n a t i o n [15] a n d t h e r e f o r e t h e i m p o s s i b i l i t y of c h e m i c a l c h a r a c t e r i z a t i o n .
BIOCHIMIE, 1979, 61, n ° 5-6.
It is w e l l k n o w n t h a t NBD-GI r e a c t s o n c y s t e i n e [22]. So, p r i o r m o d i f i c a t i o n t h e s i n g l e a n d n o n e s s e n t i a l t h i o l g r o u p of 3 - p h o s p h o g l y c e r a t e k i n a s e w a s m a s k e d w i t h PCMB. U n d e r t h e s e c o n d i t i o n s , NBD-C1 l e a d s to a t o t a l loss of a c t i v i t y . T h e t i m e
Reaction of NBD-CI with yeast 3-phosphoglycerale kinase. E x t e n t o[ N B D i n c o r p o r a t i o n .
dependent inactivation follows pseudo first-order k i n e t i c s (fig. 1A). T h e i n a c t i v a t i o n r a t e is f u n c t i o n of t h e c o n c e n t r a t i o n of r e a g e n t . A s t r a i g h t l i n e 1 000 (fig. 1B) is o b t a i n e d b y p l o t t i n g log - m i n -1
T h e v i s i b l e s p e c t r u m of t h e m o d i f i e d e n z y m e p r e s e n t s a p e a k at 475 n m a n d a s h o u l d e r at 385 n m (fig. 3) a n d t h u s is c o n s t i t u t e d b y t h e abs o r p t i o n of t y r o s i n e a n d l y s i n e N B D - d e r i v a t i v e s . From the corresponding absorption coefficient (see M e t h o d s ) d e d u c e d f r o m m o d e l c o m p o u n d s
I1/2
v e r s u s l o g (NBD-C1) (raM). T h e c o r r e s p o n d i n g s l o p e e q u a l to 1.07 also i n d i c a t e s t h a t t h e r e a c t i o n
lOO
665
A
8O 60 40
i-_" 20
0
15
30
45
0
60
0.5
1.0
~o~ [NBo_ c,] (,.M)
minutes
F16. 1. - - Kinetics o[ PGK inactivation. PGK : 71 ~tM ; 0.05 M Tris HC1 buffer (©--©) pH 7,3, (@---O) pH 8.9 ; t e m p e r a t u r e 30°C. A : Time course of i n a c t i v a t i o n ; NBD-C1 : 5.6 raM. B : D e t e r m i n a t i o n of the order of reaction with respect to the NBD-C1 concentration.
is f i r s t o r d e r a n d s u g g e s t s t h a t i n a c t i v a t i o n is a c h i e v e d b y t h e i n c o r p o r a t i o n of o n l y o n e m o l e of r e a g e n t p e r e n z y m e . T h e p H d e p e n d e n c y of t h e r a t e of i n a c t i v a t i o n is s h o w n i n f i g u r e 2. A n a p -
0.4
°. 3
1,0 @ U C 0
o
0.2 0
3 2
< o.s
"5 c 0.1
/ 0 7
B
9 600
pH
Fro. 2. - - pH rate profile f o r the inactivation. Reaction conditions were as described in figure 1 w i t h 6 mM NBD-C1.
p a r e n t p K c o r r e s p o n d i n g to t h e r e s i d u e i n v o l v e d i n t h e i n a c t i v a t i o n p r o c e s s c a n b e e v a l u a t e d at a b o u t 8.7. BIOCHIMIE, 1979, 61, n ° 5-6.
s;0
'
,;0
-
.k. nm
Fro. 3 . -
Visible spectrum o f NBD-PGK reaction products. Spectra are carried o u t on 32 ~tM PGK in 1 cm l i g h t p a t h c e l l s . ( - ) spectrum is p e r f o r m e d on a n e n z y m e sample modified as follow • 18 ttM enzyme are reacted w i t h 1.4 mM NBD-CI for ½ h o u r s at "30°C in 0.05 M Tris HC1 buffer pH 7.3. The excess of reagent is elimin a t e d as described in m e t h o d s ; (. . . . ) the sample is t h e n b r o u g h t at pH 9 a n d allowed to stay a t 25°C for 24 h.
C. R o u s t a n a n d coll.
666
[13, 20], the extent of NBD i n c o r p o r a t i o n on tyrosyl and lysyl residues is d e t e r m i n e d . To confirm the results o b t a i n e d from the s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n s , r a d i o a c t i v i t y labelling with[14CJ NBD-C1 are p e r f o r m e d . The values o b t a i n e d from several d i f f e r e n t l y labelled p r o t e i n samples are p r e s e n t e d in Table I t o g e t h e r w i t h the results f o u n d by s p e c t r o t i t r a t i o n . These t w o a n a l y t i c a l m e t h o d s give the same results and are in good agreement. T h e y p o i n t out that t h e c h e m i c a l m o d i f i c a t i o n is m a i n l y d i r e c t e d t o w a r d s tyrosine.
S t o i c h i o m e t r y of the reaction. The rate of t y r o s i n e m o d i f i c a t i o n (k~) is s i m i l a r to that o b s e r v e d for i n a c t i v a t i o n (fig. 4). This fact is in a c c o r d w i t h data of figure 6 and table I
• E
1.0
c 6 o
-g fi
5.0
N
Kinetic analysis of NBD incorporation. Changes in a b s o r p t i o n at 475 n m and 385 nm as a f u n c t i o n of t i m e s h o w quite different k i n e t i c patterns. Change at 385 nm presents an e x p o n e n tial c u r v e (fig. 4) w h e r e a s change at 475 nm is s i g m o i d a l (fig. 5).
10
20
30
40
minutes
FIG. 5. - - Effect o f p H on the t i m e course of N - L g s m e -
NBD-derivative formation.
PGK : 70 a M ; NBD-CI : 2 m M ; temperature 30°C ; 0.05 M Tris HC1. buffer, ( - - - - ) pH 7.1 (--.--.--) pH 8.0 ," (. . . . ) pH 8.4 ( ) pH 8.9. 1,0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
~ E 0,5
o
~oo
:o.3
70
E07
0
.;
w h e r e is i l l u s t r a t e d the c o r r e l a t i o n b e t w e e n the n u m b e r of O-Tyr-NBO and the loss of activity. So, a single m o d i f i e d t y r o s y l r e s i d u e w o u l d be r e s p o n s i b l e for the total i n h i b i t i o n of the enzyme. 100
t-
0
20
1' minor,s
2
40
minutes
FW,. 4 . -
Time course of tgrosine modification.
PGK : 360 IxM ; 0.05 M Tris HC1 buffer pH 7.3 ; NBD-Cl 6.2 mM. Temperature 30°C. Inset : correlation between the number of tyrosine modified versus time (O--Q) and the per cent of residual activity (©--©).
T h e r e a c t i o n of t h e NB,D-CI w i t h h y d r o x y l group of t y r o s i n e f o l l o w e d by s p e c t r o p h o t o m e t r y at 385 nm s h o w s a p s e u d o f i r s t - o r d e r kinetics (fig. 4). The c o r r e s p o n d i n g a p p a r e n t constant rate k 1 is about 0.07 rain 1 at pH 7.3. Tim timecourse for the f o r m a t i o n of N-NBD f o l l o w e d at 475 nm s h o w a lag phase and can be a n a l y z e d assuming successive r e a c t i o n s [213. kl k2 HO-E-NH 2 -+ N BDO-E-I~H2 --~ NBDO-E-NH-NBD In this p r o c e s s the value of the constant rate ratio k l / k 2 can be f o u n d equal to about 0.5 as w e l l at p H 7.3 as pH 8.9. So, the rate of m o d i f i c a t i o n of t y r o s i n e and lysine both i n c r e a s e s w i t h pH.
BIOCHIMIE, 1979, 61, n o 5-6.
,~
50
1.0
2.0
O - Tyr, _ N B O g r o u p ~ e n z y m e
FIG. 6 . -
Residual aetioity of PGK oersus tool of modified tyrosine.
Determination by radioactivity labeling (O) or speetrophotometrie titration (O).
Protection against inactivation. The effect of several substrates or analogues on the rate of the i n a c t i v a t i o n by NBD-C1 was tested. It is clear from the results in Table II that an
Reaction of NBO-CI with yeast 3-phosphoglycerate kinase. a l m o s t total p r o t e c t i o n a g a i n s t i n h i b i t i o n is o b s e r ved with substrates (3-phosphoglycerate, ATP a n d AI)P) u s e d at s a t u r a t i n g c o n c e n t r a t i o n s [23].
667
c a t i o n , the i n c o r p o r a t i o n of N B D on t y r o s i n e w a s o n l y s l i g h t a n d w a s r e l a t e d to the f e e b l e loss of activity.
TABLE I.
Comparison of the extent of NBD-incorporation determined by spectrophotometric titration and radioactive I~C labelling. NBD incorporation
Experiments
Inhibition
ATP-Mg ADP AMP Adenosine Tripolyphosphate Pyrop hosphate
22 28 30 22 30 30
Residue
0.43 0.65 1.00
0.36 0.55 0.80
0.07 0.10 0.20
mole / mole enzyme
36 57 76
0.43 0.64 0.90
Protection (p.cent) pli 7.3
10
Residue
per. cent
Concentrations (raM)
30
mole / mole enzyme
titration
Effect of various ligands on the inactivation of 3-phosphoglycerate kinase by NBD-CI.
3-phosphoglycerate
N-Lys-NBD
Spectro-
rement
TABLE II.
Ligands
O-Tyr-NBD
1~C measu-
95 95 95 95 66 10 71 79
pfl. 8.9
95 95 95 51 32 78 25
/molo enzyme
/mole enzyme
Transfer of the NBD group from tyrosine to lysine. S a m p l e s of e n z y m e i n a c t i v a t e d at p H 7.3 a n d at d i f f e r e n t l e v e l s are f r e e d f r o m e x c e s s of r e a g e n t a n d t h e n i n c u b a t e d at p H 9. T i m e - d e p e n d e n t c h a n g e in the v i s i b l e s p e c t r u m Yeas o b s e r v e d . T h i s c h a n g e c o r r e s p o n d s to an i n c r e a s e at 475 n m a n d a d e c r e a s e at 385 n m (fig. 3). T h i s r e f l e c t s a t r a n s f e r of t h e NBD g r o u p f r o m t y r o s y l o x y g e n to a n i t r o g e n g r o u p of a l y s i n e . T h e r a t e of t h i s t r a n s f e r ( t l / 2 ~ 15 h) ~vas t h e s a m e at s e v e r a l d i f f e r e n t p r o t e i n c o n c e n t r a t i o n s (0.5 m g - 1.6 m g / m l ) a n d t h e a d d i t i o n of an e x c e s s of n a t i v e e n z y m e to T y r O-NBD- d e r i v a t i v e d i d n o t i n c r e a s e t h e r a t e of
TABLE I l L
Migration of NBD-group. Modified amino acid (mole/mole enzyme)
Expt.
before transfer (a) Tyr
1 2 3
0.40 0.80 1.05
Lys 0.13 0.20 0.50
after transfer (b) Tyr 0.22 0.40 0.65
NBD-transfer (mole/mole enzyme)
Lys 0.31 0.60 0.90
0.18 0.40 0.40
(a) The enzyme was inactivated at pH 7.3 and freed from excess reagent. (b) The transfert was performed by incubating the modified enzyme at pH 9 for 24 hours.
In a d d i t i o n , an i m p o r t a n t e f f e c t is also o b s e r v e d w i t h o t h e r l i g a n d s s t u d i e d . W h e n e i t h e r 3-phosp h o g l y c e r a t e o r ADP w a s p r e s e n t d u r i n g m o d i f i -
BIOCHIMIE, 1979, 61,
n ° 5-6.
l y s i n e m o d i f i c a t i o n . T h e s e d a t a are i n d i c a t i v e of an i n t r a m o l e c u l a r p r o c e s s . T h e e x t e n t of this c o n v e r s i o n is i l l u s t r a t e d in t a b l e III. After t r a n s f e r on
668
C. Roustan and coll.
lysine res4due, the activity of 3-phosphoglycerate kinase is not restored sho~ving that the corresponding N - n i t r o b e n z o f u r a z a n PGK derivative is also completely inactive. F i n a l l y , the optical rotatory d i s p e r s i o n data in the far ultraviolet of the two i n a c t i v a t e d derivatives w e r e c o m p a r e d w i t h those of native enzyme. The value of [a]233 ,m for native phos'phoglycerate kinase, Tyr-NBD and Lys-NBD_ enzymes is - - 5 0 0 0 ° _ 200 °. Thus, there is no significant alteration i n the helix c o n f o r m a t i o n of the m o d i f i e d enzyme.
Discussion. The r e a c t i o n of NBD-C1 w i t h 3-phosphoglycerate kinase results i n the i n a c t i v a t i o n of the enzyme b e i n g of pseudo first-order t o w a r d reagent. The k i n e t i c s of i n a c t i v a t i o n show that the incorp o r a t i o n of one mole of NB~D per enzyme is sufficient to p r o d u c e the loss of activity. The modified a m i n o acid can be s p e c t r o p h o t o m e t r i c a l l y identified as tyrosine. The m o d i f i c a t i o n of only one t y r o s i n e correlates w i t h complete i n a c t i v a t i o n . This s t o i e h i o m e t r y is established either by spect r o p h o t o m e t r y or b y r a d i o a c t i v i t y measurements. The fact that the rate of this i n a c t i v a t i o n is f o u n d to he i d e n t i c a l to that of NBD-O-tyr f o r m a t i o n can be t a k e n as a n o t h e r proof for the s t o i c h i o m e t r y of the reaction. So, all these data show that the enzyme inactivation results from the m o d i f i c a t i o n of tyrosine. I n addition, a N-NBD-derivative c o r r e s p o n d i n g to a lysine m o d i f i c a t i o n can a.lso be observed. Kinetics of this r e a c t i o n are sigmoid. The pH d e p e n d e n c e rates of the reaction t o w a r d T y r o s i n e or Lysine are similar. So, these two latter results suggest a m e c h a n i s m of the NBD r e a c t i o n w i t h 3-phosphoglycerate kinase i n "which the N-NBD-clerivative formation is d e p e n d e n t on a p r i o r m o d i f i c a t i o n of the t y r o s y l group. Such a m e c h a n i s m has already been reported for lysozyme [21, 15]. In the ease of Ribonuclease, the k i n e t i c p a t t e r n is quit~e different ; the r e a c t i o n of NBD nvith the two f u n c t i o n a l groups is r a n d o m [~]. The reactive t y r o s i n e i n yeast 3-phosphoglycerate kinase w o u l d seem to possess an a p p a r e n t pK w h i c h is lower t h a n the n o r m a l value. These properties could be a c c o u n t e d for in part by the p r o x i m i t y effect b e t w e e n the t y r o s i n e and lysine residues. BIOCHIMIE, 1979, 61, n ° 5-6.
P r o t e c t i o n studies w h i c h show that substrates p r e v e n t access of reagent to the essential t y r o s i n e suggest that this anfino-acid residue lies i n the active site region of the enzyme. The results reported here c o n f i r m those obtained p r e v i o u s l y [4] using iodine and strongly suggest that the chemical m o d i f i c a t i o n occurs at the same essential residue. I n contrast, some authors [5, 6] have f o u n d that the n i t r a t i o n of one t y r o s i n e in 3-phosphoglycerate k i n a s e i n d u c e s only p a r t i a l i n a c t i v a t i o n a n d a significant p r o t e c t i o n is observed only w i t h nucleotide substrates [3]. So, is the t y r o s i n e detected b y the NBD-C1 the same one detected by t,etranitromethane ? F u r t h e r w o r k w o u l d be needed to solve this problem. Ho~,ever, the more imp o r t a n t result c o n c e r n s the t r a n s f e r of NBD. If, the 3-phosphoglycerate k i n a s e i n a c t i v a t e d at pH 7.3 is b r o u g h t to pH 9, after e l i m i n a t i o n of excess NBD-C1, the reagent migrates from t y r o s i n e to lysine. The extent of the t r a n s f e r seems to be limited to the fraction of .enzyme modified only on the t y r o s i n e a n d not to be c o n c e r n e d w i t h the fraction modified at once on t y r o s i n e a n d lysine. So, the same lysine w o u l d be modified d u r i n g the NBD-CI reaction a n d the t r a n s f e r process. Any way, the N-NBD-PGK derivative o b t a i n e d after t r a n s f e r is also inactivated, t h e r e b y c o n f i r m i n g the presence of an essential lysine in the active center of the enzyme [3]. It is c o n c l u d e d that t y r o s i n e a n d lysine residues are close on from each other in the t r i d i m e n s i o n a l structure of the active site of 3-phosphoglycerate kinase.
REFERENCES. 1. Bryant, T. N., Watson, H. C. a Wendell, P. L. (1974) Nature (London), 247, 14-17. 2. Arvidsson, L. ~ Larsson-Raznikiewiez, M. (1973) Biochim. Biophys. Aeta, 310, 430-436. 3. Markland, F. S., Bacharach, A. D. E., Weber, B. H., O'Grady, T. C., Saunders, G. C. & Umemura, N. (1975) J. Biol. Chem.. 250, 1301-1310. 4. Roustan, C., Fattoum, A. a PradeI, L. A. (1976) Biochemistry, 15, 2172-2177. 5. Hjelmgren, T., Arvidsson, L. a Larsson-Raznikiewiez, M. (1976) Biochim. Biophys. Acta, 445, 342349. 6. Meyer, M. C. a W e s t h e a d , E. W. (1976) FEBS Left., 71, 25-28.
7. Hjelmgren, T., Strld, L. &Arvidsson, L. (1976) FEBS Lett., 68, 137-140. 8. Philips, M., Roustan, C., Fattoum, A. & Pradel, L. A. (1978) Biochim. Biophys. Acta, 523, 368-376. 9. Desvages, G., Roustan, C., Brevet, A. ,¢ Pradel, L. A. (1977) C. R. Acad. Sci., s~r. D, Sci. Nat. (Paris), 284, 1461-1464. 10. Fattoum, A., Feinberg, J., Desvages, G. ,~ Roustan, C. (1977) C. R. Acad. Sci., s~r. D, Sci. Nat. (Paris), 284, 1843-1846. 11. Desvages, G., Roustan, C. ,~ Prade], L. A. (1978) in preparation.
Reaction of NBD-CI with yeast 3-phosphoglycerate kinase. 12. Bacharach, A. D. E., Markland, F. S., Pellino, A. Weber, B. H. (1977) Bioehem. Biophys. Res. Comm., 74, 165-171. 13. Ferguson, S. J., Lloyd, W. J. ~ Radda, G. K. (1975) Eur. Y. Bioehem., 54, 127-133. 14. Dalloechio, F., Signorini, M. ~ Rippa, M. (1978) Arch. Biochem. Biophys., 185, 57-60. 15. Aboderin, A. A. ~ Boedefeld, E. (1976) Biochim. Biophys. Aeta, 420, 177-186. 16. Scopes, R. K. (1971) Biochem. J., 122, 89-92. 17. Biieher, T. (1947) Biochim. Biophys. Acta, 1, 292-314. 18. Biicher, T. (1955) Methods Enzgmol., 1, 415-422
BIOCHIM1E, 1979, 61, n ° 5-6.
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19. F a t t o u m , A., R o u s t a n , C., Feinberg, J., Desvages, G. Pradel, L. A. (1978) Eur. J. Biochem., 82, 161167. 20. Ferguson, S. J., Lloyd. W. J., Lyons, M. H. ~ Ra~da, G. K. (1975) Eur. "J. Biochem., 54, 117-126. 21. Aboderin, A. A., Boedefeld, E. ~ Luisi, P. L. (1973) Biochim. Biophys. Acta, 328, 20-30. 22. Birkett, D. J., Price, N. C., Radda, G. K. ~, Salmon, A. G. (1970) FEBS Lett., 6, 346-348. 23. Scopes, R. K. (1973) in (Boyer, P. D.ed.) 3rd edn. Vol. 8, pp. 335-351, Academic Press, New York.
1979, 61, 663-669.
Spatial proximity of a tyrosyl and a lysyl residue in the active site region of yeast 3-phosphoglycerate kinase. Claude ROUSTAN , Abdellatif FATTOUM and Louise-Anne PRA~DEL.
Centre de R e c h e r c h e s de B i o c h i m i e M a c r o m o l ~ c u l a i r e du CNRS, B.P. 5051, 34033 M o n t p e l l i e r C e d e x , F r a n c e .
Rdsum~.
En conclusion, ce r6sidu 1ysyl est situ6 & proximit6 du q r o u p e m e n t t y r o s y l essentiel darts la r6qion du centre actif.
L'emploi de r6actifs c h i m i q u e s sp6cifiques darts l a modification d e la 3-PGK de l e v u r e a v a i t r6v616 l ' i m p o r t a n c e de certains r6sidus d ' a c i d e s a m i n e s tels q u e des 1ysines, arqinines, u n e tyrosine et un a c i d e q l u t a m i q u e a u sein du centre actif de cet e n z y m e . De plus d e s r6sidus t y r o s y l et g l u t a m y l a v a i e n t 6t6 localis6s d a n s la structure p r i m a i r e . La raise en 6 v i d e n c e d ' u n e proximit6 s p a t i a l e entre u n e Iysine d ' u n e p a r t et la tyrosine essentieUe d ' a u t r e p a r t a n6cessit6 l ' e m p l o i d ' u n r6actif dit d e transfert, le 7-chloro-4-nitrobenzof u r a z a n e . Ce r6actif modifie u n seul r6sidu tyrosine, e n t r a i n a n t l'inactivation totale de Fenz y m e s u i v a n t u n e r6action de p s e u d o p r e m i e r ordre. Cette modification n ' e n t r a l n e a u c u n c h a n q e m e n t si~/nificatif du contenu en h61ice a de la prot6ine. Une protection totale contre l'inactivation et la modification d e la t y r o s i n e a 6t6 o b t e n u e en p r 6 s e n c e d e s substrats. De plus I'AMP, le t r i p o l y p h o s p h a t e et le p y r o p h o s p h a t e exercent 6 q a l e m e n t un effet protecteur. En outre, & p H alcalin, u n d 6 p l a c e m e n t du spectre caract6ristique du d6riv6 tyrosine-O-nitrob e n z o f u r a z a n e est o b s e r v & I1 c o r r e s p o n d a u transfert du r6actff de l ' o x y q 6 n e de la tyrosine & l'azote d ' u n e lysine. Le d6riv6 /V-nitrobenzof u r a z a n e form6 est lui a u s s i inactif.
Summary. The effect of 7-chloro-4-nitrobenzofurazan on y e a s t 3 - p h o s p h o q l y c e r a t e k i n a s e c a u s e s a modification of one t y r o s y l r e s i d u e c o n c o m i t a n t l y with a total loss of activity of the e n z y m e . The modification is not a c c o m p a n i e d b y a n y siqnificant c o n f o r m a t i o n a l c h a n q e . A total protection a q a i n s t inactivation is o b s e r v e d with the substrates ; furthermore, AMP, t r i p o l y p h o s p h a t e a n d p y r o p h o s p h a t e afford a n effective protection. At pH 9, a shift in the a b s o r b a n c e spec~ trum of the tyrosine O - n i t r o b e n z o f u r a z a n deriv a t i v e of 3 - p h o s p h o q l y c e r a t e k i n a s e is observed. It c a n b e r e l a t e d to the transfer of the reaqent from tyrosine to lysine. The N-nitrobenzof u r a z a n d e r i v a t i v e is also c o m p l e t e l y inactive. It is c o n c l u d e d that a Iysine residue is located close to the essential t y r o s y l residue.
Key words : Tyr-Lys proximity/Active site/Yeast 3-phosphoglycerate kinase.
Introduction. A d d r e s s to w h i c h correspondence s h o u l d be s e n t : Centre de Recherches de B i o c h i m i e Macromol~culaire, CNRS, B.P. 5051, 34033 M o n t p e l l i e r Cedex, France. Abbreviations : -- PGK : 3-phosphoglycerate kinase. - - NBD-CI : 7-chloro-~-nitrobenzofurazan. - - PCMB p - c h l o r o m e r c u r i b e n z o i c acid. Code n u m b e r o[ e n z y m e s : 3-phosphoglycerate k i n a s e EC 2.7.2.3. Glyceraldehyde-3-phosphate dehtldrogenase EC 1.2.1.12.
Yeast 3-phosphoglycerate kinase (EC 2.7.2.3) is characterized in the glycolytic system by its monomeric form. The folding of its single polypeptide chain has been established from X-ray studies. However, in the absence of sufficient data on the p r i m a r y structure, the crystallographic picture [1] of the 3-phos,phoglycerate kinase molecule cannot include the description of the active
664
C. Rouslan and coll.
center. Furthermore, whereas the nucleotide bind i n g r e g i o n has b e e n a l r e a d y s p e c i f i c a l l y c h a r a c t e r i z e d , t h e 3 - p h o s p h o g l y c e r a t e b i n d i n g site is alw a y s u n c e r t a i n Ell.
(109 mCi/mM) was from C.E.A. (France) ; p-chloromereuribenzoie acid was obtained from NBC. ATP (sodium salt), ADP (sodium salt), AMP (sodium salt), adenosine, NADH (sodium salt) and 3-phosphoglyceratc were from Boehringer.
A s t u d y of the s t r u c t u r e of t h e a c t i v e site of yeast 3-phosphoglycerate kinase has been investig a t e d b y c h e m i c a l m o d i f i c a t i o n s of s e v e r a l a m i n o a c i d s s u c h as c y s t e i n e E2], ]ysine [3j, t y r o s i n e [3, 4, 5, 6 1 , a r g i n i n e [7, 81 a n d g l u t a m i c a c i d [9t. T h e l o c a l i s a t i o n in t h e p o l y p e p t i d e c h a i n of cyst e i n e [101, g l u t a m i c a c i d [113, t y r o s i n e [12] h a s been achieved.
Protein concentration. 3-phosphoglyeerate kinase concentration was determined from absorbance at 280 nm using "a l1 cm % = 5 [18] ; the concentration of modified protein samples was measured either by UV absorbanee or by the mierobiuret method. A moleeular weight of 42000 [191 was used in the calculations.
However, the spatial relationship between these r e s i d u e s in r e g i o n of t h e a c t i v e c e n t e r is still to be d e t e r m i n e d . T h e i r close p r o x i m i t y c o u l d l e a d to i n t e r a c t i o n s w h i c h w o u l d a l t e r t h e i r i n t r i n s i c p r o p e r t i e s i.e., o n e t y r o s i n e p o s s e s s e s an a b n o r m a l p K of i o n i s a t i o n [5]. I n o r d e r to d e t e r m i n e w h e t h e r t w o c r i t i c a l r e s i d u e s a r e a d j a c e n t in t h e t e r t i a r y s t r u c t u r e , t h e use of a m o n o f u n c t i o n a ] r e a g e n t w h i c h c a n u n d e r g o an i n t r a m o . l e c u l a r t r a n s f e r f r o m one r e s i d u e to a n o t h e r is of p a r t i c u l a r i n t e r e s t [13, 141. It is t h e case of 7-chloro-4n i t r o b e n z o f u r a z a n (NBD-C1) w h i c h c a n r e a c t read i l y e i t h e r w i t h t y r o s y l h y d r o x y l or a m i n o g r o u p s of l y s i n e a n d w h i c h h a s b e e n sho~vn to u n d e r g o an i n t r a m o l e c u l a r t r a n s f e r at p H 9 f r o m a t y r o s i n e to a l y s i n e in m i t o c h o n d r i a l Aq~Pase [131.
Physical measurements. Absorption speetra were done on a Cary Model 15 Speetrophotometer. Optieal rotatory dispersion measurements were performed on a Flea type Speetropol I Speetropolarimeter in 0.05 M Tris HC1 buffer pH 7.3 or 9 at room temperature using a 1 em light path cell. The protein eoneentration was 0.1-0.2 m g / m l . The 14C counting was carried out using instagel (Packard) as seientillator in Interteehnique liquid scintillation Spectrometer Model SL 30.
In this communication we present data which i d e n t i f y o n e r e a c t i v e e s s e n t i a l t y r o s i n e r e s i d u e in 3-phosphoglycerate kinase and which show the s p a t i a l p r o x i m i t y of t h i s r e s i d u e w i t h a l y s i n e at t h e a c t i v e site of t h e e n z y m e .
Chemical modi[ication. All operations, unless otherwise stated, were carried out at 30°13 and in 0.05 M Tris HC1 at pH 7.3 or 8.9. Before any modification, the - - S H group of the enzyme was masked with PCMB with an enzyme reagent molar ratio of 1:1. Then, in a typical experiment at pH 7.3, reactions with NBD-CI were initiated by addition of 75 Ixl of 80 mM reagent (in ethanol) to 1 ml of enzyme solution (71 I~M). Aliquots of the mixture were taken at different times and tested for residual enzymic activity. The reactions were stopped by precipitation with (NI4~)~SO4 (600 g/liter) and freed of the excess of reagent by exhaustive dialysis or Sephadex G-25 filtration. The extent of the reagent incorporation is determined by radioactivity measurements. In this ease, the enzyme is incubated with [14C] NBD-C1 as described above. After removing of excess of reagent, the sample was submitted to the radioaetivity counting. To identify and quantify the NBD-amino acid derivatives, the same sample is speetrophotometrieally analyzed. The NBD derivatives can be characterized by an absorption m a x i m a at 475 n m and 350 nm for amino group, 385 nm for the phenolic group. The amount of NBDlysine and NBD-tyrosine can be determined from the E M475 m n = 2fi 0 0 0 and £M3sa nm ~--- 11 600 respectively [13, 20]. The absorbanee of the lysine derivative (partieularly below 385 nm) affects the 385 nm peak. So, this contribution (e N-NBD ---- 3000) [21] has been M38~ nm taken into account for the t y r o s y l determination.
Materials and Methods.
Results.
3-phosphoglycerate kinase f r o m baker's yeast was prepared according to Scopes [16]. A specific activity of about 1300 U/nag at 30°C and pH 7.3 was determined spectrophotometrieally [17] ; glyeeraldehyde 3-phosphate dehydrogenase f r o m yeast (80 units/rag at 25°C) was obtained from Boehringer. 7-ehloro-4-nitrobenzofurazan (7-chloro-4-nitrobenzo-2-oxa-l,3-diazole or NBD-CI) was Merck reagent. The [14C] (U) NBD-C1
Inactivation o[ the enzyme.
T h e N B D d e r i v a t i v e s h a v e i n t e n s e s p e c i f i c abs o r p t i o n s in the v i s i b l e r e g i o n of t h e s p e c t r u m a n d so c a n be u s e d e f f i c i e n t l y at one ~o p e r f o r m k i n e t i c s t u d i e s a n d at the s a m e t i m e to i d e n t i f y the modified amino acid residues. Unfortunately, o n e of the d i s a d v a n t a g e s of N B D - d e r i v a t i v e s is t h e i r l a b i l i t y i n t h e c o n d i t i o n s r e q u i r e d for a m i n o a c i d d e t e r m i n a t i o n [15] a n d t h e r e f o r e t h e i m p o s s i b i l i t y of c h e m i c a l c h a r a c t e r i z a t i o n .
BIOCHIMIE, 1979, 61, n ° 5-6.
It is w e l l k n o w n t h a t NBD-GI r e a c t s o n c y s t e i n e [22]. So, p r i o r m o d i f i c a t i o n t h e s i n g l e a n d n o n e s s e n t i a l t h i o l g r o u p of 3 - p h o s p h o g l y c e r a t e k i n a s e w a s m a s k e d w i t h PCMB. U n d e r t h e s e c o n d i t i o n s , NBD-C1 l e a d s to a t o t a l loss of a c t i v i t y . T h e t i m e
Reaction of NBD-CI with yeast 3-phosphoglycerale kinase. E x t e n t o[ N B D i n c o r p o r a t i o n .
dependent inactivation follows pseudo first-order k i n e t i c s (fig. 1A). T h e i n a c t i v a t i o n r a t e is f u n c t i o n of t h e c o n c e n t r a t i o n of r e a g e n t . A s t r a i g h t l i n e 1 000 (fig. 1B) is o b t a i n e d b y p l o t t i n g log - m i n -1
T h e v i s i b l e s p e c t r u m of t h e m o d i f i e d e n z y m e p r e s e n t s a p e a k at 475 n m a n d a s h o u l d e r at 385 n m (fig. 3) a n d t h u s is c o n s t i t u t e d b y t h e abs o r p t i o n of t y r o s i n e a n d l y s i n e N B D - d e r i v a t i v e s . From the corresponding absorption coefficient (see M e t h o d s ) d e d u c e d f r o m m o d e l c o m p o u n d s
I1/2
v e r s u s l o g (NBD-C1) (raM). T h e c o r r e s p o n d i n g s l o p e e q u a l to 1.07 also i n d i c a t e s t h a t t h e r e a c t i o n
lOO
665
A
8O 60 40
i-_" 20
0
15
30
45
0
60
0.5
1.0
~o~ [NBo_ c,] (,.M)
minutes
F16. 1. - - Kinetics o[ PGK inactivation. PGK : 71 ~tM ; 0.05 M Tris HC1 buffer (©--©) pH 7,3, (@---O) pH 8.9 ; t e m p e r a t u r e 30°C. A : Time course of i n a c t i v a t i o n ; NBD-C1 : 5.6 raM. B : D e t e r m i n a t i o n of the order of reaction with respect to the NBD-C1 concentration.
is f i r s t o r d e r a n d s u g g e s t s t h a t i n a c t i v a t i o n is a c h i e v e d b y t h e i n c o r p o r a t i o n of o n l y o n e m o l e of r e a g e n t p e r e n z y m e . T h e p H d e p e n d e n c y of t h e r a t e of i n a c t i v a t i o n is s h o w n i n f i g u r e 2. A n a p -
0.4
°. 3
1,0 @ U C 0
o
0.2 0
3 2
< o.s
"5 c 0.1
/ 0 7
B
9 600
pH
Fro. 2. - - pH rate profile f o r the inactivation. Reaction conditions were as described in figure 1 w i t h 6 mM NBD-C1.
p a r e n t p K c o r r e s p o n d i n g to t h e r e s i d u e i n v o l v e d i n t h e i n a c t i v a t i o n p r o c e s s c a n b e e v a l u a t e d at a b o u t 8.7. BIOCHIMIE, 1979, 61, n ° 5-6.
s;0
'
,;0
-
.k. nm
Fro. 3 . -
Visible spectrum o f NBD-PGK reaction products. Spectra are carried o u t on 32 ~tM PGK in 1 cm l i g h t p a t h c e l l s . ( - ) spectrum is p e r f o r m e d on a n e n z y m e sample modified as follow • 18 ttM enzyme are reacted w i t h 1.4 mM NBD-CI for ½ h o u r s at "30°C in 0.05 M Tris HC1 buffer pH 7.3. The excess of reagent is elimin a t e d as described in m e t h o d s ; (. . . . ) the sample is t h e n b r o u g h t at pH 9 a n d allowed to stay a t 25°C for 24 h.
C. R o u s t a n a n d coll.
666
[13, 20], the extent of NBD i n c o r p o r a t i o n on tyrosyl and lysyl residues is d e t e r m i n e d . To confirm the results o b t a i n e d from the s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n s , r a d i o a c t i v i t y labelling with[14CJ NBD-C1 are p e r f o r m e d . The values o b t a i n e d from several d i f f e r e n t l y labelled p r o t e i n samples are p r e s e n t e d in Table I t o g e t h e r w i t h the results f o u n d by s p e c t r o t i t r a t i o n . These t w o a n a l y t i c a l m e t h o d s give the same results and are in good agreement. T h e y p o i n t out that t h e c h e m i c a l m o d i f i c a t i o n is m a i n l y d i r e c t e d t o w a r d s tyrosine.
S t o i c h i o m e t r y of the reaction. The rate of t y r o s i n e m o d i f i c a t i o n (k~) is s i m i l a r to that o b s e r v e d for i n a c t i v a t i o n (fig. 4). This fact is in a c c o r d w i t h data of figure 6 and table I
• E
1.0
c 6 o
-g fi
5.0
N
Kinetic analysis of NBD incorporation. Changes in a b s o r p t i o n at 475 n m and 385 nm as a f u n c t i o n of t i m e s h o w quite different k i n e t i c patterns. Change at 385 nm presents an e x p o n e n tial c u r v e (fig. 4) w h e r e a s change at 475 nm is s i g m o i d a l (fig. 5).
10
20
30
40
minutes
FIG. 5. - - Effect o f p H on the t i m e course of N - L g s m e -
NBD-derivative formation.
PGK : 70 a M ; NBD-CI : 2 m M ; temperature 30°C ; 0.05 M Tris HC1. buffer, ( - - - - ) pH 7.1 (--.--.--) pH 8.0 ," (. . . . ) pH 8.4 ( ) pH 8.9. 1,0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
~ E 0,5
o
~oo
:o.3
70
E07
0
.;
w h e r e is i l l u s t r a t e d the c o r r e l a t i o n b e t w e e n the n u m b e r of O-Tyr-NBO and the loss of activity. So, a single m o d i f i e d t y r o s y l r e s i d u e w o u l d be r e s p o n s i b l e for the total i n h i b i t i o n of the enzyme. 100
t-
0
20
1' minor,s
2
40
minutes
FW,. 4 . -
Time course of tgrosine modification.
PGK : 360 IxM ; 0.05 M Tris HC1 buffer pH 7.3 ; NBD-Cl 6.2 mM. Temperature 30°C. Inset : correlation between the number of tyrosine modified versus time (O--Q) and the per cent of residual activity (©--©).
T h e r e a c t i o n of t h e NB,D-CI w i t h h y d r o x y l group of t y r o s i n e f o l l o w e d by s p e c t r o p h o t o m e t r y at 385 nm s h o w s a p s e u d o f i r s t - o r d e r kinetics (fig. 4). The c o r r e s p o n d i n g a p p a r e n t constant rate k 1 is about 0.07 rain 1 at pH 7.3. Tim timecourse for the f o r m a t i o n of N-NBD f o l l o w e d at 475 nm s h o w a lag phase and can be a n a l y z e d assuming successive r e a c t i o n s [213. kl k2 HO-E-NH 2 -+ N BDO-E-I~H2 --~ NBDO-E-NH-NBD In this p r o c e s s the value of the constant rate ratio k l / k 2 can be f o u n d equal to about 0.5 as w e l l at p H 7.3 as pH 8.9. So, the rate of m o d i f i c a t i o n of t y r o s i n e and lysine both i n c r e a s e s w i t h pH.
BIOCHIMIE, 1979, 61, n o 5-6.
,~
50
1.0
2.0
O - Tyr, _ N B O g r o u p ~ e n z y m e
FIG. 6 . -
Residual aetioity of PGK oersus tool of modified tyrosine.
Determination by radioactivity labeling (O) or speetrophotometrie titration (O).
Protection against inactivation. The effect of several substrates or analogues on the rate of the i n a c t i v a t i o n by NBD-C1 was tested. It is clear from the results in Table II that an
Reaction of NBO-CI with yeast 3-phosphoglycerate kinase. a l m o s t total p r o t e c t i o n a g a i n s t i n h i b i t i o n is o b s e r ved with substrates (3-phosphoglycerate, ATP a n d AI)P) u s e d at s a t u r a t i n g c o n c e n t r a t i o n s [23].
667
c a t i o n , the i n c o r p o r a t i o n of N B D on t y r o s i n e w a s o n l y s l i g h t a n d w a s r e l a t e d to the f e e b l e loss of activity.
TABLE I.
Comparison of the extent of NBD-incorporation determined by spectrophotometric titration and radioactive I~C labelling. NBD incorporation
Experiments
Inhibition
ATP-Mg ADP AMP Adenosine Tripolyphosphate Pyrop hosphate
22 28 30 22 30 30
Residue
0.43 0.65 1.00
0.36 0.55 0.80
0.07 0.10 0.20
mole / mole enzyme
36 57 76
0.43 0.64 0.90
Protection (p.cent) pli 7.3
10
Residue
per. cent
Concentrations (raM)
30
mole / mole enzyme
titration
Effect of various ligands on the inactivation of 3-phosphoglycerate kinase by NBD-CI.
3-phosphoglycerate
N-Lys-NBD
Spectro-
rement
TABLE II.
Ligands
O-Tyr-NBD
1~C measu-
95 95 95 95 66 10 71 79
pfl. 8.9
95 95 95 51 32 78 25
/molo enzyme
/mole enzyme
Transfer of the NBD group from tyrosine to lysine. S a m p l e s of e n z y m e i n a c t i v a t e d at p H 7.3 a n d at d i f f e r e n t l e v e l s are f r e e d f r o m e x c e s s of r e a g e n t a n d t h e n i n c u b a t e d at p H 9. T i m e - d e p e n d e n t c h a n g e in the v i s i b l e s p e c t r u m Yeas o b s e r v e d . T h i s c h a n g e c o r r e s p o n d s to an i n c r e a s e at 475 n m a n d a d e c r e a s e at 385 n m (fig. 3). T h i s r e f l e c t s a t r a n s f e r of t h e NBD g r o u p f r o m t y r o s y l o x y g e n to a n i t r o g e n g r o u p of a l y s i n e . T h e r a t e of t h i s t r a n s f e r ( t l / 2 ~ 15 h) ~vas t h e s a m e at s e v e r a l d i f f e r e n t p r o t e i n c o n c e n t r a t i o n s (0.5 m g - 1.6 m g / m l ) a n d t h e a d d i t i o n of an e x c e s s of n a t i v e e n z y m e to T y r O-NBD- d e r i v a t i v e d i d n o t i n c r e a s e t h e r a t e of
TABLE I l L
Migration of NBD-group. Modified amino acid (mole/mole enzyme)
Expt.
before transfer (a) Tyr
1 2 3
0.40 0.80 1.05
Lys 0.13 0.20 0.50
after transfer (b) Tyr 0.22 0.40 0.65
NBD-transfer (mole/mole enzyme)
Lys 0.31 0.60 0.90
0.18 0.40 0.40
(a) The enzyme was inactivated at pH 7.3 and freed from excess reagent. (b) The transfert was performed by incubating the modified enzyme at pH 9 for 24 hours.
In a d d i t i o n , an i m p o r t a n t e f f e c t is also o b s e r v e d w i t h o t h e r l i g a n d s s t u d i e d . W h e n e i t h e r 3-phosp h o g l y c e r a t e o r ADP w a s p r e s e n t d u r i n g m o d i f i -
BIOCHIMIE, 1979, 61,
n ° 5-6.
l y s i n e m o d i f i c a t i o n . T h e s e d a t a are i n d i c a t i v e of an i n t r a m o l e c u l a r p r o c e s s . T h e e x t e n t of this c o n v e r s i o n is i l l u s t r a t e d in t a b l e III. After t r a n s f e r on
668
C. Roustan and coll.
lysine res4due, the activity of 3-phosphoglycerate kinase is not restored sho~ving that the corresponding N - n i t r o b e n z o f u r a z a n PGK derivative is also completely inactive. F i n a l l y , the optical rotatory d i s p e r s i o n data in the far ultraviolet of the two i n a c t i v a t e d derivatives w e r e c o m p a r e d w i t h those of native enzyme. The value of [a]233 ,m for native phos'phoglycerate kinase, Tyr-NBD and Lys-NBD_ enzymes is - - 5 0 0 0 ° _ 200 °. Thus, there is no significant alteration i n the helix c o n f o r m a t i o n of the m o d i f i e d enzyme.
Discussion. The r e a c t i o n of NBD-C1 w i t h 3-phosphoglycerate kinase results i n the i n a c t i v a t i o n of the enzyme b e i n g of pseudo first-order t o w a r d reagent. The k i n e t i c s of i n a c t i v a t i o n show that the incorp o r a t i o n of one mole of NB~D per enzyme is sufficient to p r o d u c e the loss of activity. The modified a m i n o acid can be s p e c t r o p h o t o m e t r i c a l l y identified as tyrosine. The m o d i f i c a t i o n of only one t y r o s i n e correlates w i t h complete i n a c t i v a t i o n . This s t o i e h i o m e t r y is established either by spect r o p h o t o m e t r y or b y r a d i o a c t i v i t y measurements. The fact that the rate of this i n a c t i v a t i o n is f o u n d to he i d e n t i c a l to that of NBD-O-tyr f o r m a t i o n can be t a k e n as a n o t h e r proof for the s t o i c h i o m e t r y of the reaction. So, all these data show that the enzyme inactivation results from the m o d i f i c a t i o n of tyrosine. I n addition, a N-NBD-derivative c o r r e s p o n d i n g to a lysine m o d i f i c a t i o n can a.lso be observed. Kinetics of this r e a c t i o n are sigmoid. The pH d e p e n d e n c e rates of the reaction t o w a r d T y r o s i n e or Lysine are similar. So, these two latter results suggest a m e c h a n i s m of the NBD r e a c t i o n w i t h 3-phosphoglycerate kinase i n "which the N-NBD-clerivative formation is d e p e n d e n t on a p r i o r m o d i f i c a t i o n of the t y r o s y l group. Such a m e c h a n i s m has already been reported for lysozyme [21, 15]. In the ease of Ribonuclease, the k i n e t i c p a t t e r n is quit~e different ; the r e a c t i o n of NBD nvith the two f u n c t i o n a l groups is r a n d o m [~]. The reactive t y r o s i n e i n yeast 3-phosphoglycerate kinase w o u l d seem to possess an a p p a r e n t pK w h i c h is lower t h a n the n o r m a l value. These properties could be a c c o u n t e d for in part by the p r o x i m i t y effect b e t w e e n the t y r o s i n e and lysine residues. BIOCHIMIE, 1979, 61, n ° 5-6.
P r o t e c t i o n studies w h i c h show that substrates p r e v e n t access of reagent to the essential t y r o s i n e suggest that this anfino-acid residue lies i n the active site region of the enzyme. The results reported here c o n f i r m those obtained p r e v i o u s l y [4] using iodine and strongly suggest that the chemical m o d i f i c a t i o n occurs at the same essential residue. I n contrast, some authors [5, 6] have f o u n d that the n i t r a t i o n of one t y r o s i n e in 3-phosphoglycerate k i n a s e i n d u c e s only p a r t i a l i n a c t i v a t i o n a n d a significant p r o t e c t i o n is observed only w i t h nucleotide substrates [3]. So, is the t y r o s i n e detected b y the NBD-C1 the same one detected by t,etranitromethane ? F u r t h e r w o r k w o u l d be needed to solve this problem. Ho~,ever, the more imp o r t a n t result c o n c e r n s the t r a n s f e r of NBD. If, the 3-phosphoglycerate k i n a s e i n a c t i v a t e d at pH 7.3 is b r o u g h t to pH 9, after e l i m i n a t i o n of excess NBD-C1, the reagent migrates from t y r o s i n e to lysine. The extent of the t r a n s f e r seems to be limited to the fraction of .enzyme modified only on the t y r o s i n e a n d not to be c o n c e r n e d w i t h the fraction modified at once on t y r o s i n e a n d lysine. So, the same lysine w o u l d be modified d u r i n g the NBD-CI reaction a n d the t r a n s f e r process. Any way, the N-NBD-PGK derivative o b t a i n e d after t r a n s f e r is also inactivated, t h e r e b y c o n f i r m i n g the presence of an essential lysine in the active center of the enzyme [3]. It is c o n c l u d e d that t y r o s i n e a n d lysine residues are close on from each other in the t r i d i m e n s i o n a l structure of the active site of 3-phosphoglycerate kinase.
REFERENCES. 1. Bryant, T. N., Watson, H. C. a Wendell, P. L. (1974) Nature (London), 247, 14-17. 2. Arvidsson, L. ~ Larsson-Raznikiewiez, M. (1973) Biochim. Biophys. Aeta, 310, 430-436. 3. Markland, F. S., Bacharach, A. D. E., Weber, B. H., O'Grady, T. C., Saunders, G. C. & Umemura, N. (1975) J. Biol. Chem.. 250, 1301-1310. 4. Roustan, C., Fattoum, A. a PradeI, L. A. (1976) Biochemistry, 15, 2172-2177. 5. Hjelmgren, T., Arvidsson, L. a Larsson-Raznikiewiez, M. (1976) Biochim. Biophys. Acta, 445, 342349. 6. Meyer, M. C. a W e s t h e a d , E. W. (1976) FEBS Left., 71, 25-28.
7. Hjelmgren, T., Strld, L. &Arvidsson, L. (1976) FEBS Lett., 68, 137-140. 8. Philips, M., Roustan, C., Fattoum, A. & Pradel, L. A. (1978) Biochim. Biophys. Acta, 523, 368-376. 9. Desvages, G., Roustan, C., Brevet, A. ,¢ Pradel, L. A. (1977) C. R. Acad. Sci., s~r. D, Sci. Nat. (Paris), 284, 1461-1464. 10. Fattoum, A., Feinberg, J., Desvages, G. ,~ Roustan, C. (1977) C. R. Acad. Sci., s~r. D, Sci. Nat. (Paris), 284, 1843-1846. 11. Desvages, G., Roustan, C. ,~ Prade], L. A. (1978) in preparation.
Reaction of NBD-CI with yeast 3-phosphoglycerate kinase. 12. Bacharach, A. D. E., Markland, F. S., Pellino, A. Weber, B. H. (1977) Bioehem. Biophys. Res. Comm., 74, 165-171. 13. Ferguson, S. J., Lloyd, W. J. ~ Radda, G. K. (1975) Eur. Y. Bioehem., 54, 127-133. 14. Dalloechio, F., Signorini, M. ~ Rippa, M. (1978) Arch. Biochem. Biophys., 185, 57-60. 15. Aboderin, A. A. ~ Boedefeld, E. (1976) Biochim. Biophys. Aeta, 420, 177-186. 16. Scopes, R. K. (1971) Biochem. J., 122, 89-92. 17. Biieher, T. (1947) Biochim. Biophys. Acta, 1, 292-314. 18. Biicher, T. (1955) Methods Enzgmol., 1, 415-422
BIOCHIM1E, 1979, 61, n ° 5-6.
669
19. F a t t o u m , A., R o u s t a n , C., Feinberg, J., Desvages, G. Pradel, L. A. (1978) Eur. J. Biochem., 82, 161167. 20. Ferguson, S. J., Lloyd. W. J., Lyons, M. H. ~ Ra~da, G. K. (1975) Eur. "J. Biochem., 54, 117-126. 21. Aboderin, A. A., Boedefeld, E. ~ Luisi, P. L. (1973) Biochim. Biophys. Acta, 328, 20-30. 22. Birkett, D. J., Price, N. C., Radda, G. K. ~, Salmon, A. G. (1970) FEBS Lett., 6, 346-348. 23. Scopes, R. K. (1973) in (Boyer, P. D.ed.) 3rd edn. Vol. 8, pp. 335-351, Academic Press, New York.
