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Effects of activators on chemically modified yeast hexokinase
BIOCHIMIE, 1976, 58, 51-59.
Effects of activators on chemically modified yeast hexokinase. L u i s C a r l o s MENEZES a n d J u l i o PUDLES <>. I n s t i t u t d e B i o c h i m i e , U n i o e r s i t ~ d e P a r i s - S u d , C e n t r e d'Orsa!t, 91~05 O r s a y ( F r a n c e ) . S u m m a r y . - - Enzymic studies p e r f o r m e d w i t h chemically modified yeast h e x o k i n a s e (ATP : D-hexose-6-phosphotransferase) confirm previous r e s u l t s indicating t h a t the ~olfhydryl, i m i d a z o l a n d m o s t of the reactive a m i n o groups do not seem to be directly implicated i n the enzyme active site. On the o t h e r h a n d the modifieation of these f u n c t i o n a l groups of the e n z y m e does not affect the t r a n s i t i o n between the acidic inactive f o r m to a n active e n z y m e form a f t e r deprotonation. The c h e m i c a l l y modified f o r m s of h e x o k i n a s e a n d the native enzyme are affected in the same w a y b y a c t i v a t o r s (citrate, o-malate, 3-phosphoglycerate a n d Pi) w h e n the activity w a s m e a s u r e d at pH 6.6. Moreover the loss of enzyme activity observed in the course of the chemical modifications is accompanied b y a n increase of the a c t i v a t i o n effect. This increase m u s t be related to some r e o r g a n i z a t i o n of the enzyme active site in presence of the effectors, since t h e same effect was observed w h e n h e x o k i n a s e was d e n a t u r e d w i t h 3M u r e a at pH 7.5. However no increase i n the a c t i v a t i o n effect was observed ~vhen the d e n a t u r a t i o n 'was carried o u t at pH 6.5 At t h i s pH the loss in activity a n d the change of optical a b s o r p t i o n a t 286 n m were m u c h slower t h a n at pH 7.5, vehieh indicates a great difference i n the p r o t e i n s t r u c t u r e between these pHs.
INTRODUCTION. K o s o w a n d R o s e [1] r e p o r t e d t h a t t h e r a t e of the ATP : glucose transphosphorylase reaction c a t a l y z e d b y y e a s t h e x o k i n a s e , is i n h i b i t e d b y l o w e r i n g t h e p H of t h e r e a c t i o n b e l o w 7.0, at s u b o p t i m a l c o n c e n t r a t i o n s of A T P . T h i s e f f e c t of a c i d i t y is o v e r c o m e b y a c t i v a t o r s s u c h as, o r t h o phosphate, citrate, malate, 3-phosphoglycerate a n d r i b o s i d e t r i p h o s p h a t e . T h e y also o b s e r v e d t h a t i n t h e a c i d r a n g e , A T P a p p e a r s to s e r v e b o t h as a n a c t i v a t o r a n d as a s u b s t r a t e w i t h t h e r e s u l t that l/v versus 1/[ATP] plots are non linear. T h e i r r e s u l t s i n d i c a t e t h a t H + is a n u n 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 t o A T P as t h e v a r i a ble substrate and that two protons are required f o r t h e i n h i b i t i o n w i t h a m e a n p K o f 7.3. In this report the kinetic behaviour between p H 6.5 a n d o H 8.0 of y e a s t h e x o k i n a s e c h e m i c a l l y m o d i f i e d b y g r o u p s p e c i f i c r e a g e n t s w i l l b e desc r i b e d as w e l l as t h e e f f e c t of a c t i v a t o r s o n t h e s e enzymic forms. E n z y m e : Yeast h e x o k i n a s e or A T P : D-hexose-6p h o s p h o t r a n s f e r a s e (E.G. 2.7.1.1). A b b r e v i a t i o n s used are : TNBS, 2, 4, 6 - t r i n i t r o h e n z e n e - l - s u l f o n a t e ; DTNB, 5, 5'-dithiobis ( 2 - n i t r o b e n zoic acid). (*) Dedicated to t h e Memory of Huguette RobichonSzulmajster. To ~vhom all correspondence s h o u l d be addressed.
T h e d i f f e r e n t f o r m s of e n z y m e s t u d i e d h a d r e s p e c t i v e l y t h e i r imi.dazol, s u l f h y d r y l a n d a m i n o groups modified.
EXPERIMENTAL --
PROCEDURE.
~MATERIALS.
Baker's yeast hexokinase (ATP : hexose-f-phosphotransferase) from Boehringer Mannheim GmbH (Mannheim, Germany), was repurified by chromatography through CM-cellulose column [for details s e e ref. 2]. P y r u v a t e k i n a s e a n d g l u c o s e - 6 - p h o s phate dehydrogenase were purchased from Boehringer. U r e a , o b t a i n e d f r o m P r o l a b o as r e a g e n t g r a d e , w a s r e c r y s t a l l i z e d f r o m 9,5 p e r c e n t e t h a n o l . 5.5'-dithio-bis (2-nitrobenzoic acid) (DTNB) and ~-mercaptoethanol were obtained from Aldrich C h e m . Co. I n c . ; A T P d i s o d i u m s a l t a n d N A D P w e r e p u r c h a s e d f r o m P L , B i o c h e m i c a l s Co. P h o s phoenolpyruvate and 3-phosphoglycerate were obtained from Boehringer. L-malic acid from Merck and citric acid from Prolabo. 2-(N-morphol i n o ) e t h a n e s u l f o n i c a c i d (MES) a n d t r i s - ( h y d r o x y methyl)-qnethyl-2 anainoethane sulfonic acid (TES) were obtained from Serva. Ethoxyformic anhydride was obtained from Garlo Erba. 2,4,6-trinitrobenzene sulfonate disodium salt (TNBS) was purc h a s e d f r o m N u t r i t i o n a l B i o c h e m i c a l s Co.
52
L . C. M e n e z e s
All other c h e m i c a l s w e r e of a n a l y t i c a l r e a g e n t grade. Solutions w e r e p r e p a r e d in d e i o n i z e d distilled w a t e r . --
~E,THO
DS.
Enzymic assay -- Hexokinase activity under the v a r i e t y of c o n d i t i o n s that w e r e s t u d i e d was f o l l o w e d in the p r e s e n c e o,f an excess of the coup l i n g e n z y m e glucose-6-P d e h y d r o g e n a s e and NAD,P ~ i t h a Gary 118 C s p e e t r o p h o t o m e t e r . F o r the Km studies p y r u v a t e kinase and p h o s p h o e n o l p y r u v a t e w e r e incl,uded, to r e g e n e r a t e ATP, as d e s c r i b e d by K o s o w and Rose [1].
In the a c t i v a t i o n studies of the m o d i f i e d hexokinase, the a m o u n t o'f e n z y m e taken for assay was always i n c r e a s e d to c o m p e n s a t e for the degree of i n a c t i v a t i o n so that the actual rates in the assays w e r e c o m p a r a b l e . P r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d by a b s o r p t i o n m e a s u r e m e n t at 2,8.0 nm. A value of 0.9'2 w a s taken for the a b s o r b a n c e of a 1 m g / m l solution of h e x o k i n a s e in a 1 cm cnvette at 280 nm, as i n d i c a t e d by Lazarus et al [3]. --Amino a c i d a n a l y s i s of the photooxidize.d p r o t e i n was p e r f o r m e d as detailed in a p r e v i o u s r e p o r t [2]. -- Modification of amino group with 2,4,6-trinit r o b e n z e n e s n l f o n a t e . - - To the e n z y m e (0.25 mg in 0.9 m l ) in 0.1 M p o t a s s i u m p h o s p h a t e buffer (pH 8.0) 0.1 ml of a 70 mM aqueous solution of TNBS was added. T h e final v o l u m e w a s 1 ml. The r e a c t i o n was f o l l o w e d at 344 nm in a Safas double beam r e c o r d i n g s p e c t r o p h o t o m e l e r at 2.3 °. The extent of m o d i f i c a t i o n w a s d e t e r m i n e d by u s i n g a m o l a r e x t i n c t i o n value o.f 1'0,5.0.0 cm-XM-1 determ i n e d w i t h a s t a n d a r d solution e-TN~P-c~-acetyilysine at the same p H an, d it is in quite a g r e e m e n t w i t h the value obLa:in,ed by Goldfarf [4]. Aliquots of 25 vl c,f a 55 mM aquaeous solution of glycine, con.raining 40 m ~ 2 - m e r c a p t o e t h a n o l . -- Modification of the sulfhgdryl groups with 5 , 5 ' - d i t h i o b i s ( 2 - n i t r o b e n z o a t e ) ( D T N B ) . - - To the e n z y m e (500 ~tg) in 50 mM Tris-HC1 buffer (pH 8.0), c o n t a i n i n g 0.1 M EDTA, a DTNB solution in the same buffer w a s a d d e d to a final conc e n t r a t i o n of 50 ~M. T h e final v o l u m e of the reaction m i x t u r e w a s 1.0 ml.
T h e course of the r e a c t i o n w a s f o l l o w e d at 412 nm and at 23 °. T h e m o l a r e x t i n e t i o n coefficient of TNB at 412 n m is 13,6,0,0 era-aM -x [5]. Aliquots of 5 ~1 w e r e r e m o v e d at different time i n t e r v a l s and i m m e d i a t e l y used for the e n z y m i c assay. BIOCHIMIE, 1976, 58, n ° 1-2.
and J. Pudles. --Modification of the histidine residues. -T h e h i s t i d i n e residues of yeast h e x o k i n a s e w e r e m o d i f i e d e i t h e r by p h o t o o x i d a t i o n in p r e s e n c e of m e t h y l e n e blue (at p H 6.0 or 7.5) or by e t h o x y f o r m y l a t i o n w i t h e t h o x y f o r m i c a n h y d r i d e . Details for these m o d i f i c a t i o n s w e r e d e s c r i b e d in t w o p r e c e d i n g r e p o r t s [2, 6]. .-- I n a c t i v a t i o n o f y e a s t h e x o k i n a s e b y 3 M urea, at p H 7.5 a n d 6.5. - - H e x o k i n a s e (1 m g / m l ) w a s d e n a t u r e d by i n c u b a t i o n at 25 ° in a solution of 3 3I urea in 10 mM p o t a s s i u m p h o s p h a t e (pH 7.5) or in 10 fiaM 2-(.N-morpholino) ethane s u l f o n i c acid (pH 6.5), both buffers c o n t a i n i n g 0.1 M NaC1. Aliquots of 10 ,M w e r e r e m o v e d at different time i n t e r v a l s and diluted in 2~0 M of urea solutions at the c o n c e n t r a t i o n an, d p H at w h i c h the i n a c t i v a tion ha.d been c a r r i e d out.
F r o m these diluted solutions, aliquots w e r e taken (between 2 to 50 ~1) and the r e s i d u a l activity m e a s u r e d i m m e d i a t e l y at p H 6.5, in a final v o l u m e of 1 ml of the r e a c t i o n m i x t u r e (without urea). In a parallel e x p e r i m e n t u n d e r the same conditions, aliquots w e r e taken a n d assayed in the presence of the activator. The kinetics of d e n a t u r a t i o n of the e n z y m e in 3 M urea w a s also f o l l o w e d at 286 nm in a Cary 118 C double beam s p e c t r o p h o t o m e t e r , using <> w i t h 2 × 0.4375 cm light paths. The e x p e r i m e n t a l c o n d i t i o n s w e r e the same as those d e s c r i b e d above. R e n a t u r a t i o n w a s o b s e r v e d by d i l u t i n g 50 ~1 of the d e n a t u r a t e d e n z y m e in 3 M u r e a solution in 1 m l e i t h e r of 10 mM p o t a s s i u m p h o s p h a t e buffer (pH 7.5) or 10 mM 2-(morpholino) ethanesulfonic acid (pH 6.5), b o t h buffers c o n t a i n i n g 0.1 M NaCI. At different time intervals, aliquots of 2-5 ~1 w e r e taken and the e n z y m e a c t i v i t y i m m e d i a t e l y measm-ed w i t h o u t a d d i t i o n a l dilution. RESULTS. - - E f f e c t o f tile c h e m i c a l m o d i f i c a t i o n o n t h e e n z y m e a e t i o i t y at p H 6.6 ~ P r e v i o u s studies on the t r i n i t r o p h e n y l a t i o n of p r o t e i n s h a v e s h o w n that 2,4,6-trinitrobenzenes.ulfonate is a s p e c i f i c reagent of the e-amino groups of the p r o t e i n [7-~.
The effect of this r e a g e n t on the e n z y m e activ i t y of yeast h e x o k i n a s e in the course of the m o d i f i c a t i o n of the a m i n o groups is s h o w n in figure 1. The m o d i f i c a t i o n o¢ the first 4 a m i n o groups p e r e n z y m e suhunit causes the loss of m o r e
Chemically modified yeast hexokinase.
53
,of
t h a n 50 p e r c e n t th,e e ' n z y m e a c t i v i t y , t h e n t h e activity decreases slowly for the other amino groups which were trinitrophenylated. After mod i f i c a t i o n of 28 o u t of t h e 37 a m i n o g r o u p s d e t e r m i n e d p e r e n z y m e s u b u n i t , still 5 p e r c e n t o f t h e residual activity was retained.
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Fi6. 1. - - lnactioation of yeast h e x o k i n a s e as a [unction of the n u m b e r of m o d i f i e d lysine residues. Hexokinase was t r i n i t r o p h e n y l a t e d and the reaction stopped at different t i m e intervals as described in the E x p e r i m e n t a l Procedure section. After reaction w i t h 2,4,6-trinitrobenzenesulfonate, the enzyme was appropriately diluted and the hexokinase activity was assayed by incubation of the enzyme (160 to 2500 ng) at 23 °, in a final volume of 1 ml ~vith 2-(N-morpho]ino) e t h a n e s n l f o n i e acid, pH 6.6, 100 mM ; glucose 1 mM ; ATP 0,05 mM ; MgC12 5 mM ; NADP 0,5 mM and glueose-6-P dehydrogenase 0,7 unit, The activity was assayed in presence (A) an in absence of 1 mM citrate (~). In the insert is r e p r e s e n t e d the variation of the activation factor ~vith citrate in function of the n u m b e r of t r i n i t r o p h e n y l a t e d amino group. Activation factor is defined as the ratio between the enzymic activity m e a s u r e d in presence and in absence of citrate.
2. - - I n a c t i v a t i o n of yeast hexohinase as a / u n c t i o n o/ the n u m b e r o[ cgsteine residues modified. The reaction of 5,5-dithiobis(2-nitrobenzoate) w i t h hexokinase was carried as described in the Experim e n t a l Procedure section. Enzymic activity was assayed b o t h in presence (A) and in absence (A) of 1 mC citrate. The activity assays were carried with 50 lo 125 ng of hexokinase, u n d e r the same conditions as those in figure 1. In the insert is represented the variation of the activation factor w i t h citrate, in f u n c t i o n of the n u m b e r of s u l f h y d r y l groups modified in the enzyme.
30( J~
7
250 200
150
100
T h e e f f e c t of t h e m o d i f i c a t i o n of t h e s u f f h y d r y l g r o u p s b y D T N B is s h o w n i n f i g u r e 2. I n t,his c a s e , it c a n b e s e e n t h a t f o r t h e f i r s t s u l f h y d r y l g r o u p n m d f f i e d o n l y a s l i g h t l o s s of t h e a c t i v i t y w a s observed ; with the increase on the modification, steeper becomes the inactivation process. Howe v e r a f t e r m o d i f i c a t i o n of 3.7 s u l f h y d r y l g r o u p s out of t h e 4 d e t e r m i n e d p e r e n z y m e s u b u n i t , 20 p e r c e n t of ,the r e s i d u a l a c t i v i t y w a s still left. F a s e l l a a n d H a m a n e s [10] o b t a i n e d s i m i l a r r e s u l t s b y t h e m o d i f i c a t i o n of t h e s u l h y d r y l g r o u p s o f h e x o k i n a s e w i t h P C ~ B ( a c t i v i t y m e a s u r e d at p H 8.0). The histidyl residues were modified either by p h o t o o x i d a t i o n i n p r e s e n c e o f m e t h y l e n e b l u e at p H 7.5 o r 6.0, o r b y e t h o x y f o r m y l a t i o n w i t h
BIOCHIMIE, 1976, 58, n ° 1-2.
Groups (no. perenzyme subunit)
FIG.
. "-~-'~'''''---~ 20 25
----.....
50
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t ~z photoox,d~t,on t,me,m,~
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photooxidation
time (rain)
FIG. 3. - - Inaclioation of yeast h e x o k i n a s e by photooxidation in presence of m e t h y l e n e blue. P h o t o o x i d a t i o n was carried out by i r r a d i a t i n g w i t h a 300 'watt l a m p (Mazdasol) 0.6 ml samples at a protein c o n c e n t r a t i o n of 0.25 m g / m l , in pyrex tubes (1 × 12 cm). The solvent w a s 5 mM potassium phosphate buffer, pH 7.5, containing 0.1 mM EDTA and 0.l M NaC1. Methylene blue yeas added to a final conc e n t r a t i o n of 0.01 per cent. The t e m p e r a t u r e was m a i n t a i n e d at 17 ° (for details see ref. 2). Enzymic activity w a s assayed in presence (A) and in absence (A) of 1 mM citrate. The activity assays were carried w i t h 75 to 1000 ng of hexokinase, in the same conditions as those in figure 1. In the i n s e r t is r e p r e s e n t e d the v a r i a t i o n of the activation factor w i t h citrate, in f u n c t i o n of the time of he×okinase photooxidation.
L. C. M e n e z e s a n d J. P u d l e s .
54
e t h o x y f o r m i c a n h y d r i d e at p H 7.5, as d e s c r i b e d p r e v i o u s l y [2, 6]. T h e i n a c t i v a t i o n b y b o t h t e c h n i q u e ~vas f o l l o w e d b y n m a s u r i n g t h e a c t i v i t y a t p H 6,6 a n d w a s i d e n t i c a l to o u r p r e v i o u s r e s u l t s
300
in which the activity was (fig. 3 a n d 4).
o - -
•
250
blocked hi.tidal res,dues no o e t e n z me s u u n , t ~
20( o
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- - Effects of the activators in the course of the chemical modification of hexokinase. - - I t is
15(
i n t e r e s t i n g to o b s e r v e t h a t i n t h e c o u r s e of t h e trinitrophenylation, the activation factor inc r e a s e s f o r t h e f i r s t 14 a m i n o g r o u p s m o d i f i e d , t h e n it b e g i n s t o d e c r e a s e w i t h f u r t h e r m o d i f i c a t i o n u n t i l it r e a c h e s n e a r l y t h e s a m e l e v e l of a c t i v a t i o n as o b s e r v e d f o r t h e n a t i v e e n z y m e (fig. 1 a n d i n s e r t ) . ] ' h i s i n c r e a s e of t h e a c t i v a t i o n f a c t o r w a s also o b s e r v e d w i t h m a l a t e , 3 - P - g l y c e r a l e a n d p h o s p h a t e ( t a b l e I). F o r t h e s e l a s t a c t i v a t o r s t h e e f f e c t `was d e t e r m i n e d w h e n t h e r e s i d u a l a c t i v i t y w a s of 5 p e r c e n t .
1oo
50
Blocked
Fro. 4. --
at pH
At t h e e n d of t h e p h o t o d y n a n l i c treatment, w h e n t h e e n z y m e h a d loss 9.0, p e r c e n t of t h e a c t i v i t y (fig. 3), 5 h i s t i d y l , 2 c y s t e i n y l , 2 m e t h i o n y l and 2 tryptophyl residues per enzyme subunit had b e e n oxi,dized f r o m r e s p e c t i v e l y 9, 4, 11 a n d 4. H o w e v e r b y e t h o x y f o r m y l a t i o n w e m o d i f i e d spec i f i c a l l y all tile 9 h i s t i d y l r e s i d u e s . As c a n b e s e e n i n f i g u r e 4, still 40 p e r c e n l of t h e e n z y m e a c t i v i t y w a s left a f t e r a c y l a t i o n of t h e s e r e s i d u e s .
4--4
ze
measured
Histidyl
Residues(no.
per enzyme subunit)
I n a c t i v a t i o n of yeast hexokinase as a f u n c -
t i o n of the number of histidyl residues ethoxyformylated.
To the enzyme (0.85 m g i n 0.5 ml) in 0.1 M potass i u m p h o s p h a t e buffer (pH 7.5) c o n t a i n i n g 0.1 mM EDTA, 5 m l of a 175 mM e t h a n o l solution of ethoxyf o r m i c a n h y d r i d e ~vas added. The t e m p e r a t u r e xvas m a i n t a i n e d at 0% Aliquots of 20 ~1 `were removed at different t i m e i n t e r v a l s a n d the r e a c t i o n stopped by adding. 20 ul of a 0.1 M glyeine solution, pH 8.4. The enzymic activity ~vas assayed i n presence (A) a n d in absence (A) of 1 mM citrate. The activity assays ~vere carried ~ 4 t h 160 to 3,20 ng of enzyme, in t h e same c o n d i t i o n s as t h o s e i n figure 1. I n t h e i n s e r t is r e p r e s e n t e d the v a r i a t i o n of the a c t i v a t i o n f a c t o r "with citrate in f u n c t i o n of the n u m b e r of h i s t i d y l residues e t h o x y f o r m y l a t e d .
O n t h e o t h e r h a n d w h e n "the a c t i v i t y w a s m e a s u r e d at p H 8.0, n o a c t i v a t i o n w a s o b s e r v e d i n p r e s e n c e of c i t r a t e (see t a b l e I), i n d i c a t i n g t h a t t h e m o d i f i e d e n z y m e , s i m i l a r l y to t h e n a t i v e o n e , w a s s e n s i t i v e to t h e a c t i v a t o r s o n l y at a c i d p H . I n f i g u r e s 2, 3 a n d 4 a n d r e s p e c t i v e i n s e r t s , t h e e f f e c t s of c i t r a t e i n t h e c o ~ r s e of t h e m o d i f i c a t i o n of t h e e n z y m e b y DT.NB, p h o t o o x i d a t i o n at p H 7.5
TABLE [[.
Effect of activators on the relative rates of the native and chemically m o d i f i e d hexokinase. Functional group modified Additions
none pH6.6
imidazol (a)
pH 8.0 pH6.6
sulfhydryl (b)
amino (c)
pH8.0
pH6.6
pH8.0
pit 6.6 pH 8.0
13.5
42
5.0
22.5
3.6(d) 14.9(d) 0.7(e) 2.4 (e) 6.9(d) 15.3(d) 2.2 (e) 2 . 4 ( 0 1.6(e) 2.4(e) 2.2 (e) 2.3(e) 0.9(e)
None ................
32.5
100
Citrate 1 mM . . . . . . . .
71.5
93
37
43
18.5
22
L-malate I mM . . . . . . 3-P-glycerate 1 m M . . . Pi 20 mM . . . . . . . . . . .
62 68 39
100 94 34
27 35 22
38 36 15
15 13 9
22.5 22
(a) 9 i m i d a z o l groups modified per e n z y m e s u b u n i t `with e t h o x y f o r m i e anhydride. b) 3.7 s u l f h y d r y l groups modified per e n z y m e s u b u n i t ~vith DTNB. (c) a m i n o groups modified 'with TNBS ; (d) 14 a m i n o groups ; (e) 28 a m i n o groups per e n z y m e s u b u n i t . F o r detail see E x p e r i m e n t a l section a n d fig. 3.
BIOCHIMIE, 1976, 58, n ° 1-2.
Chemically
modified
yeast hexokinase.
and ethoxyformylation are shown. It can be seen t h a t f o r a n y of t h e s e m o d i f i c a t i o n s , t h e d e c r e a s e i n a c t i v i t y w a s f o l l o w e d b y a n i n c r e a s e of "the activation factor. Moreover, this effect was on/y o b s e r v e d at p H 6,6 a n d n o t at p H 8.0, a n d a l w a y s a t l o w c o n c e n t r a t i o n of A T P . A s i m i l a r e f f e c t w a s observed with malate, 3-P-glycerate and phosp h a t e ( t a b l e I).
r a t e of i n a c t i v a t i o n at t h e f o r m e r p H w a s l o w e r , and moreover we don't have for the moment any evidence indicating that the same residues were o x i d i z e d at b o t h p H v a l u e s .
--. E f f e c t of p i t on M g A T P d e p e n d e n c e of the chemically m o d i f i e d hexokinase. - - To determine if t h e m o d i f i c a t i o n of t h e a m i n o , s u l f h y d r y l a n d h i s t i d y l g r o u p s h a d a n y e f f e c t o n t h e b i n d i n g of MgATP or in the transition between an inactive f o r m of t h e e n z y m e i n t h e a c i d i c r e g i o n a n d a n active form in the alkaline region, studies were performed on the MgATP concentration depen-
It w a s q u i t e i n t e r e s t i n g to n o t i c e t h a t i n t h e c o u r s e of t h e p h o t o d y n a m i c t r e a t m e n t a t p H 6.0 w e d i d n o t o b s e r v e a n y i n c r e a s e of t h e a c t i v a t i o n factor in presence of citrate.
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Fro. 5 (upper set of curves). - - Double reciprocal plots of velocity of yeast hexokinase as a function of MgATP concentration, between pH 6.5 to 7.9, for the native and modified hexokinase. Each tube contained : enzyme 95 to 500, ng, glucose 2 m M ; MgC12 5 m M ; NADP 0.5 mM ; glueose-6-P-dehydrogenase 0.7' u n i t ; P - e n o l p y r u v a t e 1 mM ; p y r u v a t e kinase 1.5 u n i t s a n d buffer 100 mM in a final volume of I ml. Buffers used were 2-(N-morpholino) e t h a n e s,ulfonie acid, pH 6.5 ( I ) or H - T r i s - ( h y d r o x y m e t h y D m e t h y l - 2 ' - a m i n o - e t h a n e s u l f o n i c acid pH 6.9 (4k), pH 7.0'5 (©), pH 7.2, (A),, pH 7.3 (i~), pH 7.5 (@) a n d pH 7.9 ( ). In the figure are r e p r e s e n t e d the d a t a o b t a i n e d ~vith : A - - n a t i v e enzyme ; B - - t r i n i t r o p h e n y l a t e d enzyme, r e t a i n i n g 5 p e r cent of the activity a n d h a v i n g 26 a m i n o groups, modified per enzyme s u b u n i t ; C - - p h o t o o x i d i z e d enzyme, w h i c h r e t a i n s 20 p e r cent of t h e a c t i v i t y (4 histidyls, 1 cvsteinyl, 2 m e t h i o n y l a n d 1 t r y p t o p h y l residues photooxidized p e r enzyme s u b u n i t ) ; D ~ " e t h o x y ¢ o r m y l a t e d enzyme, w h i c h r e t a i n s 40 p e r cent of t h e activity (9 h i s t i d y l s residues blocked p e r e n z y m e s u b u n i t ) ; E - - hexokinase modified b y reaction w i t h 5,5'-dithiohis(2-nitrobenz,o,ic acid), h a v i n g 3.7 cysteinyl groups' blocked p e r enzyme s u h u n i t a n d r e t a i n i n g 20 per cent of the original activity. Lower set of curves : pH effect on m a x z m u m velocity for the first hyperbolic region of the A T P dependence curve. The d a t a f r o m the u p p e r set of double reciprocal plots are represented, showing the ¥ m e x t r a p o l a t e d front the hyperbo],ic low ,~TP region, Vm', as a f u n c t i o n of pH. Curves F, G, H, I a n d J corresponds respectively to the n a t i v e (©), t r i n i t r o p h e n y l a t e d (A), p h o t o oxidized (A), e t h o x y f o r m y l a t e d (@) a n d DTNB modified~ ([~) hexokinase. This result was unexpected, since we had prev i o u s l y o b s e r v e d t h a t i n t h e c o u r s e of t h e p h o t o i n a c t i v a t i o n at p H 6.0 o r a t p H 7.5, f o r a n i d e n t i c a l r e s i d u a l a c t i v i t y t h e s a m e a m o u n t of a m i n o a c i d r e s i d u e s w e r e o x i d i z e d [2]. H o w e v e r , t h e
BIOCHIMIE, 1976, 58, n ° 1-2.
d e n c e of t h e h e x o k i n a s e a n d 7.9.
a c t i v i t y b e t w e e n p H 6.5
For these experiments, the residual activity for the different forms of the enzyme were respecti-
56
L . C. M e n e z e s
v e l y 5 p e r c e n t f o r t h e t r i n i t r o p h e n y l a t e d , 20 p e r c e n t f o r t h e D T N B m o d i f i e d , 10 p e r c e n t f o r t h e p h o t o o x i , d i z e d a n d 40 p e r c e n t f o r t h e e t h o x y formylaied enzyme.
0.8
and J. PudIes. - - E f f e c t o f 3 M urea. - - T h e r e s u l t s of t h e c h e m i c a l m ( ~ d i f i c a l i o n s e e m e d to s u g g e s t t h a t t h e i n c r e a s e of t h e a c t i v a t i o n f a c t o r i n p r e s e n c e of t h e a l l o s t e r i c e f f e c t o r s w a s d u e to a c o n f o r m a t i o n a l c h a n g e . W e c h e c k e d if t h i s e f f e c t w o u l d a p p e a r i n t h e c o u r s e of t h e d e n a t u r a t i o n of i h e enzyme by a non covalent bond forlning agent as urea.
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pH Fro. 6. - - R e p l o t of the data represented in figure 5 (lower set of curves). Vm is the Vmax at pH 7.9. The several lines corresp o n d to t h e n a t i v e e n z y m e (O) a n d to the t r i n i t r o p h c n y l a t e d (&), photooxidized (A), e t h o x y f o r m y l a t e d (O) a n d DTNB modified (O) bexokinase.
F i g u r e s 5 ( u p p e r s e t o'f c u x v e s ) sho~v t h e d o u b l e r e c i p r o c a l p l o t s b e t w e e n p H 6.5 a n d 7.9 i n f u n c t i o n of t h e M g A T P c o n c e n t r a t i o n f o r t h e native and modified enzymes. In figures 5 (lower set of curves) are represented the pH effects on the extrapolaled low ATP V m v a l u e s , V ' m , as i n d i c a t e d b y K o s o w a n d R o s e [1]. T h e p H p r o f i l e s o f t h e a c t i v i t y ~vere q u a l i t a tively identical for the native and chemically modified forms of the enzyme. Figure 6 shows the representation o,f - - l o g "Vm ( ~ ~ 1) as a f u n c t i o n o f p H , a c c o r d i n g to t h e e q u a t i o n d e v e l o p e d b y K o s o w a n d R o s e [1] f o r all t h e c h e m i c a l l y m o d i f i e d f o r m s o f t h e e n z y m e . I n all c a s e s a s i n g l e l i n e w a s o b t a i n e d w i t h a s l o p e v a r y i n g b e t w e e n 2.2 to 2.4, w h i c h a g r e e s w i t h t h e resu,lts of t h e s e a n t o r s f o r t h e n a t i v e e n z y m e , indicating that also for the modified form of the enzyme 2 protons are required for the pH transition. No s i g n i f i c a n t d i f f e r e n c e s w e r e f o u n d f o r t h e Km values for MgATP between the native and chemically modified enzymes. BIOCHIMIE, 1976, 58, n ° 1-2.
~= m
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Minutes
FIG. 7. - - Denaturation of yeast h e x o k i n a s e by 3 M urea at pH 7.5, and s u b s e q u e n t r e n a t u r a t i o n by dilution. Hexokinase w a s i n c u b a t e d at pH 7.5, in 3 M u r e a a n d aliquots ~vere taken, at different t i m e i n t e r v a l s for enzymic assays as described in E x p e r i m e n t a l Procedure Section. At the arrow, d e n a t u r e d enzyme was diluted 21 t i m e s i n 10 mM p o t a s s i u m p h o s p h a t e buffer (pH 7.5). The activity assays were carried out at pH 6.5 i n presence (•) or i n absence (A) of 1 mM citrate, in the same conditions as those in figure 1. The a m o u n t of e n z y m e t a k e n for assay varied f r o m 95 to 2380 ng, so t h a t actual rates i n the assays were comparable. In the i n s e r t is r e p r e s e n t e d the v a r i a t i o n of the a c t i v a t i o n factor 'with citrate (ratio between the enzymic activity in presence a n d absence of citrate) as a f u n c t i o n of t h e t i m e of i n c u b a t i o n of t h e h e x o k i nase i n 3 M u r e a and, subsequently, the t i m e a f t e r dilution. The a r r o w indicates the t i m e at w h i c h t h e d e n a t u r e d e n z y m e ~vas diluted.
F i g u r e 7 s h o ' w s t h a t indee.d, h i g h e r w a s t h e d e g r e e of i n a c t i v a t i o n b y 3 M u r e a a t p H 7.5, higher also was the activation effect by citrate. When the enzyme was reactivated by dilution (2,1 t i m e s ) t h e a c t i v a t i o n e f f e c t r e t u r n s n e a r l y to a n o r m a l level. T h e r e c o v e r y o f t h e i n i t i a l a c t i v i t y w a s 70 p e r c e n t . I t is i n t e r e s t i n g to r e m a r k t h a t w h e n t h e a e n a t u r a t i o n w a s c a r r i e d o u t i n 3 M u r e a at p H 6.5, the activation factor with citrate remained unc h a n g e d (fig. 8).
Chemically modified yeast hexokinase. On t h e o t h e r h a n d , b o t h t h e loss in e n z y m i c a c t i v i t y a n d the c h a n g e i n a b s o r b a n c e at 286 n m w e r e m u c h s l o w e r w h e n the e n z y m e w a s d e n a t u r e d in 3 M u r e a at p H 6.5 t h a n at p H 7.5 (fig. 9). A l t h o u g h the s t a b i l i t y of t h e e n z y m e w a s h i g h e r at p H 6.5, t h e r e c o v e r y of t h e i n i t i a l a c t i v i t y a f t e r
57
dilution was not higher than when the enzyme w a s r e a c t i v a t e d a f t e r u r e a t r e a t m e n t at p H 7.5. W e c o u l d n o t o b s e r v e a n y effect of c i t r a t e o n t h e r a t e of r e n a t u r a t i o n of t h e e n z y m e u n d e r o u r experimental conditions. DISCUSSION.
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Minutes
Fro. 8. - - D e n a t u r a t i o n of yeast h e x o k i n a s e by 3 M urea at p H ,6.5 a n d s u b s e q u e n t r e n a t u r a t i o n by dilution.
Hexokinase was incubated in 3 M urea, at pH 6.5. as described in the Experimental Procedure Section. At the arrow, the denatured enzyme was diluted 21 times in 10 mM 2-(N-morpholino) ethanesullonic acid (pH 6.5). The enzymic assays ~vere carried out at pH 6.5, in presence (A) or absence (A) of 1 mM citrate, under the same conditions as in fig. 1. The a m o u n t of enzyme taken for assay varied f r o m 95 to 2380 rag, so that actual rates in the assays were comparable. In the insert is represented the variation of the activation factor with citrate, as a function of the time of incubation of hexokinase in 3 M urea.
M o r e r e c e n t l y , L a z a r u s e t al [11] r e p o r t e d that a f t e r m o d i f i c a t i o n of t h e f o u r s u l f h y d r y l g r o u p s per enzyme subunit with methylmercuryiodide, t h e t w o less r e a c t i v e o n e c o u l d be e s s e n t i a l f o r the enzyme activity. T h e r o l e of the a m i n o g r o u p s in y e a s t h e x o k i n a s e w a s less s t u d i e d . G r i l l o [12] s h o w e d t h a t pyridoxal phosphate, which reacts reversibly w i t h t h e a m i n o g r o u p s , h a s an i n h i b i t o r y effect o n y e a s t h e x o k i n a s e , a n d t h a t it is n o t c o m p e t i t i v e t o w a r d s the s u b s t r a t e s A T P a n d D-glucose. I n this r e p o r t w e h a v e s h o w n t h a t t h e m o d i f i c a t i o n of a n y of t h e s e f u n c t i o n a l g r o u p s p r o m o t e s a loss of e n z y m e a c t i v i t y . H o w e v e r , e v e n a f t e r e x t e n s i v e m o d i f i c a t i o n t h e e n z y m e still r e t a i n s some activity. T h e s e r e s u l t s as w e l l as p r e v i o u s w o r k [2, 6, 10, 11, 12 a n d 13] s h o w that the i m i d a z o l , s u l f h y d r y l a n d t h e m o s t a c c e s s i b l e a m i n o g r o u p s to T N B S s e e m to be i m p o r t a n t for the s t a b i l i s a t i o n of t h e s t r u c t u r e of y e a s t h e x o k i n a s e . H o w e v e r , if t h e s e g r o u p s do not p l a y a d i r e c t r o l e in the c a t a l y t i c process, nontheless we cannot exclude them from being implicated either :
too
~
P r e v i o u s s t u d i e s f r o m this l a b o r a t o r y [2, 6] h a v e s h o w n t h a t the h i s t i d y l r e s i d u e s w e r e n o t i n v o l v e d e i t h e r in the c a t a l y t i c o r t h e s u b s t r a t e b i n d i n g site w h e n t h e a c t i v i t i e s w e r e d e t e r m i n e d at a l k a l i n e p H values. I n the c a s e of t h e s u l f h y d r y l g r o u p s , t h e r e s u l t s of d i f f e r e n t a u t h o r s w e r e controversial. Fasella and Hammes L10] had s h o w n t h a t the loss of a c t i v i t y of h e x o k i n a s e a f t e r m o d i f i c a t i o n of t h e s e g r o u p s w i t h PCMB, w a s d u e o n l y to a d e s t a b i l i z a t i o n of the e n z y m e c o n f o r m a tion.
7~
1) in t h e t r a n s i t i o n b e t w e e n t h e a c i d i c i n a c t i v e f o r m of the e n z y m e to an a c t i v e f o r m u p o n the a d d i t i o n of a c t i v a t o r s o r i n c r e a s e of p H ; 2) o r in the i n t e r a c t i o n b e t w e e n the t e r n a r y complex (enzymic-substrates) with the activators.
.,,1
[
i
t° MINUTES
Fro. 9. - - D e n a t u r a t i o n o f yeast hexokinase by 3 M urea at pH ~.5 and 7.5 as m o n i t o r e d by the decrease in absorbance at 286 rim.
A ~ represents the enzyme absorption at the end of the denaturation process and At the enzyme absorption at different time intervals, pH 6.5 (O) and pH 7.5 (n) (see Methods). BIOCHIMIE, 1976, 58, n ° 1-2.
T o d e t e r m i n e if t h e k i n e t i c p r o p e r t i e s of m o d i f i e d f o r m of h e x o k i n a s e w e r e d i f f e r e n t a n a l o g o u s w i t h t h o s e of t h e n a t i v e e n z y m e , studied these properties under the conditions which the enzyme was highly modified.
the or we in
We observe that none of the chemical modificat i o n s h a d a n y s i g n i f i c a n t effect on t h e K m v a l u e s
58
L. C. M e n e z e s a n d J. Pudles.
for MgATP as c o m p a r e d w i t h the native enzyme. This seems to i n d i c a t e that the s u l f h y d r y l , histidyI a n d most of the a m i n o groups of the e n z y m e are not i n v o l v e d in the MgATP b i n d i n g site. On the other h a n d all these m o d i f i e d f o r m s of the e n z y m e s h o w the same c o m p l e x k i n e t i c b e h a v i o u r b e l o w p i t 7.3 at s u b - o p t i m a l c o n c e n t r a t i o n of ATP, as the native enzyme. The p H activities profiles, w e r e i d e n t i c a l also w h e n the data o b t a i n e d for the ¥ ' m w e r e plotted as a f u n c t i o n of pH. In all cases, h i g h e r was the degree of the chenfical m o d i f i c a t i o n and i n a c t i v a t i o n , h i g h e r was also the a c t i v a t i o n f a c t o r in the p r e s e n c e of citnate, D-malate, 3 - p h o s p h o g l y c e r a t e or Pi, w h e n the e n z y m e a c t i v i t y was m e a s u r e d at p H 6.6 at s u b - o p t i m a l c o n c e n t r a t i o n s of ATP. T h e i n c r e a s e d r e s p o n s e to the activators in the course of the c h e m i c a l m o d i f i c a t i o n s of h e x o k i nase, i n d i c a t e s that the loss in e n z y m i c a c t i v i t y w a s not d u e to the f o r m a t i o n of c o m p l e t e l y inactive f o r m of the enzyule, but r a t h e r to the formation of m o d i f i e d f o r m s of the e n z y m e still p a r tially active. These results suggest that the infidazol and sulfh y d r y l groups are not i m p l i c a t e d in the t r a n s i t i o n b e t w e e n the a c i d i c i n a c t i v e f o r m of the e n z y m e to an active f o r m after d e p r o t o n a t i o n . H o w e v e r w e c a n n o t c o m p l e t e l y e x c l u d e the p a r t i c i p a t i o n in this p r o c e s s of the less accessible a m i n o groups, w h i c h w e w e r e unable to t r i n i t r o p h e n y l a t e u n d e r our experimental conditions. W h e n the activities of the m o d i f i e d enzymes w e r e m e a s u r e d at p H 8,0 at s u b - o p t i m a l c o n c e n t r a t i o n of ATP no a c t i v a t i o n effect was observed, as h a d been r e p o r t e d p r e v i o u s l y by K o s o w a n d Rose for the native e n z y m e [1]. These results i n d i c a t e 'that the i n c r e a s e d effect of the activators w e r e only r e l a t e d to the p r o t o n ated f o r m of the enzymes as w a s d e s c r i b e d for the n o r m a l a c t i v a t i o n effect w i t h the native enzyme. It is also i n t e r e s t i n g to note that for the native enzyme, the rate at p H 6..6 u n d e r the i n f l u e n c e of the activators d i d not r e a c h the level of the activity w h e n the assay w a s p e r f o r m e d at pH 8.0 and at low A T P c o n c e n t r a t i o n s . H o w e v e r , f o r the chem i c a l l y m o d i f i e d f o r m s of the enzyme, c i t r a t e c o u l d raise the a c t i v i t y to a level as h i g h as that m e a s u r e d at p H 8.0 (see table I). Kosow and Rose h a d r u l e d out the p o s s i b i l i t y that the a c t i v a t i o n effect due to the effectors o r to the d e p r o t o n a t i o n of the p r o t e i n , could be related to a change on the state of a g g r e @ t i o n of the enzyme. O~ur results seem to c o n f i r m this data,
BIOCHIMIE, 1976, 58, n ° 1-2.
since the p h o t o o x i d i z e d ( u n p u b l i s h e d results) or e t h o x y f o r m y l a t e d h e x o k i n a s e [6J as w e l l as the e n z y m e h a v i n g the s u l f h y d r y l groups m o d i f e d [11] w e r e c o m p l e t e l y d i s s o c i a t e d u n d e r the c o n d i t i o n s in w h i c h the k i n e t i c p r o p e r t i e s w e r e studied, w h i c h suggest that the d i s s o c i a t e d f o r m of the e n z y m e is still sensitive to the a c t i v a t o r s or to the p H effect. The i n c r e a s e of the a c t i v a t i o n f a c t o r in the course of a c h e m i c a l m o d i f i c a t i o n h a d been rep o r t e d by R o s c h l a u and Hess [14]. These a u t h o r s h a v e s h o w n that in the course of the t r i n i t r o p h e n y l a t i o n of one r e a c t i v e s-amino group of yeast p y r u v a t e kinase, the e n z y m e loses a c t i v i t y but at the same time the r e s p o n s e to the allosteric effector ( f r u c t o s e - l , 6 - d i p h o s p h a t e ) increases. In our case the i n c r e a s e of the activation effect was o b t a i n e d not only in the course of the m o d i f i c a tion of such different groups as thiol, a m i n o a n d histidyl, but also by urea t r e a t m e n t of the e n z y m e at p H 7.5. These activation effects w h i c h are o b s e r v e d by s u c h different treatments, i n d i c a t e s that cautions must be taken, on e x p l a i n i n g the i m p l i c a t i o n of a s p e c i f i c a m i n o acid r e s i d u e in this k i n d of process, based on an u n i q u e m o d i f i c a t i o n . ,In our p o i n t of view, the a c t i v a t i o n effects observed, could be r e l a t e d to a p a r t i a l r e v e r s a l of the d e n a t u r i n g effect, r e s u l t i n g in a better f i t t i n g of the substrates on the e n z y m e a c t i v e site than in the d e n a t u r e d form. U n e x p e c t e d w e r e the results obtained in the course of the d e n a t u r a t i o n of the enzyme by 3 M u r e a at p H 6.5, or d u r i n g the p h o t o o x i d a t i o n at p H 6.0. U n d e r these c o n d i t i o n s the activation factor w i t h citrate r e m a i n e d c o n s t a n t t h r o u g h all the i n a c t i v a t i o n process. It is quite possible that u n d e r d e n a t u r i n g conditions at acid or aIkaline pH the c o n f o r m a t i o n a l f o r m s of the e n z y m e are d.ifferent. We h a d a l r e a d y o b s e r v e d that h e x o k i n a s e w a s m o r e stable to p h o t o o x i d a t i o n [21 or e t h o x y f o r m y l a t i o n [63 w h e n these m o d i f i c a t i o n s w e r e c a r r i e d out b e l o w p H 7.0. Moreover the i n c r e a s e d stability of the e n z y m e to the urea t r e a t m e n t at p H 6.5 as c o m p a r e d to p H 7.5, could suggest that the p r o t o n a t i o n of some s p e c i f i c groups on the e n z y m e structure, p r o m o t e s an im,portant effect on the p r o t e i n c o n f o r m a t i o n . Our attemps to o b s e r v e a s t r u c t u r a l effect i n d u c e d by c i t r a t e on the p h o t o o x i d i z e d e n z y m e in p r e s e n c e of o-glucose and B-v-methylene ATP or lyxose and ATP [15], w e r e n~nsuccessful, w h e n
Chemically modified yeast hexokinase. followed by fluorescence or by differential speclroscopy. I n c o n c l u s i o n , t h e s e r e s u l t s s h o w t h a t it t h e c h e m i c a l m o d i f i c a t i o n of t h e i m i d a z o l , s u , l f h y d r y l a n d a m i n o g r o u p s of y e a s t h e x o k i n a s e p r o m o t e s t h e i n a c t i v a t i o n of t h e e n z y m e , n e v e r t h e l e s s t h i s loss in the activity does not affect the activators b i n d i n g site o r t h e i o n i z a b l e g r o u p o f p K 7.3 i n volved in the t r a n s i t i o n b e t w e e n the a c i d i c ina c t i v e f o r m of t h e e n z y m e a n d a n a c t i v e e n z y m e from after deprotonation. M o r e o v e r , t h e s e r e s u l t s also i n d i c a t e b y e x c l u sion, that a ionizable group with a highly displaced p K ( c a r b o x y l or t y r o s y l group) seems to be i m p l i c a t e d in these m o l e c u l a r events. F u r t h e r w o r k is b e i n g c a r r i e d o u t to d e t e r m i n e t h e r o l e of these groups.
Aeknawledgments. This ~work was supported f r o m the Centre National de la Recherche Scientifique (E.R. 142) and F o n d a t i o n pour la Recherche Mddicale Franqaise. We are grateful to Drs J. v a n Heijenoort and G. Nemethy for c o m m e n t s i n the p r e p a r a t i o n of this manuscript. R~SUM~. Des dtudes enzymatiques effectu6es avec r h e x o k i n a s e de levure (ATP : o-hexose 6-phosphotransf6rase), modifi6e c h i m i q u e m e n t , ont confirm6 des r6sultats ant6rieurs i n d i q u a n t que le sulfhydryl, l'imidazol et ta p l u p a r t des groupes amin6s ne s e m h l e n t pas intervenir d i r e e t e m e n t au niveau du site aetif. D'autre p a r t la modification de ces groupes fonctionnels n'affecte pas la t r a n s i t i o n entre la f o r m e acide inactive et la f o r m e active de l ' e n z y m e apr6s d6protonation.
BIOCHIMIE, 1976, 58, n ° 1-2.
59
L'hexokinase modifi6e par diff6rents r~actifs est aetiv6e, de la m~me faqon que l ' e n z y m e native, p a r le citrate, le o-malate, le 3:phosphoglyc6rate et le Pi, q u a n d l'activit6 est mesur6e h pH 6,6. De plus, la perte d'activit6 e n z y m a t i q u e observ6e au cours des modifications chimiques est aecompagn6e d'une a u g m e n t a t i o n de l'effet d'aetivation. Cette a u g m e n t a t i o n dolt r6sulter d ' u n e r6organisation du site actif de l ' e n z y m e en pr6sence des effecteurs, puisque cet effet est aussi observ6 q u a n d l'hexokinase est d6natur6e par l'ur6e 3M, pH 7,5. Cependant nous n'avons observ6 aueune a u g m e n t a t i o n de l'effet d'activation q u a n d la d~naturation est faite h pH 6.5. A c e pH la perte d'activit6 et le c h a n g e m e n t d ' a b s o r p t i o n optique h 286 n m sont beaueoup plus lents qu'h pH 7,5, ce qui indique une grande diff6rence dans la structure de la prot~ine entre ces pit. REFERENCES. 1. Kosow, D. P. & Rose, I. A. (1971) J. Biol. Chem., 246, 2618-2625. 2. Menezes, L. C., Grouselle, M. & Pudles, J. (1972) Eur. J. Biochem., 30, 81-92. 3. Lazarus, N. R., Ramel, A. H., Rustum, Y. M. & Barnard, E. A. (1966) Biochemistry, 5, 4003-4016. 4. Goldfarf, A. R. (1966) Biochemistry, 5, 2570-2578. 5. Ellman, G. L. (1959) Arch. Biochem. Biophys., 82, 70-77. 6. Grouselle, M., Thiam, A. A. & Pudles, J. (1973) Eur. J. Biochem., 39, 431-441. 7. Loverde, A. ~ S t r i t t m a t e r (1968) J. Biol. Chem., 243, 5779-5787. 8. F r i e d m a n , R. B. & Radda, G. K. (1969) Biochem. J., 114, 611-619. 9. Goldin, B. R. & Frieden, C. (1971) Biochemistry, '10, 3527-3534. 10. Fasella, P. ~ Hammes, G. G. (1963) Arch. Biochem. Biophys., Jl00, 295-297. 11. Lazarus, N. R., Dereehin, M. & Barnard, E. A. (1968) Biochemistry, 7, 2390-2400. 12. Grillo, M. A. (1968) Enzymologia, 34, 7-19. 13. Kaji, A. (1966) Biochim. Biophys. Acta, 122, 43-56. 14. R6schlau, P. ~ Hess, B. (1972) Hoppe Seyler's E. Physiol. Chem., 353, 944-948. 15. Delafuente, G., Lagunas, R. & Sols, A. (1970) Etzr. J. Biochem., 16, 226-233.
Effects of activators on chemically modified yeast hexokinase. L u i s C a r l o s MENEZES a n d J u l i o PUDLES <>. I n s t i t u t d e B i o c h i m i e , U n i o e r s i t ~ d e P a r i s - S u d , C e n t r e d'Orsa!t, 91~05 O r s a y ( F r a n c e ) . S u m m a r y . - - Enzymic studies p e r f o r m e d w i t h chemically modified yeast h e x o k i n a s e (ATP : D-hexose-6-phosphotransferase) confirm previous r e s u l t s indicating t h a t the ~olfhydryl, i m i d a z o l a n d m o s t of the reactive a m i n o groups do not seem to be directly implicated i n the enzyme active site. On the o t h e r h a n d the modifieation of these f u n c t i o n a l groups of the e n z y m e does not affect the t r a n s i t i o n between the acidic inactive f o r m to a n active e n z y m e form a f t e r deprotonation. The c h e m i c a l l y modified f o r m s of h e x o k i n a s e a n d the native enzyme are affected in the same w a y b y a c t i v a t o r s (citrate, o-malate, 3-phosphoglycerate a n d Pi) w h e n the activity w a s m e a s u r e d at pH 6.6. Moreover the loss of enzyme activity observed in the course of the chemical modifications is accompanied b y a n increase of the a c t i v a t i o n effect. This increase m u s t be related to some r e o r g a n i z a t i o n of the enzyme active site in presence of the effectors, since t h e same effect was observed w h e n h e x o k i n a s e was d e n a t u r e d w i t h 3M u r e a at pH 7.5. However no increase i n the a c t i v a t i o n effect was observed ~vhen the d e n a t u r a t i o n 'was carried o u t at pH 6.5 At t h i s pH the loss in activity a n d the change of optical a b s o r p t i o n a t 286 n m were m u c h slower t h a n at pH 7.5, vehieh indicates a great difference i n the p r o t e i n s t r u c t u r e between these pHs.
INTRODUCTION. K o s o w a n d R o s e [1] r e p o r t e d t h a t t h e r a t e of the ATP : glucose transphosphorylase reaction c a t a l y z e d b y y e a s t h e x o k i n a s e , is i n h i b i t e d b y l o w e r i n g t h e p H of t h e r e a c t i o n b e l o w 7.0, at s u b o p t i m a l c o n c e n t r a t i o n s of A T P . T h i s e f f e c t of a c i d i t y is o v e r c o m e b y a c t i v a t o r s s u c h as, o r t h o phosphate, citrate, malate, 3-phosphoglycerate a n d r i b o s i d e t r i p h o s p h a t e . T h e y also o b s e r v e d t h a t i n t h e a c i d r a n g e , A T P a p p e a r s to s e r v e b o t h as a n a c t i v a t o r a n d as a s u b s t r a t e w i t h t h e r e s u l t that l/v versus 1/[ATP] plots are non linear. T h e i r r e s u l t s i n d i c a t e t h a t H + is a n u n 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 t o A T P as t h e v a r i a ble substrate and that two protons are required f o r t h e i n h i b i t i o n w i t h a m e a n p K o f 7.3. In this report the kinetic behaviour between p H 6.5 a n d o H 8.0 of y e a s t h e x o k i n a s e c h e m i c a l l y m o d i f i e d b y g r o u p s p e c i f i c r e a g e n t s w i l l b e desc r i b e d as w e l l as t h e e f f e c t of a c t i v a t o r s o n t h e s e enzymic forms. E n z y m e : Yeast h e x o k i n a s e or A T P : D-hexose-6p h o s p h o t r a n s f e r a s e (E.G. 2.7.1.1). A b b r e v i a t i o n s used are : TNBS, 2, 4, 6 - t r i n i t r o h e n z e n e - l - s u l f o n a t e ; DTNB, 5, 5'-dithiobis ( 2 - n i t r o b e n zoic acid). (*) Dedicated to t h e Memory of Huguette RobichonSzulmajster. To ~vhom all correspondence s h o u l d be addressed.
T h e d i f f e r e n t f o r m s of e n z y m e s t u d i e d h a d r e s p e c t i v e l y t h e i r imi.dazol, s u l f h y d r y l a n d a m i n o groups modified.
EXPERIMENTAL --
PROCEDURE.
~MATERIALS.
Baker's yeast hexokinase (ATP : hexose-f-phosphotransferase) from Boehringer Mannheim GmbH (Mannheim, Germany), was repurified by chromatography through CM-cellulose column [for details s e e ref. 2]. P y r u v a t e k i n a s e a n d g l u c o s e - 6 - p h o s phate dehydrogenase were purchased from Boehringer. U r e a , o b t a i n e d f r o m P r o l a b o as r e a g e n t g r a d e , w a s r e c r y s t a l l i z e d f r o m 9,5 p e r c e n t e t h a n o l . 5.5'-dithio-bis (2-nitrobenzoic acid) (DTNB) and ~-mercaptoethanol were obtained from Aldrich C h e m . Co. I n c . ; A T P d i s o d i u m s a l t a n d N A D P w e r e p u r c h a s e d f r o m P L , B i o c h e m i c a l s Co. P h o s phoenolpyruvate and 3-phosphoglycerate were obtained from Boehringer. L-malic acid from Merck and citric acid from Prolabo. 2-(N-morphol i n o ) e t h a n e s u l f o n i c a c i d (MES) a n d t r i s - ( h y d r o x y methyl)-qnethyl-2 anainoethane sulfonic acid (TES) were obtained from Serva. Ethoxyformic anhydride was obtained from Garlo Erba. 2,4,6-trinitrobenzene sulfonate disodium salt (TNBS) was purc h a s e d f r o m N u t r i t i o n a l B i o c h e m i c a l s Co.
52
L . C. M e n e z e s
All other c h e m i c a l s w e r e of a n a l y t i c a l r e a g e n t grade. Solutions w e r e p r e p a r e d in d e i o n i z e d distilled w a t e r . --
~E,THO
DS.
Enzymic assay -- Hexokinase activity under the v a r i e t y of c o n d i t i o n s that w e r e s t u d i e d was f o l l o w e d in the p r e s e n c e o,f an excess of the coup l i n g e n z y m e glucose-6-P d e h y d r o g e n a s e and NAD,P ~ i t h a Gary 118 C s p e e t r o p h o t o m e t e r . F o r the Km studies p y r u v a t e kinase and p h o s p h o e n o l p y r u v a t e w e r e incl,uded, to r e g e n e r a t e ATP, as d e s c r i b e d by K o s o w and Rose [1].
In the a c t i v a t i o n studies of the m o d i f i e d hexokinase, the a m o u n t o'f e n z y m e taken for assay was always i n c r e a s e d to c o m p e n s a t e for the degree of i n a c t i v a t i o n so that the actual rates in the assays w e r e c o m p a r a b l e . P r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d by a b s o r p t i o n m e a s u r e m e n t at 2,8.0 nm. A value of 0.9'2 w a s taken for the a b s o r b a n c e of a 1 m g / m l solution of h e x o k i n a s e in a 1 cm cnvette at 280 nm, as i n d i c a t e d by Lazarus et al [3]. --Amino a c i d a n a l y s i s of the photooxidize.d p r o t e i n was p e r f o r m e d as detailed in a p r e v i o u s r e p o r t [2]. -- Modification of amino group with 2,4,6-trinit r o b e n z e n e s n l f o n a t e . - - To the e n z y m e (0.25 mg in 0.9 m l ) in 0.1 M p o t a s s i u m p h o s p h a t e buffer (pH 8.0) 0.1 ml of a 70 mM aqueous solution of TNBS was added. T h e final v o l u m e w a s 1 ml. The r e a c t i o n was f o l l o w e d at 344 nm in a Safas double beam r e c o r d i n g s p e c t r o p h o t o m e l e r at 2.3 °. The extent of m o d i f i c a t i o n w a s d e t e r m i n e d by u s i n g a m o l a r e x t i n c t i o n value o.f 1'0,5.0.0 cm-XM-1 determ i n e d w i t h a s t a n d a r d solution e-TN~P-c~-acetyilysine at the same p H an, d it is in quite a g r e e m e n t w i t h the value obLa:in,ed by Goldfarf [4]. Aliquots of 25 vl c,f a 55 mM aquaeous solution of glycine, con.raining 40 m ~ 2 - m e r c a p t o e t h a n o l . -- Modification of the sulfhgdryl groups with 5 , 5 ' - d i t h i o b i s ( 2 - n i t r o b e n z o a t e ) ( D T N B ) . - - To the e n z y m e (500 ~tg) in 50 mM Tris-HC1 buffer (pH 8.0), c o n t a i n i n g 0.1 M EDTA, a DTNB solution in the same buffer w a s a d d e d to a final conc e n t r a t i o n of 50 ~M. T h e final v o l u m e of the reaction m i x t u r e w a s 1.0 ml.
T h e course of the r e a c t i o n w a s f o l l o w e d at 412 nm and at 23 °. T h e m o l a r e x t i n e t i o n coefficient of TNB at 412 n m is 13,6,0,0 era-aM -x [5]. Aliquots of 5 ~1 w e r e r e m o v e d at different time i n t e r v a l s and i m m e d i a t e l y used for the e n z y m i c assay. BIOCHIMIE, 1976, 58, n ° 1-2.
and J. Pudles. --Modification of the histidine residues. -T h e h i s t i d i n e residues of yeast h e x o k i n a s e w e r e m o d i f i e d e i t h e r by p h o t o o x i d a t i o n in p r e s e n c e of m e t h y l e n e blue (at p H 6.0 or 7.5) or by e t h o x y f o r m y l a t i o n w i t h e t h o x y f o r m i c a n h y d r i d e . Details for these m o d i f i c a t i o n s w e r e d e s c r i b e d in t w o p r e c e d i n g r e p o r t s [2, 6]. .-- I n a c t i v a t i o n o f y e a s t h e x o k i n a s e b y 3 M urea, at p H 7.5 a n d 6.5. - - H e x o k i n a s e (1 m g / m l ) w a s d e n a t u r e d by i n c u b a t i o n at 25 ° in a solution of 3 3I urea in 10 mM p o t a s s i u m p h o s p h a t e (pH 7.5) or in 10 fiaM 2-(.N-morpholino) ethane s u l f o n i c acid (pH 6.5), both buffers c o n t a i n i n g 0.1 M NaC1. Aliquots of 10 ,M w e r e r e m o v e d at different time i n t e r v a l s and diluted in 2~0 M of urea solutions at the c o n c e n t r a t i o n an, d p H at w h i c h the i n a c t i v a tion ha.d been c a r r i e d out.
F r o m these diluted solutions, aliquots w e r e taken (between 2 to 50 ~1) and the r e s i d u a l activity m e a s u r e d i m m e d i a t e l y at p H 6.5, in a final v o l u m e of 1 ml of the r e a c t i o n m i x t u r e (without urea). In a parallel e x p e r i m e n t u n d e r the same conditions, aliquots w e r e taken a n d assayed in the presence of the activator. The kinetics of d e n a t u r a t i o n of the e n z y m e in 3 M urea w a s also f o l l o w e d at 286 nm in a Cary 118 C double beam s p e c t r o p h o t o m e t e r , using <
The effect of this r e a g e n t on the e n z y m e activ i t y of yeast h e x o k i n a s e in the course of the m o d i f i c a t i o n of the a m i n o groups is s h o w n in figure 1. The m o d i f i c a t i o n o¢ the first 4 a m i n o groups p e r e n z y m e suhunit causes the loss of m o r e
Chemically modified yeast hexokinase.
53
,of
t h a n 50 p e r c e n t th,e e ' n z y m e a c t i v i t y , t h e n t h e activity decreases slowly for the other amino groups which were trinitrophenylated. After mod i f i c a t i o n of 28 o u t of t h e 37 a m i n o g r o u p s d e t e r m i n e d p e r e n z y m e s u b u n i t , still 5 p e r c e n t o f t h e residual activity was retained.
30(
QI ,,
,oo[
;,. /
¢
250
-
:~ •- >
_.
/*
~ ~
~
coo
~..~
~.°--~ ~od,f,=,~ ~lmyd,yl 9muv~
/.'-f--"'--,.,,,
\
"~
100~ , . . . . . . ~ 50
/ ~ •>
,ook
I "
,
.0,,,,.',
I
I ,
~ ,~ " - - T
5
Modified
10
Amino
I
I Modified Sufhydryl
..... ", ..... ','.. .... ~ ..... '2.,,,
"'~'~°
50
1
15
J0
Group= (no. p e r enzyme=ul0ulit)
Fi6. 1. - - lnactioation of yeast h e x o k i n a s e as a [unction of the n u m b e r of m o d i f i e d lysine residues. Hexokinase was t r i n i t r o p h e n y l a t e d and the reaction stopped at different t i m e intervals as described in the E x p e r i m e n t a l Procedure section. After reaction w i t h 2,4,6-trinitrobenzenesulfonate, the enzyme was appropriately diluted and the hexokinase activity was assayed by incubation of the enzyme (160 to 2500 ng) at 23 °, in a final volume of 1 ml ~vith 2-(N-morpho]ino) e t h a n e s n l f o n i e acid, pH 6.6, 100 mM ; glucose 1 mM ; ATP 0,05 mM ; MgC12 5 mM ; NADP 0,5 mM and glueose-6-P dehydrogenase 0,7 unit, The activity was assayed in presence (A) an in absence of 1 mM citrate (~). In the insert is r e p r e s e n t e d the variation of the activation factor ~vith citrate in function of the n u m b e r of t r i n i t r o p h e n y l a t e d amino group. Activation factor is defined as the ratio between the enzymic activity m e a s u r e d in presence and in absence of citrate.
2. - - I n a c t i v a t i o n of yeast hexohinase as a / u n c t i o n o/ the n u m b e r o[ cgsteine residues modified. The reaction of 5,5-dithiobis(2-nitrobenzoate) w i t h hexokinase was carried as described in the Experim e n t a l Procedure section. Enzymic activity was assayed b o t h in presence (A) and in absence (A) of 1 mC citrate. The activity assays were carried with 50 lo 125 ng of hexokinase, u n d e r the same conditions as those in figure 1. In the insert is represented the variation of the activation factor w i t h citrate, in f u n c t i o n of the n u m b e r of s u l f h y d r y l groups modified in the enzyme.
30( J~
7
250 200
150
100
T h e e f f e c t of t h e m o d i f i c a t i o n of t h e s u f f h y d r y l g r o u p s b y D T N B is s h o w n i n f i g u r e 2. I n t,his c a s e , it c a n b e s e e n t h a t f o r t h e f i r s t s u l f h y d r y l g r o u p n m d f f i e d o n l y a s l i g h t l o s s of t h e a c t i v i t y w a s observed ; with the increase on the modification, steeper becomes the inactivation process. Howe v e r a f t e r m o d i f i c a t i o n of 3.7 s u l f h y d r y l g r o u p s out of t h e 4 d e t e r m i n e d p e r e n z y m e s u b u n i t , 20 p e r c e n t of ,the r e s i d u a l a c t i v i t y w a s still left. F a s e l l a a n d H a m a n e s [10] o b t a i n e d s i m i l a r r e s u l t s b y t h e m o d i f i c a t i o n of t h e s u l h y d r y l g r o u p s o f h e x o k i n a s e w i t h P C ~ B ( a c t i v i t y m e a s u r e d at p H 8.0). The histidyl residues were modified either by p h o t o o x i d a t i o n i n p r e s e n c e o f m e t h y l e n e b l u e at p H 7.5 o r 6.0, o r b y e t h o x y f o r m y l a t i o n w i t h
BIOCHIMIE, 1976, 58, n ° 1-2.
Groups (no. perenzyme subunit)
FIG.
. "-~-'~'''''---~ 20 25
----.....
50
~a
\
t ~z photoox,d~t,on t,me,m,~
\ ,
photooxidation
time (rain)
FIG. 3. - - Inaclioation of yeast h e x o k i n a s e by photooxidation in presence of m e t h y l e n e blue. P h o t o o x i d a t i o n was carried out by i r r a d i a t i n g w i t h a 300 'watt l a m p (Mazdasol) 0.6 ml samples at a protein c o n c e n t r a t i o n of 0.25 m g / m l , in pyrex tubes (1 × 12 cm). The solvent w a s 5 mM potassium phosphate buffer, pH 7.5, containing 0.1 mM EDTA and 0.l M NaC1. Methylene blue yeas added to a final conc e n t r a t i o n of 0.01 per cent. The t e m p e r a t u r e was m a i n t a i n e d at 17 ° (for details see ref. 2). Enzymic activity w a s assayed in presence (A) and in absence (A) of 1 mM citrate. The activity assays were carried w i t h 75 to 1000 ng of hexokinase, in the same conditions as those in figure 1. In the i n s e r t is r e p r e s e n t e d the v a r i a t i o n of the activation factor w i t h citrate, in f u n c t i o n of the time of he×okinase photooxidation.
L. C. M e n e z e s a n d J. P u d l e s .
54
e t h o x y f o r m i c a n h y d r i d e at p H 7.5, as d e s c r i b e d p r e v i o u s l y [2, 6]. T h e i n a c t i v a t i o n b y b o t h t e c h n i q u e ~vas f o l l o w e d b y n m a s u r i n g t h e a c t i v i t y a t p H 6,6 a n d w a s i d e n t i c a l to o u r p r e v i o u s r e s u l t s
300
in which the activity was (fig. 3 a n d 4).
o - -
•
250
blocked hi.tidal res,dues no o e t e n z me s u u n , t ~
20( o
8.0
- - Effects of the activators in the course of the chemical modification of hexokinase. - - I t is
15(
i n t e r e s t i n g to o b s e r v e t h a t i n t h e c o u r s e of t h e trinitrophenylation, the activation factor inc r e a s e s f o r t h e f i r s t 14 a m i n o g r o u p s m o d i f i e d , t h e n it b e g i n s t o d e c r e a s e w i t h f u r t h e r m o d i f i c a t i o n u n t i l it r e a c h e s n e a r l y t h e s a m e l e v e l of a c t i v a t i o n as o b s e r v e d f o r t h e n a t i v e e n z y m e (fig. 1 a n d i n s e r t ) . ] ' h i s i n c r e a s e of t h e a c t i v a t i o n f a c t o r w a s also o b s e r v e d w i t h m a l a t e , 3 - P - g l y c e r a l e a n d p h o s p h a t e ( t a b l e I). F o r t h e s e l a s t a c t i v a t o r s t h e e f f e c t `was d e t e r m i n e d w h e n t h e r e s i d u a l a c t i v i t y w a s of 5 p e r c e n t .
1oo
50
Blocked
Fro. 4. --
at pH
At t h e e n d of t h e p h o t o d y n a n l i c treatment, w h e n t h e e n z y m e h a d loss 9.0, p e r c e n t of t h e a c t i v i t y (fig. 3), 5 h i s t i d y l , 2 c y s t e i n y l , 2 m e t h i o n y l and 2 tryptophyl residues per enzyme subunit had b e e n oxi,dized f r o m r e s p e c t i v e l y 9, 4, 11 a n d 4. H o w e v e r b y e t h o x y f o r m y l a t i o n w e m o d i f i e d spec i f i c a l l y all tile 9 h i s t i d y l r e s i d u e s . As c a n b e s e e n i n f i g u r e 4, still 40 p e r c e n l of t h e e n z y m e a c t i v i t y w a s left a f t e r a c y l a t i o n of t h e s e r e s i d u e s .
4--4
ze
measured
Histidyl
Residues(no.
per enzyme subunit)
I n a c t i v a t i o n of yeast hexokinase as a f u n c -
t i o n of the number of histidyl residues ethoxyformylated.
To the enzyme (0.85 m g i n 0.5 ml) in 0.1 M potass i u m p h o s p h a t e buffer (pH 7.5) c o n t a i n i n g 0.1 mM EDTA, 5 m l of a 175 mM e t h a n o l solution of ethoxyf o r m i c a n h y d r i d e ~vas added. The t e m p e r a t u r e xvas m a i n t a i n e d at 0% Aliquots of 20 ~1 `were removed at different t i m e i n t e r v a l s a n d the r e a c t i o n stopped by adding. 20 ul of a 0.1 M glyeine solution, pH 8.4. The enzymic activity ~vas assayed i n presence (A) a n d in absence (A) of 1 mM citrate. The activity assays ~vere carried ~ 4 t h 160 to 3,20 ng of enzyme, in t h e same c o n d i t i o n s as t h o s e i n figure 1. I n t h e i n s e r t is r e p r e s e n t e d the v a r i a t i o n of the a c t i v a t i o n f a c t o r "with citrate in f u n c t i o n of the n u m b e r of h i s t i d y l residues e t h o x y f o r m y l a t e d .
O n t h e o t h e r h a n d w h e n "the a c t i v i t y w a s m e a s u r e d at p H 8.0, n o a c t i v a t i o n w a s o b s e r v e d i n p r e s e n c e of c i t r a t e (see t a b l e I), i n d i c a t i n g t h a t t h e m o d i f i e d e n z y m e , s i m i l a r l y to t h e n a t i v e o n e , w a s s e n s i t i v e to t h e a c t i v a t o r s o n l y at a c i d p H . I n f i g u r e s 2, 3 a n d 4 a n d r e s p e c t i v e i n s e r t s , t h e e f f e c t s of c i t r a t e i n t h e c o ~ r s e of t h e m o d i f i c a t i o n of t h e e n z y m e b y DT.NB, p h o t o o x i d a t i o n at p H 7.5
TABLE [[.
Effect of activators on the relative rates of the native and chemically m o d i f i e d hexokinase. Functional group modified Additions
none pH6.6
imidazol (a)
pH 8.0 pH6.6
sulfhydryl (b)
amino (c)
pH8.0
pH6.6
pH8.0
pit 6.6 pH 8.0
13.5
42
5.0
22.5
3.6(d) 14.9(d) 0.7(e) 2.4 (e) 6.9(d) 15.3(d) 2.2 (e) 2 . 4 ( 0 1.6(e) 2.4(e) 2.2 (e) 2.3(e) 0.9(e)
None ................
32.5
100
Citrate 1 mM . . . . . . . .
71.5
93
37
43
18.5
22
L-malate I mM . . . . . . 3-P-glycerate 1 m M . . . Pi 20 mM . . . . . . . . . . .
62 68 39
100 94 34
27 35 22
38 36 15
15 13 9
22.5 22
(a) 9 i m i d a z o l groups modified per e n z y m e s u b u n i t `with e t h o x y f o r m i e anhydride. b) 3.7 s u l f h y d r y l groups modified per e n z y m e s u b u n i t ~vith DTNB. (c) a m i n o groups modified 'with TNBS ; (d) 14 a m i n o groups ; (e) 28 a m i n o groups per e n z y m e s u b u n i t . F o r detail see E x p e r i m e n t a l section a n d fig. 3.
BIOCHIMIE, 1976, 58, n ° 1-2.
Chemically
modified
yeast hexokinase.
and ethoxyformylation are shown. It can be seen t h a t f o r a n y of t h e s e m o d i f i c a t i o n s , t h e d e c r e a s e i n a c t i v i t y w a s f o l l o w e d b y a n i n c r e a s e of "the activation factor. Moreover, this effect was on/y o b s e r v e d at p H 6,6 a n d n o t at p H 8.0, a n d a l w a y s a t l o w c o n c e n t r a t i o n of A T P . A s i m i l a r e f f e c t w a s observed with malate, 3-P-glycerate and phosp h a t e ( t a b l e I).
r a t e of i n a c t i v a t i o n at t h e f o r m e r p H w a s l o w e r , and moreover we don't have for the moment any evidence indicating that the same residues were o x i d i z e d at b o t h p H v a l u e s .
--. E f f e c t of p i t on M g A T P d e p e n d e n c e of the chemically m o d i f i e d hexokinase. - - To determine if t h e m o d i f i c a t i o n of t h e a m i n o , s u l f h y d r y l a n d h i s t i d y l g r o u p s h a d a n y e f f e c t o n t h e b i n d i n g of MgATP or in the transition between an inactive f o r m of t h e e n z y m e i n t h e a c i d i c r e g i o n a n d a n active form in the alkaline region, studies were performed on the MgATP concentration depen-
It w a s q u i t e i n t e r e s t i n g to n o t i c e t h a t i n t h e c o u r s e of t h e p h o t o d y n a m i c t r e a t m e n t a t p H 6.0 w e d i d n o t o b s e r v e a n y i n c r e a s e of t h e a c t i v a t i o n factor in presence of citrate.
/ A
30(]
00
< ~.~ 100
1l)
B
.//j" •
20
30
30
G
.
/=
20
I0
3O
10
( MgATP mM )
20
30
10
I MgATP mM )
20
3'0
(MgATP mM)
y,
50
/
4O
/
30 E
20
10
#MgAYPraM)
60
• = f l
(MgATPmM~
×
55
7 .i ./
/
20
/ J
65
/
73
77
pH
65
69
73
o//ef
/ / 77
65
pH
f
II
/
/
J
69
J
/
/
o/ i/
69
73
pH
77
65
69
73
77
&5
pH
69
73
77
pH
Fro. 5 (upper set of curves). - - Double reciprocal plots of velocity of yeast hexokinase as a function of MgATP concentration, between pH 6.5 to 7.9, for the native and modified hexokinase. Each tube contained : enzyme 95 to 500, ng, glucose 2 m M ; MgC12 5 m M ; NADP 0.5 mM ; glueose-6-P-dehydrogenase 0.7' u n i t ; P - e n o l p y r u v a t e 1 mM ; p y r u v a t e kinase 1.5 u n i t s a n d buffer 100 mM in a final volume of I ml. Buffers used were 2-(N-morpholino) e t h a n e s,ulfonie acid, pH 6.5 ( I ) or H - T r i s - ( h y d r o x y m e t h y D m e t h y l - 2 ' - a m i n o - e t h a n e s u l f o n i c acid pH 6.9 (4k), pH 7.0'5 (©), pH 7.2, (A),, pH 7.3 (i~), pH 7.5 (@) a n d pH 7.9 ( ). In the figure are r e p r e s e n t e d the d a t a o b t a i n e d ~vith : A - - n a t i v e enzyme ; B - - t r i n i t r o p h e n y l a t e d enzyme, r e t a i n i n g 5 p e r cent of the activity a n d h a v i n g 26 a m i n o groups, modified per enzyme s u b u n i t ; C - - p h o t o o x i d i z e d enzyme, w h i c h r e t a i n s 20 p e r cent of t h e a c t i v i t y (4 histidyls, 1 cvsteinyl, 2 m e t h i o n y l a n d 1 t r y p t o p h y l residues photooxidized p e r enzyme s u b u n i t ) ; D ~ " e t h o x y ¢ o r m y l a t e d enzyme, w h i c h r e t a i n s 40 p e r cent of t h e activity (9 h i s t i d y l s residues blocked p e r e n z y m e s u b u n i t ) ; E - - hexokinase modified b y reaction w i t h 5,5'-dithiohis(2-nitrobenz,o,ic acid), h a v i n g 3.7 cysteinyl groups' blocked p e r enzyme s u h u n i t a n d r e t a i n i n g 20 per cent of the original activity. Lower set of curves : pH effect on m a x z m u m velocity for the first hyperbolic region of the A T P dependence curve. The d a t a f r o m the u p p e r set of double reciprocal plots are represented, showing the ¥ m e x t r a p o l a t e d front the hyperbo],ic low ,~TP region, Vm', as a f u n c t i o n of pH. Curves F, G, H, I a n d J corresponds respectively to the n a t i v e (©), t r i n i t r o p h e n y l a t e d (A), p h o t o oxidized (A), e t h o x y f o r m y l a t e d (@) a n d DTNB modified~ ([~) hexokinase. This result was unexpected, since we had prev i o u s l y o b s e r v e d t h a t i n t h e c o u r s e of t h e p h o t o i n a c t i v a t i o n at p H 6.0 o r a t p H 7.5, f o r a n i d e n t i c a l r e s i d u a l a c t i v i t y t h e s a m e a m o u n t of a m i n o a c i d r e s i d u e s w e r e o x i d i z e d [2]. H o w e v e r , t h e
BIOCHIMIE, 1976, 58, n ° 1-2.
d e n c e of t h e h e x o k i n a s e a n d 7.9.
a c t i v i t y b e t w e e n p H 6.5
For these experiments, the residual activity for the different forms of the enzyme were respecti-
56
L . C. M e n e z e s
v e l y 5 p e r c e n t f o r t h e t r i n i t r o p h e n y l a t e d , 20 p e r c e n t f o r t h e D T N B m o d i f i e d , 10 p e r c e n t f o r t h e p h o t o o x i , d i z e d a n d 40 p e r c e n t f o r t h e e t h o x y formylaied enzyme.
0.8
and J. PudIes. - - E f f e c t o f 3 M urea. - - T h e r e s u l t s of t h e c h e m i c a l m ( ~ d i f i c a l i o n s e e m e d to s u g g e s t t h a t t h e i n c r e a s e of t h e a c t i v a t i o n f a c t o r i n p r e s e n c e of t h e a l l o s t e r i c e f f e c t o r s w a s d u e to a c o n f o r m a t i o n a l c h a n g e . W e c h e c k e d if t h i s e f f e c t w o u l d a p p e a r i n t h e c o u r s e of t h e d e n a t u r a t i o n of i h e enzyme by a non covalent bond forlning agent as urea.
0.6 04
......... i j
I .
EIE
0
"~ O~ _o
-0.2
i
-0.4
zB / 250
/ iqo
i .... -o
21o ,, oI 11o m,n.t~s
zlo
~o
200
-0.6 -0.8
z7
j:
, -
150
-
6.5
67
69
71
73
Z5
X7
pH Fro. 6. - - R e p l o t of the data represented in figure 5 (lower set of curves). Vm is the Vmax at pH 7.9. The several lines corresp o n d to t h e n a t i v e e n z y m e (O) a n d to the t r i n i t r o p h c n y l a t e d (&), photooxidized (A), e t h o x y f o r m y l a t e d (O) a n d DTNB modified (O) bexokinase.
F i g u r e s 5 ( u p p e r s e t o'f c u x v e s ) sho~v t h e d o u b l e r e c i p r o c a l p l o t s b e t w e e n p H 6.5 a n d 7.9 i n f u n c t i o n of t h e M g A T P c o n c e n t r a t i o n f o r t h e native and modified enzymes. In figures 5 (lower set of curves) are represented the pH effects on the extrapolaled low ATP V m v a l u e s , V ' m , as i n d i c a t e d b y K o s o w a n d R o s e [1]. T h e p H p r o f i l e s o f t h e a c t i v i t y ~vere q u a l i t a tively identical for the native and chemically modified forms of the enzyme. Figure 6 shows the representation o,f - - l o g "Vm ( ~ ~ 1) as a f u n c t i o n o f p H , a c c o r d i n g to t h e e q u a t i o n d e v e l o p e d b y K o s o w a n d R o s e [1] f o r all t h e c h e m i c a l l y m o d i f i e d f o r m s o f t h e e n z y m e . I n all c a s e s a s i n g l e l i n e w a s o b t a i n e d w i t h a s l o p e v a r y i n g b e t w e e n 2.2 to 2.4, w h i c h a g r e e s w i t h t h e resu,lts of t h e s e a n t o r s f o r t h e n a t i v e e n z y m e , indicating that also for the modified form of the enzyme 2 protons are required for the pH transition. No s i g n i f i c a n t d i f f e r e n c e s w e r e f o u n d f o r t h e Km values for MgATP between the native and chemically modified enzymes. BIOCHIMIE, 1976, 58, n ° 1-2.
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FIG. 7. - - Denaturation of yeast h e x o k i n a s e by 3 M urea at pH 7.5, and s u b s e q u e n t r e n a t u r a t i o n by dilution. Hexokinase w a s i n c u b a t e d at pH 7.5, in 3 M u r e a a n d aliquots ~vere taken, at different t i m e i n t e r v a l s for enzymic assays as described in E x p e r i m e n t a l Procedure Section. At the arrow, d e n a t u r e d enzyme was diluted 21 t i m e s i n 10 mM p o t a s s i u m p h o s p h a t e buffer (pH 7.5). The activity assays were carried out at pH 6.5 i n presence (•) or i n absence (A) of 1 mM citrate, in the same conditions as those in figure 1. The a m o u n t of e n z y m e t a k e n for assay varied f r o m 95 to 2380 ng, so t h a t actual rates i n the assays were comparable. In the i n s e r t is r e p r e s e n t e d the v a r i a t i o n of the a c t i v a t i o n factor 'with citrate (ratio between the enzymic activity in presence a n d absence of citrate) as a f u n c t i o n of t h e t i m e of i n c u b a t i o n of t h e h e x o k i nase i n 3 M u r e a and, subsequently, the t i m e a f t e r dilution. The a r r o w indicates the t i m e at w h i c h t h e d e n a t u r e d e n z y m e ~vas diluted.
F i g u r e 7 s h o ' w s t h a t indee.d, h i g h e r w a s t h e d e g r e e of i n a c t i v a t i o n b y 3 M u r e a a t p H 7.5, higher also was the activation effect by citrate. When the enzyme was reactivated by dilution (2,1 t i m e s ) t h e a c t i v a t i o n e f f e c t r e t u r n s n e a r l y to a n o r m a l level. T h e r e c o v e r y o f t h e i n i t i a l a c t i v i t y w a s 70 p e r c e n t . I t is i n t e r e s t i n g to r e m a r k t h a t w h e n t h e a e n a t u r a t i o n w a s c a r r i e d o u t i n 3 M u r e a at p H 6.5, the activation factor with citrate remained unc h a n g e d (fig. 8).
Chemically modified yeast hexokinase. On t h e o t h e r h a n d , b o t h t h e loss in e n z y m i c a c t i v i t y a n d the c h a n g e i n a b s o r b a n c e at 286 n m w e r e m u c h s l o w e r w h e n the e n z y m e w a s d e n a t u r e d in 3 M u r e a at p H 6.5 t h a n at p H 7.5 (fig. 9). A l t h o u g h the s t a b i l i t y of t h e e n z y m e w a s h i g h e r at p H 6.5, t h e r e c o v e r y of t h e i n i t i a l a c t i v i t y a f t e r
57
dilution was not higher than when the enzyme w a s r e a c t i v a t e d a f t e r u r e a t r e a t m e n t at p H 7.5. W e c o u l d n o t o b s e r v e a n y effect of c i t r a t e o n t h e r a t e of r e n a t u r a t i o n of t h e e n z y m e u n d e r o u r experimental conditions. DISCUSSION.
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Fro. 8. - - D e n a t u r a t i o n of yeast h e x o k i n a s e by 3 M urea at p H ,6.5 a n d s u b s e q u e n t r e n a t u r a t i o n by dilution.
Hexokinase was incubated in 3 M urea, at pH 6.5. as described in the Experimental Procedure Section. At the arrow, the denatured enzyme was diluted 21 times in 10 mM 2-(N-morpholino) ethanesullonic acid (pH 6.5). The enzymic assays ~vere carried out at pH 6.5, in presence (A) or absence (A) of 1 mM citrate, under the same conditions as in fig. 1. The a m o u n t of enzyme taken for assay varied f r o m 95 to 2380 rag, so that actual rates in the assays were comparable. In the insert is represented the variation of the activation factor with citrate, as a function of the time of incubation of hexokinase in 3 M urea.
M o r e r e c e n t l y , L a z a r u s e t al [11] r e p o r t e d that a f t e r m o d i f i c a t i o n of t h e f o u r s u l f h y d r y l g r o u p s per enzyme subunit with methylmercuryiodide, t h e t w o less r e a c t i v e o n e c o u l d be e s s e n t i a l f o r the enzyme activity. T h e r o l e of the a m i n o g r o u p s in y e a s t h e x o k i n a s e w a s less s t u d i e d . G r i l l o [12] s h o w e d t h a t pyridoxal phosphate, which reacts reversibly w i t h t h e a m i n o g r o u p s , h a s an i n h i b i t o r y effect o n y e a s t h e x o k i n a s e , a n d t h a t it is n o t c o m p e t i t i v e t o w a r d s the s u b s t r a t e s A T P a n d D-glucose. I n this r e p o r t w e h a v e s h o w n t h a t t h e m o d i f i c a t i o n of a n y of t h e s e f u n c t i o n a l g r o u p s p r o m o t e s a loss of e n z y m e a c t i v i t y . H o w e v e r , e v e n a f t e r e x t e n s i v e m o d i f i c a t i o n t h e e n z y m e still r e t a i n s some activity. T h e s e r e s u l t s as w e l l as p r e v i o u s w o r k [2, 6, 10, 11, 12 a n d 13] s h o w that the i m i d a z o l , s u l f h y d r y l a n d t h e m o s t a c c e s s i b l e a m i n o g r o u p s to T N B S s e e m to be i m p o r t a n t for the s t a b i l i s a t i o n of t h e s t r u c t u r e of y e a s t h e x o k i n a s e . H o w e v e r , if t h e s e g r o u p s do not p l a y a d i r e c t r o l e in the c a t a l y t i c process, nontheless we cannot exclude them from being implicated either :
too
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P r e v i o u s s t u d i e s f r o m this l a b o r a t o r y [2, 6] h a v e s h o w n t h a t the h i s t i d y l r e s i d u e s w e r e n o t i n v o l v e d e i t h e r in the c a t a l y t i c o r t h e s u b s t r a t e b i n d i n g site w h e n t h e a c t i v i t i e s w e r e d e t e r m i n e d at a l k a l i n e p H values. I n the c a s e of t h e s u l f h y d r y l g r o u p s , t h e r e s u l t s of d i f f e r e n t a u t h o r s w e r e controversial. Fasella and Hammes L10] had s h o w n t h a t the loss of a c t i v i t y of h e x o k i n a s e a f t e r m o d i f i c a t i o n of t h e s e g r o u p s w i t h PCMB, w a s d u e o n l y to a d e s t a b i l i z a t i o n of the e n z y m e c o n f o r m a tion.
7~
1) in t h e t r a n s i t i o n b e t w e e n t h e a c i d i c i n a c t i v e f o r m of the e n z y m e to an a c t i v e f o r m u p o n the a d d i t i o n of a c t i v a t o r s o r i n c r e a s e of p H ; 2) o r in the i n t e r a c t i o n b e t w e e n the t e r n a r y complex (enzymic-substrates) with the activators.
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Fro. 9. - - D e n a t u r a t i o n o f yeast hexokinase by 3 M urea at pH ~.5 and 7.5 as m o n i t o r e d by the decrease in absorbance at 286 rim.
A ~ represents the enzyme absorption at the end of the denaturation process and At the enzyme absorption at different time intervals, pH 6.5 (O) and pH 7.5 (n) (see Methods). BIOCHIMIE, 1976, 58, n ° 1-2.
T o d e t e r m i n e if t h e k i n e t i c p r o p e r t i e s of m o d i f i e d f o r m of h e x o k i n a s e w e r e d i f f e r e n t a n a l o g o u s w i t h t h o s e of t h e n a t i v e e n z y m e , studied these properties under the conditions which the enzyme was highly modified.
the or we in
We observe that none of the chemical modificat i o n s h a d a n y s i g n i f i c a n t effect on t h e K m v a l u e s
58
L. C. M e n e z e s a n d J. Pudles.
for MgATP as c o m p a r e d w i t h the native enzyme. This seems to i n d i c a t e that the s u l f h y d r y l , histidyI a n d most of the a m i n o groups of the e n z y m e are not i n v o l v e d in the MgATP b i n d i n g site. On the other h a n d all these m o d i f i e d f o r m s of the e n z y m e s h o w the same c o m p l e x k i n e t i c b e h a v i o u r b e l o w p i t 7.3 at s u b - o p t i m a l c o n c e n t r a t i o n of ATP, as the native enzyme. The p H activities profiles, w e r e i d e n t i c a l also w h e n the data o b t a i n e d for the ¥ ' m w e r e plotted as a f u n c t i o n of pH. In all cases, h i g h e r was the degree of the chenfical m o d i f i c a t i o n and i n a c t i v a t i o n , h i g h e r was also the a c t i v a t i o n f a c t o r in the p r e s e n c e of citnate, D-malate, 3 - p h o s p h o g l y c e r a t e or Pi, w h e n the e n z y m e a c t i v i t y was m e a s u r e d at p H 6.6 at s u b - o p t i m a l c o n c e n t r a t i o n s of ATP. T h e i n c r e a s e d r e s p o n s e to the activators in the course of the c h e m i c a l m o d i f i c a t i o n s of h e x o k i nase, i n d i c a t e s that the loss in e n z y m i c a c t i v i t y w a s not d u e to the f o r m a t i o n of c o m p l e t e l y inactive f o r m of the enzyule, but r a t h e r to the formation of m o d i f i e d f o r m s of the e n z y m e still p a r tially active. These results suggest that the infidazol and sulfh y d r y l groups are not i m p l i c a t e d in the t r a n s i t i o n b e t w e e n the a c i d i c i n a c t i v e f o r m of the e n z y m e to an active f o r m after d e p r o t o n a t i o n . H o w e v e r w e c a n n o t c o m p l e t e l y e x c l u d e the p a r t i c i p a t i o n in this p r o c e s s of the less accessible a m i n o groups, w h i c h w e w e r e unable to t r i n i t r o p h e n y l a t e u n d e r our experimental conditions. W h e n the activities of the m o d i f i e d enzymes w e r e m e a s u r e d at p H 8,0 at s u b - o p t i m a l c o n c e n t r a t i o n of ATP no a c t i v a t i o n effect was observed, as h a d been r e p o r t e d p r e v i o u s l y by K o s o w a n d Rose for the native e n z y m e [1]. These results i n d i c a t e 'that the i n c r e a s e d effect of the activators w e r e only r e l a t e d to the p r o t o n ated f o r m of the enzymes as w a s d e s c r i b e d for the n o r m a l a c t i v a t i o n effect w i t h the native enzyme. It is also i n t e r e s t i n g to note that for the native enzyme, the rate at p H 6..6 u n d e r the i n f l u e n c e of the activators d i d not r e a c h the level of the activity w h e n the assay w a s p e r f o r m e d at pH 8.0 and at low A T P c o n c e n t r a t i o n s . H o w e v e r , f o r the chem i c a l l y m o d i f i e d f o r m s of the enzyme, c i t r a t e c o u l d raise the a c t i v i t y to a level as h i g h as that m e a s u r e d at p H 8.0 (see table I). Kosow and Rose h a d r u l e d out the p o s s i b i l i t y that the a c t i v a t i o n effect due to the effectors o r to the d e p r o t o n a t i o n of the p r o t e i n , could be related to a change on the state of a g g r e @ t i o n of the enzyme. O~ur results seem to c o n f i r m this data,
BIOCHIMIE, 1976, 58, n ° 1-2.
since the p h o t o o x i d i z e d ( u n p u b l i s h e d results) or e t h o x y f o r m y l a t e d h e x o k i n a s e [6J as w e l l as the e n z y m e h a v i n g the s u l f h y d r y l groups m o d i f e d [11] w e r e c o m p l e t e l y d i s s o c i a t e d u n d e r the c o n d i t i o n s in w h i c h the k i n e t i c p r o p e r t i e s w e r e studied, w h i c h suggest that the d i s s o c i a t e d f o r m of the e n z y m e is still sensitive to the a c t i v a t o r s or to the p H effect. The i n c r e a s e of the a c t i v a t i o n f a c t o r in the course of a c h e m i c a l m o d i f i c a t i o n h a d been rep o r t e d by R o s c h l a u and Hess [14]. These a u t h o r s h a v e s h o w n that in the course of the t r i n i t r o p h e n y l a t i o n of one r e a c t i v e s-amino group of yeast p y r u v a t e kinase, the e n z y m e loses a c t i v i t y but at the same time the r e s p o n s e to the allosteric effector ( f r u c t o s e - l , 6 - d i p h o s p h a t e ) increases. In our case the i n c r e a s e of the activation effect was o b t a i n e d not only in the course of the m o d i f i c a tion of such different groups as thiol, a m i n o a n d histidyl, but also by urea t r e a t m e n t of the e n z y m e at p H 7.5. These activation effects w h i c h are o b s e r v e d by s u c h different treatments, i n d i c a t e s that cautions must be taken, on e x p l a i n i n g the i m p l i c a t i o n of a s p e c i f i c a m i n o acid r e s i d u e in this k i n d of process, based on an u n i q u e m o d i f i c a t i o n . ,In our p o i n t of view, the a c t i v a t i o n effects observed, could be r e l a t e d to a p a r t i a l r e v e r s a l of the d e n a t u r i n g effect, r e s u l t i n g in a better f i t t i n g of the substrates on the e n z y m e a c t i v e site than in the d e n a t u r e d form. U n e x p e c t e d w e r e the results obtained in the course of the d e n a t u r a t i o n of the enzyme by 3 M u r e a at p H 6.5, or d u r i n g the p h o t o o x i d a t i o n at p H 6.0. U n d e r these c o n d i t i o n s the activation factor w i t h citrate r e m a i n e d c o n s t a n t t h r o u g h all the i n a c t i v a t i o n process. It is quite possible that u n d e r d e n a t u r i n g conditions at acid or aIkaline pH the c o n f o r m a t i o n a l f o r m s of the e n z y m e are d.ifferent. We h a d a l r e a d y o b s e r v e d that h e x o k i n a s e w a s m o r e stable to p h o t o o x i d a t i o n [21 or e t h o x y f o r m y l a t i o n [63 w h e n these m o d i f i c a t i o n s w e r e c a r r i e d out b e l o w p H 7.0. Moreover the i n c r e a s e d stability of the e n z y m e to the urea t r e a t m e n t at p H 6.5 as c o m p a r e d to p H 7.5, could suggest that the p r o t o n a t i o n of some s p e c i f i c groups on the e n z y m e structure, p r o m o t e s an im,portant effect on the p r o t e i n c o n f o r m a t i o n . Our attemps to o b s e r v e a s t r u c t u r a l effect i n d u c e d by c i t r a t e on the p h o t o o x i d i z e d e n z y m e in p r e s e n c e of o-glucose and B-v-methylene ATP or lyxose and ATP [15], w e r e n~nsuccessful, w h e n
Chemically modified yeast hexokinase. followed by fluorescence or by differential speclroscopy. I n c o n c l u s i o n , t h e s e r e s u l t s s h o w t h a t it t h e c h e m i c a l m o d i f i c a t i o n of t h e i m i d a z o l , s u , l f h y d r y l a n d a m i n o g r o u p s of y e a s t h e x o k i n a s e p r o m o t e s t h e i n a c t i v a t i o n of t h e e n z y m e , n e v e r t h e l e s s t h i s loss in the activity does not affect the activators b i n d i n g site o r t h e i o n i z a b l e g r o u p o f p K 7.3 i n volved in the t r a n s i t i o n b e t w e e n the a c i d i c ina c t i v e f o r m of t h e e n z y m e a n d a n a c t i v e e n z y m e from after deprotonation. M o r e o v e r , t h e s e r e s u l t s also i n d i c a t e b y e x c l u sion, that a ionizable group with a highly displaced p K ( c a r b o x y l or t y r o s y l group) seems to be i m p l i c a t e d in these m o l e c u l a r events. F u r t h e r w o r k is b e i n g c a r r i e d o u t to d e t e r m i n e t h e r o l e of these groups.
Aeknawledgments. This ~work was supported f r o m the Centre National de la Recherche Scientifique (E.R. 142) and F o n d a t i o n pour la Recherche Mddicale Franqaise. We are grateful to Drs J. v a n Heijenoort and G. Nemethy for c o m m e n t s i n the p r e p a r a t i o n of this manuscript. R~SUM~. Des dtudes enzymatiques effectu6es avec r h e x o k i n a s e de levure (ATP : o-hexose 6-phosphotransf6rase), modifi6e c h i m i q u e m e n t , ont confirm6 des r6sultats ant6rieurs i n d i q u a n t que le sulfhydryl, l'imidazol et ta p l u p a r t des groupes amin6s ne s e m h l e n t pas intervenir d i r e e t e m e n t au niveau du site aetif. D'autre p a r t la modification de ces groupes fonctionnels n'affecte pas la t r a n s i t i o n entre la f o r m e acide inactive et la f o r m e active de l ' e n z y m e apr6s d6protonation.
BIOCHIMIE, 1976, 58, n ° 1-2.
59
L'hexokinase modifi6e par diff6rents r~actifs est aetiv6e, de la m~me faqon que l ' e n z y m e native, p a r le citrate, le o-malate, le 3:phosphoglyc6rate et le Pi, q u a n d l'activit6 est mesur6e h pH 6,6. De plus, la perte d'activit6 e n z y m a t i q u e observ6e au cours des modifications chimiques est aecompagn6e d'une a u g m e n t a t i o n de l'effet d'aetivation. Cette a u g m e n t a t i o n dolt r6sulter d ' u n e r6organisation du site actif de l ' e n z y m e en pr6sence des effecteurs, puisque cet effet est aussi observ6 q u a n d l'hexokinase est d6natur6e par l'ur6e 3M, pH 7,5. Cependant nous n'avons observ6 aueune a u g m e n t a t i o n de l'effet d'activation q u a n d la d~naturation est faite h pH 6.5. A c e pH la perte d'activit6 et le c h a n g e m e n t d ' a b s o r p t i o n optique h 286 n m sont beaueoup plus lents qu'h pH 7,5, ce qui indique une grande diff6rence dans la structure de la prot~ine entre ces pit. REFERENCES. 1. Kosow, D. P. & Rose, I. A. (1971) J. Biol. Chem., 246, 2618-2625. 2. Menezes, L. C., Grouselle, M. & Pudles, J. (1972) Eur. J. Biochem., 30, 81-92. 3. Lazarus, N. R., Ramel, A. H., Rustum, Y. M. & Barnard, E. A. (1966) Biochemistry, 5, 4003-4016. 4. Goldfarf, A. R. (1966) Biochemistry, 5, 2570-2578. 5. Ellman, G. L. (1959) Arch. Biochem. Biophys., 82, 70-77. 6. Grouselle, M., Thiam, A. A. & Pudles, J. (1973) Eur. J. Biochem., 39, 431-441. 7. Loverde, A. ~ S t r i t t m a t e r (1968) J. Biol. Chem., 243, 5779-5787. 8. F r i e d m a n , R. B. & Radda, G. K. (1969) Biochem. J., 114, 611-619. 9. Goldin, B. R. & Frieden, C. (1971) Biochemistry, '10, 3527-3534. 10. Fasella, P. ~ Hammes, G. G. (1963) Arch. Biochem. Biophys., Jl00, 295-297. 11. Lazarus, N. R., Dereehin, M. & Barnard, E. A. (1968) Biochemistry, 7, 2390-2400. 12. Grillo, M. A. (1968) Enzymologia, 34, 7-19. 13. Kaji, A. (1966) Biochim. Biophys. Acta, 122, 43-56. 14. R6schlau, P. ~ Hess, B. (1972) Hoppe Seyler's E. Physiol. Chem., 353, 944-948. 15. Delafuente, G., Lagunas, R. & Sols, A. (1970) Etzr. J. Biochem., 16, 226-233.