Volume 266, number 1,2 17-20
FEBS 08499
June 1990
An X-ray solution scattering study of the cofactor and activator induced structural changes in yeast pyruvate decarboxylase (PDC) G . H / i b n e r 1, S. K S n i g 1, A . S c h e l l e n b e r g e r 1 a n d M . H . J . K o c h 2
Martin-Luther-Universit~it Halle-Wittenberg, Biotechnikum, Wissenschaftsbereich Enzymologie/Enzymtechnologie, Domplatz 1, Halle/S. 4020, DDR and 2European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o DESY, Notkestrasse 85, D-2000 Hamburg 52, FRG Received 14 February 1990; revised version received 24 April 1990 Structure and activation pattern of pyruvate decarboxylase (PDC) from yeast was studied by synchrotron radiation X-ray solution scattering. The results give a direct proof that the reversible deactivation of PDC at pH 8.0 is accompanied by the dissociation of the tetrameric holoenzyme into dimeric halves. The kinetics of this process was followed. At pH 6.5 the dimeric halves reassociate to a tetramer even in the absence of cofactors. The changes of the scattering pattern upon binding of the substrate-like activator pyruvamide indicate that the structure expands in the course of the enzyme activation. Pyruvate decarboxylase; X-ray solution scattering
1. I N T R O D U C T I O N P y r u v a t e d e c a r b o x y l a s e is a n e n z y m e f r o m the c y t o s o l t h a t catalyzes t h e d e c a r b o x y l a t i o n o f 2 - o x o a c i d s to the corresponding aldehydes. The enzyme extracted f r o m yeast is a n trz6'2 t e t r a m e r [1] with a m o l e c u l a r m a s s o f 240 k D a [2]. In p h y s i o l o g i c a l c o n d i t i o n s a r o u n d p H 6.0 t h e t w o c o f a c t o r s t h i a m i n e p y r o p h o s p h a t e ( T P P ) a n d M g 2+ are b o u n d in a q u a s i - i r r e v e r s i b l e m a n n e r . A t p H - v a l u e s a b o v e 7.0 t h e c o f a c t o r s a r e r e l e a s e d a n d a n a p o e n z y m e is f o r m e d . P r e v i o u s studies [3] suggested t h a t s i m u l t a n e o u s l y t h e t e t r a m e r i c e n z y m e dissociates in t w o d i m e r i c halves. R e c o m b i n a t i o n o f the a p o e n z y m e i n t o t h e h o l o e n z y m e t a k e s p l a c e at p H values b e l o w 7.0 in t h e presence o f T P P a n d M g 2+. T h e a c t i v i t y o f P D C is r e g u l a t e d b y the s u b s t r a t e as i n d i c a t e d b y a s i g m o i d a l d e p e n d e n c e o f the rate o n s u b s t r a t e c o n c e n t r a t i o n [ 4 - 7 ] a n d a lag p h a s e in the a p p e a r a n c e o f p r o d u c t s c o r r e s p o n d i n g to e n z y m e activat i o n [8]. C o n s e r v a t i o n o f the v a r i o u s r e g u l a t o r y states o f t h e e n z y m e b y c r o s s - l i n k i n g [9] a n d r e t a r d a t i o n o f substrate activation by immobilization of the enzyme o n a solid m a t r i x [10] suggest t h a t a s t r u c t u r a l c h a n g e o c c u r s d u r i n g the t r a n s i t i o n f r o m the inactive to the s u b s t r a t e - a c t i v a t e d f o r m . In the p r e s e n t s o l u t i o n X - r a y
s c a t t e r i n g s t u d y we h a v e investigated the s t r u c t u r a l changes during enzyme dissociation and recombination as well as d u r i n g s u b s t r a t e a c t i v a t i o n .
2. M A T E R I A L S A N D M E T H O D S PDC was extracted from yeast from the brewery Wernesgriin [1]. The enzyme is homogeneous as indicated by SDS-PAGE, with a specific activity of 50-60 units/mg. The activity was determined spectrophotometrically using alcohol dehydrogenase (ADH; Boehringer-Mannheim) and NADH (AWD Dresden) as complementary enzyme system [11]. Pyruvamide was synthesized according to Vogel and Schinz [12]. TPP was obtained from Serva (Heidelberg). All other chemicals were of analytic grade. Protein concentrations were~ determined at 280 nm using an extinction coefficient of 281 000 M -1 .cm-I. The X-ray solution scattering measurements were made at 7°C on the X33 camera [13] of the EMBL in HASYLAB at the storage ring DORIS of the Deutsches Elektronen Synchrotron at Hamburg using the standard data acquisition and evaluation systems [14,15]. Static scattering patterns were recorded during 3 min in the range 1.5 x l0 -2 _< s -< 2.2 x 10- ~nm- 1, where s = 2sin0/A,Ais the wavelength and 20 the scattering angle. The dissociation of PDC was followed with a time resolution of 30 s.
3. R E S U L T S A N D D I S C U S S I O N
Correspondence address: A. Schellenberger, Martin-Luther-Univer-
3.1. Solution X-ray scattering patterns
sit'it Halle-Wittenberg, Biotechnikum, Wissenschaftsbereich Enzymologie/Enzymtechnologie, Domplatz 1, Halle/S. 4020, DDR
S o l u t i o n X - r a y scattering p a t t e r n s o f P D C were o b t a i n e d in 0.1 M s o d i u m citrate b u f f e r , p H 6.2, in t h e p r o t e i n c o n c e n t r a t i o n r a n g e 1 - 7 . 2 m g P D C / m l . In this r a n g e the f o r w a r d scattering is p r o p o r t i o n a l to the p r o -
Abbreviations: PDC,
pyruvate decarboxylase;
TPP,
thiamin-
pyrophosphate
Published by Elsevier Science Publishers B. II. (Biomedical Division) 00145793/90/$3.50 © 1990 Federation of European Biochemical Societies
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Volume 266, number 1,2
FEBS LETTERS
June 1990
R (nm) -/..2
+
-4.0 4- + + + + +
3.8
+
+
+ +
+
+ +
+
+ +
++ ++ + +++ +
++
+
+4.+
+ +++ +
++ + '+ 4- +-F
+ + +
+4-F
,4-++ -F
++,+
+
+
+
3.6
% octivit
: I (0)
10(
1.0
0 0
0
~o
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°
0
°o"~** ° 0.5
o
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0 °~ °°
10 I
°
° ' ° o ° ° ° '°'°°~c~'°
20 I
~°°o0
,0¢~0'
30
time (min)
Fig. I. Time dependence of the forward scattering (o), the enzymatic activity (o) and the apparent radius of gyration during the dissociation of PDC in glycine/phosphate buffer pH 8.0. [PDC] = 7.2 mg/ml.
tein concentration and the radius of gyration has a constant value of 4.1 +_ 0.1 nm, in agreement with previous observations [16]. A concentration-dependent aggregation o f PDC can thus be excluded. Even after exposures o f 30 min corresponding approximately to a dose of 0.5 Mrad no loss of enzymic activity could be detected. 3.2. Dissociation of holo-PDC into apoenzyme and recombination of the apoenzyme with the cofactors Release o f T P P and Mg 2+ from holo-PDC was initiated by changing the pH from 6.2 to 8.0. The kinetics o f this process can be monitored by the decrease in activity (Fig. 1) which has a rate constant of 6.7 x 10 -4 s-]. The decrease in activity is paralleled by a reduction o f 50°70 o f the forward scattering and of the radius of gyration by about 0.45 nm which occur at the same rate. This indicates that under the conditions used the formation o f apo-PDC is accompanied by a change in quaternary structure. The reduction of the forward scattering corresponds to the dissociation o f the holoenzyme in two dimeric halves as previously inferred [3]. The distance between the centres o f the dimers in the tetramer calculated from the radii o f gyration using the 18
parallel axis theorem is 3.7 nm in agreement with previous observations [16]. After dissociation, adjusting the pH to 6.5 results in a rapid recovery o f the forward scattering to its initial level, although the sample does not present measurable enzymic activity. Reactivation only occurs after addition o f excess T P P and Mg 2+. The most significant changes in the scattering curves occur at s > 0.1 nm-1. Thus, at pH 6.5 the dimeric halves reassociate to a tetramer even in the absence o f cofactors. This tetramer then recombines with the cofactors to give the enzymatically active holoenzyme. These results are in agreement with previous observations which had shown that the recombination rate is independent of the apoenzyme concentration and that protein association processes play no role in the recombination [10]. Further, it had been shown that at pH 6.2 the apoenzyme bound to a solid matrix can be recombined with the cofactors to the active form. This would not be expected if the enzyme existed under the form of dimeric halves at this pH. Whereas, as illustrated in Fig. 2, the scattering for s < 0.1 nm -1 hardly changes after recombination with
Volume 266, number 1,2
FEBS LETTERS ~og [I(s)]
June 1990
Log l(s)
3.0 ~jQ,~ zs
0.01
B~~~ A
0.02
0.03
o
s2(nm-21
Fig. 2. Guinier plot of the scattering patterns of PDC (A and C), after addition of 50 mM pyruvamide (B) or 10 mM T P P + 10 mM Mg 2+, respectively (D). The two sets of curves are displaced by one logarithmic unit for better visualization. [PDC] = 7.2 mg/ml. Inset: plot of log I(s) vs s for A and B.
the cofactors, there are significant differences between the pattern of apo- and holo-PDC at larger angles. These differences indicate, in agreement with circular dichroism studies [17], that upon binding of the cofactors, conformational changes take place in the subunits and/or their interfaces.
tion can be conserved by appropriate cross-linking in the presence of substrate [19].
3.3. Activation o f PDC with the substrate-like activator pyruvamide Comparison of the scattering pattern of PDC in the presence and absence of pyruvamide (Fig. 2) indicates that significant changes occur at larger angles. The intensity in the low angle region is not affected by addition of pyruvamide, but there is an increase of the radius of gyration of PDC from 4.1_+0.1 nm to 4.3 _+0.05 nm in the pyruvamide-activated enzyme. This indication of a structural change upon activation corresponding to an expansion of the enzyme correlates well with the accelerated 1H/3H exchange in the presence of pyruvate [18] as well as the enhanced reactivity of SH-groups of activated PDC with SH-reagents [19]. The observation of structural changes upon activation is also in line with previous studies indicating that fixation of PDC to a solid matrix strongly delays activation by the substrate [10] but that an active conforma-
[1] Sieber, M., K6nig, S., Hfibner, G. Schellenberger, A. (1983) Biomed. Biochem. Acta 42, 343-349. [2] Freisler, H. and Ullrich, J. (1977) Hoppe Seylers Z. Physiol. Chem. 358, 318. [3] Gounaris, A.D., Turkenkopf, l., Buckwald, Sh. and Xoung, A. (1971) 3. Biol. Chem. 246, 1302-1309. [4] Davies, D.D. (1967) Biochem. 3. 104, 50 P. [5] Hfibner, G., Fischer, G. and Schellenberger, A. (1970) Z. Chem. 10, 436-437. [6] Boiteux, A. and Hess, B. (1970) FEBS Lett. 9, 293-296. [7] Ullrich, J. and Donner, I. (1970) Hoppe Seylers Z. Physiol. Chem. 351, 1026-1029. [8] Hiibner, G., Weidhase, R. and Schellenberger, A. (1978) Eur. J. Biochem. 92, 173-181. [9] K6nig, S. (1985) Dissertation, Martin-Luther-Universit~it, HalleWittenberg. [10] Nafe, G., Hiibner, G., Fischer, G., Neef, H. and Schellenberger, A. (1972) Acta Biol. Med Germ. 29, 581-594. [11] Holzer, H., Schultz, G., Villar-Palasi, C. and 3iintgen-Sell. J. (1956) Biochem. Z. 327, 331-334. [12] Vogel, E. and Schinz, H. (1950) Helv. Chim. Acta 33, 116-130. [13] Koch, M.H.J. and Bordas, J. (1983) Nucl. Instrum. Methods 208,461-469.
REFERENCES
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FEBS L E T T E R S
[14] Boulin, C., Kempf, R., Koch, M.H.J. and McLaughlin, S.M. (1986) Nucl. Instrum. Methods A249, 399-407. [15] Boulin, C., Kempf, R., Gabriel, A. and Koch, M.H.J. 0988) Nucl. Instrum. Methods A269, 312-320. [16] Mfiller, J.J., Damaschun, G. and Hiibner, G. (1979) Acta Biol. Med. Germ. 38, 1-10.
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[17] Ullrich, J. and Wollmer, A. (1971) Hoppe Seylers Z. Physiol. Chem. 352, 1635-1644. [18] Printz, M.P. and Gounaris, A.D. (1972) J. Biol. Chem. 247, 7109-7115. [19] K6nig, S., Htibner, G. and Schellenberger, A. (1989) Biomed. Biochim. Acta in press.