78 Biochimica et Biophysica Acta, 992 (1989) 78-86 Elsevier
BBAGEN 23144
Localization and kinetics of pyruvate-metabolizing enzymes
in relation to aerobic alcoholic fermentation in
Saccharomyces
cerevisiae
CBS 8066 and Candida utilis CBS 621
H e n d r i k v a n U r k ~, D i c k S c h i p p e r 2, G u i d o J. B r e e d v e l d 1, P a u l R . M a k 1, W . A l e x a n d e r S c h e f f e r s : a n d J o h a n n e s P. v a n D i j k e n
! Department of Microbiology and Enzymology, Delft University of Technology, Delft and 2 Gist-brocades, Delft (The Netherlands) (Received I December 1988)
(Revised manuscript received 27 February 1989)
Key words: Crabtree effect; Yeast: Pyruvate; Pyruvate decarhoxylase; Alcoholic fermentation; (Saccharomyces); (Candida)
The role of pyruvate metabolism in the triggering of aero'Mc, alcoholic fermentation in Saccharomyces cerecisiae has been studied. Since Candlda utilis does not exhibit a Crabtree effect, this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate ceJrboxylase 0EC 6.4.1.1), the pyruvate dehvdrogenase complex and pyruvate decarbox)lase 0EC 4.1.1.1) in cells of glucos~Mimited chemostat cultures of the two yeasts were compared. in contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to he a cytosulic enzyme in both yeasts. This implies that for anabulic p r o c e s s ~ transport of C4-dicarboxylic acids into the mitnehondria is required. Isolated mitnebondria from both yeasts exldbited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitnehondria for pyruvate oxidation and the in situ activity of rite pyruvate dehydrogenase complex was similar in both types of mituchondria. Tim activity of the cytosolic enzyme pyruvate decarboxylase in S. cer~islae from glucose-limited chemostat cultures was 8-fuld that in C. utills. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeasts was competitively inhibited by inorganic phosphate. The enzyme of S. cerevbiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarbuxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 3tP-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glyculytic flux and a high level of this enzyme are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.
Introduction
In the metabolism of sugars by facultafively fermen- tative microorganisms, various pathways diverge at the level of pyruvate. Pyruvate may be oxidized in the tficarboxylic acid cycle or converted to fermentation products. Pyruvate is also a key intermediate in anabolic processes. Its carboxylation to oxaloacetate via pyruvate carboxylase is an anaplerotic process for the generation of tricarboxylic acid cycle intermediates [1]. The divi-
Abbreviation: Mes, 4-morpholineethanesulphonic acid. Correspondence: J.P. van Dijken, Department of Microbiology and Enzymology, Delft University of Technology, Julianalaan 67, 2628 BC Delft. The Netherlands.
0304-4165/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical
sion of pyruvate over the various pathways depends on its intracellular concentration and the activities and kinetic properties of the pyruvate-metabolizing enzymes [2]. In eukaryotic organisms an additional factor is involved in the division of the flow of pyruvate over the different routes, namely metabolic compartmentation [3].
U p o n transition from glucose limitation to glucose excess under aerobic conditions, the yeasts S. cerevisiae and C utilis show a different behaviour [4,5]: S. cere- visiae immediately starts to ferment (i.e., exhibits the short-term Crabtree effect [4]) whereas C utilis :loes not excrete fermentation products. Also the rate of accumu- lation of biomass is lfigher in cultures of C utilis [5].
Since the metabolism at the level of pyruvate plays a crucial role with respect to the above differences be- Division)
tween the two yeasts, a study was inifiaU~! on quantita- tive aspects of pyruvate metabolism. Of special interest was the localization of pyruvate carboxylase, in view of reported differences between S. cerevisiae and C. utilis [6-9]. Moreover, the regulation of f.he enzyme pyruvate decarboxylase by inorganic phosphate and other possi- ble effectors was studied, since Boiteux and Hess [10] reported inhibition of this enzyme by phosphate. The results of our study suppo~ the conclusion that the triggering of aerobic alcoholic fermentation in S. cere-
visiae after a glucose pulse to glucose-limited cultures
does not primarily result from a bottleneck in the oxidation of pyruvate.
Materials and Meflmds
Microorganisms and growth conditions
Candida utilis CBS 621 and Saccharomyces cerevisiae
CBS 8066 were grown aerobically [5] in glucose-limit~[ chemostat cultures at D = 0.2 h - ! . The glucose con- centration in the reservoir medium was 12.5 g / l . The mineral medium contained per litre: 5 g ( N H 4 ) 2 S O 4, 3 g KH2PO 4, 0.5 g MgSO4 • 7 H20, 15 mg EDTA, 4.5 rag ZnSO4- 7 H20, 0.3 mg CoCi 2 • 6 H2G, 1 mg MnCl 2 • 4 HeO, 0.3 mg CuSO4- 5 H20, 4.5 mg CaC! 2 - 2 HzO, 3 mg FeSO4.7 H20, 0.4 mg NaMoO 4- 2 H20, 1 mg HsBO s, 0.1 mg KI, 0.05 ,all silicone antifoam (BDH), 0.05 mg biotin, 1 mg calcium pantothenate, 1 mg nico- tinic acid, 25 mg inositol, 1 mg thiamin-HCl, 1 mg pyridoxine-HCl and 0.2 mg p-aminobenzoic acid. In case of $. cerevisiae the medium contained 1 m i / l Tween 80 (Merck), since this greatly stabilized sI:,hero- plasts during fractionation experiments,
Cell fractionation
Isolation of mitochondria from C. utilis was per- formed according to the procedure described by Bruinenberg et al. [11]. For S. cerevisiae a sfighl modifi- cation was required: 2 g (dry weight) of calls were preincubated for 30 min in 20 ml of a solution con- taining 50 mM dithiothreitol and 5 mM tet,zasodium EDTA. After harvesting of cells (2 g of dry weight) or, in case of $. cerevisiae, after the preincubat[on in the dithiothreitol/EDTA solution, cells were wa::hed in 20 ml ice-cold buffer A (25 mM potassium phosphate (ptl 7.5)/1 mM E D T A / 1 mM MgCI2) containing 2 M sorbitol as an osmotic stabilizer. Throughout the proce- dure a Sorvall RC-SB centrifuge was use,t with the SS-34 rotor at 0 to 4 ° C. Washed cells were ~:ollected by centrifugation (10 min, 37 500 x g) and resuspended in 10 ml of buffer A containing 2 M sorbitol, gently wanned up to 3 7 ° C and mixed with 10 ml of the same buffer at 3 7 ° C to which Zyrt~olyase (Kii~in Brewery, Japan) had been added. In ore er to obtain a sufficient amount of spheroplasts within 1 h of incubation at
79 37°C, 2000 U and 250 U of Zymolyase (60000 or 100000) were needed for S. cerevisiae and C. utilis, respectively. Spberoplasts were then collected by cen- trifugation (10 min, 22500 × g ) and washed twice in ice-cold buffer A containing 2 M sorbitol. Then spheroplasts were resuspended in 20 ml of buffer A containing 2 M sorbitol, and dialysed at room temper- ature against 4 1 buffer without sorbitol until the sorbi- tol concentration was 0.65 M. This occurred within 100 min [11]. After centrifugation of the dialysed suspension (10 rain, 22500 z g) spheroplasts were resuspended in buffer A containing 0.65 M sorbitol. Spheroplasts were disrupted by 10 strokes in a Potter-Elvehjem homo- genizer.
The suspension containing broken spheroplasts was then centrifuged in order to remove unbroken sphero- plasts (15 rain, 15000 × g). The supernatant of this step is referred to as the total fraction (T) and used for fractionation. From the total fraction two particulate fractions and a soluble fraction were obtained by two centrifugation steps. Fraction P~ is the particulate frac- tion obtained after cerarifugation for 10 rain at 37 500 x g, and consisted mainly of mitochondria [11]. The superuatant of this step was again centrifuged (20 min, 75 000 x g), resulting in a particulate fraction (P2) which consisted of membrane vesicles [11] and a soluble frac- tion (S). The pellets PI and P2 were resaspended in buffer A containing 0.65 M sorbitol and 1 m g / m l bovine serum albumin (BSA, fatty acid free, Sigma). As shown previously, the above procedure, of which dialy- sis is a key step, is optimal for the isolation of intact mitochondria that exhibit respiratory control [11]. Only with this method most of the N A D H dehydrogenase activity is recovered in the mitochondriai fraction. The distribution of cytochrome c oxidase, which is generally considered to be a suitable mitoehondrial marker en- zyme, is useless in this respect, since this enzyme is always quantitatively recovered in the mitochondrial fraction, whether the organelles are intact or not [11]. In our studies these observations which were reported for
C. utilis were confirmed for S. cerevisiae as well. The
final mitochondrial (Pl) fraction was not washed, since this leads to reduction in respiratory control values. However, contamination of the cytoplasmic marker en- zyme glucose 6-phosphate dehydrogenase was always less than 5~ of the total activity present in the total (T) fraction.
Purification of pvruvate decarboxylase
For the isolation of pyruvate decarboxylase, cells were cultured in shake flasks on 5~ glucose and 1~ yeast extract at 30°C. This cultivation method was adopted, since (most probably due to oxygen limitation) this resulted in high levels of pyruvate decarboxylase in both yeasts. Cells were harvested after 24 h, washed with distilled water and stored at - 1 5 ° C . The enzyme
was purified as follows (all purification steps were car- tied out at 4°C):
(1) Preparation of ceil-free extract. Frozen cells (7.5 g
wet weight) were resuspended in a buffer (buffer B) containing: 75 mM potassium phosphate (pH
6.5)/5
mM MgSO4/1 mM thiamine pyrophosphate/1 mM 2-mercaptoethanol. Cells were disrupted by three pas- sages through a French Pressure Cell (Amicon) at 1000 atm. Cell debris was removed by centrifugation for 20 rain at 75 000 × g in a Sorvall RC-5B centrifuge with an SS 34 rotor. The supernatant was poured over nylon wool to remove floating fat.
(2) Streptomycin sulphate precipitation. 15~ strepto-
mycin sulphate solution in buffer B was added to the cell-free extract, to obtain a streptomycin/protein ratio of 2:1. After stirring for 40 rain the precipitate was removed by centrifugation (20 rain at 75000 × g).
(3) Ammonium sulphate precipitation. Pyruvate decar-
boxylase precipitated at 50-60~ and 40-60~ am- monium sulphate saturation for S. cerevisiae and C.
utilis, respectively. The pellets were resuspended in 1.5
ml buffer C, containing: 15 mM potassium phosphate (pH 6.3)/2 mM MgSO2/5 mM thiamine pyrophos- phate/2 mM 2-mercaptoethanol. The fraction (total volume 3 ml) was dialysed overnight against 500 ml buffer C. After 5 h the buffer was refreshed.
(4) DEAE ion-exchange chromatography. The dialysed
fraction was loaded on a DEAE-Sephacel column (210 × 15 mm) which had been equilibrated with buffer C. The enzyme was eluted with a linear gradient of 0-1 M NaCi in buffer C. The total gradient volume was 250 ml and the flow rate was 28 ml/h. Pyruvate decrboxylase from S. cerevisiae eluted at 0.15-0.25 M NaCI and that from C. utilis at 0.20-0.35 M NaCI. Because pyruvate decarbo×ylase proved to be unstable at high salt con- centration, the NaCI concentration of the eluate con- taining purified pyruvate decarboxylase was lowered to 0.05 M. This was done in successive concentration steps via filtration (Amicon PTGC CX-10 filter) and dilution in buffer C. After this step the fraction was con- centrated to 1 ml by filtration.
(5) Gel filtration. Gel filtration of the enzyme from
the previous step was carried out on a Fractogel (Merck) TSK HW-50 column (260 × 15 ram) equilibrated with buffer C. Elution was carried out with the same buffer. Fractions containing pyruvate decarboxylase were pooled and concentrated to 2.5 ml. The enzyme was stored in 200 tti portions at - 1 5 ° C .
The purification steps are summarized in Tables 11 and llI. The partially purified preparations were devoid of pyruvate dehydrogenase, NAD-linked acetaldehyde dehydrogenase and NADH oxidase activity (tested un- der the conditions of the pyruvate decarboxylase assay) and were therefore suited for kinetic analysis.
Polarographic measurements
Oxygen consumption by isolated mitochondria was measured as described by [11] with a Clark-type oxygen electrode. Pyruvate, DL-malate or L-malate were used as substrates at various concentrations in the presencx of ADP (0.17 mM).
Enzyme assays
Enzyme assays were carried out at 30°C with a Hitachi 100-60 spectrophotometer at 340 rim, unless stated otherwise. Fractions were sonicated for 3 min with an MSE-150 W sonifier (MSE, London, U.K.), unless mentioned otherwise. The assay mixtures for the individual enzymes are described below.
Alcohol dehydrogenase (EC 1.1.1.1): 50 mM potas- sium phosphate buffer (pH 7.5)/0.4 ram NAD +. The reaction was started with 100 mM ethanol.
Malate dehydrogenase (EC 1.1.1.37) according to Flury [12]: 100 mM potassium phosphate buffer (pH 7.5)/0.15 mM NADH. The reaction was started with 1 mM oxaloacetate.
Pyruvate carboxylase (EC 6.4.1.1) was assayed via two methods, namely by measuring oxaloacetate forma- tion with malate dehydrogenase (method I), or via de- tection of CoA formation with DTNB (5,5"-dithiobis(2- nitrobenzoic acid), e = 13.6 1- mmo1-1- cm -1) (method II). Both methods yielded identical results. The com- position of the reaction mixture for method I was: 100 mM Tris-HC! buffer (pH 7.8)/6.7 mM MgSO4/40 mM KHCO3/0.15 mM N A D H / 9 0 pM acetylCoA/1.5 mM pyrazole/10 mM pyruvate/6 U malate dehydrogenase. The reaction was started with 3.3 mM ATP. The assay was carried out under anaerobic conditions. Method II: This was based on the assay as described by Martin and Denton [13]. The assay mixture consisted of: 100 mM Tris-HCl buffer (pH 7.8)/5 mM MgSOJ10 mM K C I / 2 0 mM KHCO3/0.1 mM D T N B / 9 0 /LM acetylCoA/10 mM pyruvate/1.1 U c~trate synthase. The reaction was started with 3.3 mM ATP.
Pyruvate decarboxylase (EC 4.1.1.1): 10 mM potas- sium phosphate (pH 6.5)/5 mM MgCl~'0.15 mM NADH/0.2 mM thiamine pyrophosphate/50 mM pyruvate/88 U alcohol dehydrogenase. The reaction was started with the sample. Activities were corrected by determining blank activities without pyruvate.
Pyruvate dehydrogenase complex was measured in situ, in intact mitochondria. The assay was carried out with [1-14C]pyruvate. For the assay 25 ml Warburg flasks were used. The flasks were closed with gas-tight septa. The assay mixture (1 ml) contained: 25 mM potassium phosphate buffer (pH 7.0)/5 mM MgC12/0.65 M sorbitol/5 mM DL-malate or 2 mM L-malate/i mM ADP. The centre well contained 0.2 ml 1 M KOH. After 5 rain of incubation in a shaking
waterbath (Julabo, 90 strokes per rain) at 30°C, the reaction was started by adding 5 mM (approx. 0.25/tCi) pyruvate with a Hamilton syringe. The reaction was stopped by adding 0.25 ml 30% (w/v) trichloroacetic acid with a syringe. After 30 rain of equilibration the septum was removed and 0.18 ml of the KOH solution in the centre well was taken for scintillation counting, using Aquasol (New England Nuclear, Dupont) as a solvent. Separate flasks were used to obtain a time curve of the enzymatic reaction. Because the mitochon- drial fractions were not purified and therefore con- tained some cytosolic pyruvate decarboxylase activity, a correction had to be made. This was performed by measuring the 14CO2 production from [1-t4C]pyruvate at 5 and 15 mM pyruvate, both in the mitochondrial (Pi) and eytosolic (S) fractions. These concentrations are saturating and non-inhibitory to the pyruvate dehy- drogenase complex, as was established from oxygen consumption experiments with mitochondria. Pyruvate decarboxylase activity, however, is enhanced at 15 mM pyruvate, due to the low affinity of the enzyme for its substrate [10,14]. From the difference in 14CO2 produc- tion at the two pyruvate concentrations the contribution of contaminating pyruvate decarboxylase may be calcu- lated. It was established in this way that in mitochon- drial preparations of both yeasts 20% of the ~4CO2 production could be due to contamination of the pre- paration with cytosolic pyruvate deearboxylase. The pyruvate dehydrogenase activities reported in the results section represent corrected values.
Tricarboxylic acid cycle activity in intact mito- chondria: The overall tricarboxylic acid cycle activity in isolated mitochondria was measured in an identical manner as the activity of the pyruvate dehydrogenase complex. However, instead of [1-14C[pyruvate, [2- 14C]pyruvate was used as a substrate, the rationale of this assay being that the second carbon atom in the pyruvate molecule is released only after one fuU turn through the tricarboxylic acid cycle.
~I P_NMR measurements
Cells from chemostat cultures grown at D = 0.2 h-1 were harvested (5 rain 5000 rpm in a Sorvall GSA rotor at 4°C). The pellets were washed in buffer D, contain- ing: 50 mM Mes (pH 5.5); ~ mM KH2PO4; 0.9 mM K2HPO4; 4 mM MgSO4; 1.7 mM NaCl; 38 mM (NH4)2SO 4 and I m l / l of a vitamin solution [11]. Cells were resuspended in buffer D to a concentration of 50-65 g dry weight/l and kept on ice until used (within 60-300 rain). 3tp-NMR spectra, with a time resolution of 30 s, were obtained on a Bruker AM 360 WB NMR-spectrometer essentially as described by Nicolay et al. [15]. An N M R tube of 20 n u n was used, and pure oxygen was bubbled through the suspension at a flow rate of 370 m l / m i n as described by Gillies et al. [16]. The tube contained 20 ml cell suspension, 1 ml of D20
81 and 50 t~l silicone antifoam. After temperature equi- libration for 15 min at 30°C, the measurements were started, following the addition of glucose to a con- centration of 185 raM. It can be calculated that during the experiments in the NMR-tube oxygen-limited con- ditions must occur. However, because this 3~p-NMR method is the only way to specifically determine the cytosolic phosphate concentration, this method was chosen to obtain an indication of the change of the cytosolic phosphate concentration. Moreover, den Hol- lander et al. [17] showed that the phosphate concentra- tions of aerobic and anaerobic glycolyzing cells do not differ significantly. In their case, oxygen limitation did not occur because the yeast cells they used exhibited low oxygen consumption rates [16].
Protein determination
Protein in the fractions was determined according to the Lowry method.
Chemicals
[1-14C]Pyruvate and [2-14C]pyruvate were from New England Nuclear. DTNB was from Janssen Chimica (Beerse, Eelgium), Dr-malate from BDH, and L-malate from Sigma. Enzymes and other biochemicals were from Boehiinger.
Results
Subcellular localization and activities of pyruvate-metabo- iizing enzymes
Subcelhilar fractiona~ion of S. cerevisiae showed that pyruvate carboxylase is a cytosolic enzyme in this organism, in confirmation with earlier studies [6,8]. However, in contrast to the report by Evans et al. [9], also in C. utilis the enzyme was exclusively recovered in the soluble fraction (Table I). The activity of the en- zyme was approximately the same in both organisms.
In both organisms, most of the pyruvate decarboxy- lase activity was recovered in the soluble fraction. The level of this enzyme was 8-fold higher in S. cerevisiae. The product of the decarboxylation, acetaldehyde, can be reduced to ethanol. Because a lack of cytosolic ethanol dehydrogenase could explain the short-term Crabtree effect in C utilis, this enzyme was also determined. As is shown in Table I, also C utilis contains a high activity of this enzyme.
From the in situ measurement of pyruvate dehydro- genase in intact mitochondria it can be concluded that the pyruvate dehydrogenase activity was similar in both yeasts (Table I).
Kinetics of oxidation of carboxylic acids by isolated mito- chondria
In view of the cytosolic localization of pyruvate carboxylase it was of interest to study the oxidation of
82 TABLE l
En:yme activities and specific activities as measured in the several isolated fractions isolated from S. cerevisiae and C. utilis
Protein content of the several fractions. Enzyme Saccharomyces cerevisiae
fraction T fraction Pl fraction P2 fraction S total recovery
tot. act. a spec. act. b tot. act. a spec. act. b tot. act) spec. act. b tot. acL a spec. acL b (70)
Pyruvate carboxylase 1.78 0,024 0 d 0 0 0 1.62 0,045
Pyruvate decarboxylase 16.28 0.22 0.38 0.02 0.005 0 15.84 0.44
Pyrnva~e dehydrogenase - c _ 1.71 0 . 0 9 . . . .
Ethanol dehydrogenase 156.88 2.12 24,70 1.30 4.70 0.47 144.00 4.00 Malate dehydrogenase 254.56 3.44 127.30 6.70 50.90 5.10 50.40 1,40
Protein content (rag) 74 19 10 36
Candida utilis 91 100 _ 1 1 0 90 88 Pyruvate carboxylase 0.75 0.017 0 0 0 0 0.93 0.032 Pyruvate decarboxylase 1.76 0.04 0.20 0,025 0.015 0.003 1.60 0.055 Pyruvate dehydrogenase - - 0.88 0 . 1 1 . . . . Ethanol dehydrogenase 43.34 0.99 8.32 1.04 3.90 0.78 30.74 1.06 Malate dehydrogenase 319.00 7.25 92.00 11.50 19.15 3.83 116.00 4.00
Protein content (rag) 44 8 5 29
124 103 _ 99 71 95 Total activity in/~mol/min,
b specific activity in p.mol/min per mg protein. c -, not determined.
d 0, not detectable.
C4-dicarboxylic acids b y mitochondria. Repleni~ht~ent o f tricarboxylic acid cycle intermediates is likely to occur mainly via t r a n s p o r t of malate into the mito- c h o n d r i a (Fig. 1), since a high malate d e h y d r o g e n a s e activity is present in the cytosol (Table I) and, m o r e - over, the equilibrium o f the reaction oxaloacetate + N A D H + H + ~ malate + N A D + is in f a v o u r o f malate production.
T h e oxidation of malate by m i t o c h o n d r i a of b o t h yeasts was determined, in the presence of a fixed con- centration of pyruvate, as a function of the malate concentration. T h e results (Fig. 2) s h o w that the affinity o f the m i t o c h o n d r i a o f b o t h yeasts for malate w a s
GLUCOSE
pyRUVATE~ACETALDEHYD E
OAA . . . . 41"-OAA ACETYL CoA-.e-' | ETHANOL
/
[ mitochondrion /
Fig. 1,. S::.hematie representation of pyruvate metabolism in yeasts. (OA~, oxaloacetate).
a p p r o x i m a t e l y the same. Also the affinity o f the mito- c h o n d r i a for pyruvate, tested at fixed malate concentra- tions, w a s similar (Fig. 3). T h e affinity c o n s t a n t for p y r u v a t e w a s a b o u t 0.3 raM, w h i c h is near the K m value that h a s b e e n r e p o r t e d for the p y r u v a t e d e h y d r o - genase c o m p l e x [14] a n d the K m value c f p y r u v a t e t r a n s p o r t b y m i t o c h o n d r i a f r o m various sources [18]. T h e s e results m a k e clear that, if the in vivo rates o f p y r u v a t e o r malate oxidation b y the t w o yeasts w o u l d be different, this could only result f r o m differences in the intracelhilar c o n c e n t r a t i o n o f these substrates.
A l t h o u g h the rate o f p y r u v a t e - d e p e n d e n t oxygen
I 2o0
pyruvate
÷malate
~
-" oxidation100- ~
rn f n m o i 0 2 I , ~ ,I
o lo 0'.2, o'.50 o.~, ioo
L - m a l a t e (mM)
Fig. 2. t.-Malate oxidation by isolated mitochondria as a function of the malate concentration at a fixed pyruvate concentration of 5 mM.
TABLE II
Purification of pyruvate decarboxylase from C. utilis
Step Total T o t a l Specific Purifi- Recovery activity protein activity cation (%) (I.U.) ( r a g ) (t.U./mg) factor l Cell-free extract 1251 329 3.8 1.0 100 2 Streptomycin precipitation 1069 264 4.0 1.1 85 3 (~H4)2SO 4 precipitation 725 92 7.9 2.1 58 4 DEAE ion exchange 365 22 16.6 4.4 29 5 Gel filtration 249 8 31.1 8.2 20 TABLE ill
Purification of pyruvate decarboxylase from S. cerevisiae Step Total T o t a l Specific Purifi- Recovery
activity protein activity cation (~o) (I.U.) ( r a g ) (l.U./mg) factor 1 Cell-free extract 465 222 2.1 1.0 100 2 Streptomycin precipitation 408 162 2.5 1.2 88 3 (]qH4)2SO 4 precipitation 294 60 4.9 2.3 63 4 DEAE ion exchange 130 11 11.8 5.6 28 5 Gel filtration 79 3 26.3 12.5 17
consumption was similar in b o t h yeasts, this does not necessarily imply that the overall activity of the tri- carboxylic acid cycle would be the same in b o t h organisms, since the observed oxygen consumption could b e due to partial oxidation of the substrate only. It was therefore attempted to determine the overall tricarbox- ylic acid cycle activity via measurement of t 4 c o 2 pro-
I 200 t
0
0.2s 0.50 0.75 1.00
pyruvate (mM)
Fig. 3. Pyruvate oxidation by isolated mitochondria as a function of the pyruvate concentration at a fixed DL-malate concentration of 5
raM. @, S. cereoisiae; D, C. utilis.
83 TABLE IV
Pyruvate.dependent oxygen consumption and CO: production from labelled pyruvate by isolated mitochondria
S. cerevisiae C. utilis Pyruvate-dgpendent
oxygen consumption a 190 160
14CO2 from [l-14C]pyruvate b 90 110 14CO2 from [2-14C]pyrovate b 4 2 a in nmol 02 per min per mg protein.
b nmo114CO, per rain per mg protein.
duction from [2-t4C]pyruvate. However, with mito- chondria of b o t h yeasts only very low rates of 14CO 2 production (Table IV) were observed, which is in sharp contrast with the high rates of pyruvate-dependent oxygen consumption and the production of t4CO2 from [1-]4C]pyruvate. These results thus show that pyruvate- dependent oxygen consumption by mitochondria is not a reliable measure for the overall tricarboxylic acid cycle activity. The cause of the low t4CO2 production from [2-14C]pyruvate is not known. It may be caused by excrelion of malate and citrate by the mitochondria [191.
Properties o f pyruvate decarboxylase
The results presented above indicate that the differ- ent behaviour of C. utilis and S. cerevisiae upon transi- tion from glucose limitation to glucose excess (see Intro- duction) is unlikely to be explained o n the basis of differences in their capacities for pyruvate oxidation or carboxylation. Although in C. utilis a significant level of pyruvate decarboxylase is present, in this yeast al- coholic fermentation does not occur after a glucose pulse. Apart from the fact that the activity of this enzyme is much lower than in S. cerevisiae, the absence of alcoholic fermentation in C. utilis could also be due to the kinetic properties of its pyruvate decarboxylase. Therefore, the kinetics of pyruvate decarboxylase of b o t h yeasts were examined.
The enzyme of b o t h yeasts was partially purified and was free of interfering enzymes (see Materials and Methods). The purification is summarized in 'Fables II and III.
The effect of several compounds on the enzyme activity was tested. Sodium citrate, tested up to 10 mM; sodium acetate, ATP, ADP, AMP, glucose 6-phospahte and fructose 1,6-bisphosphate, tested up to 5 mM; and phosphoenol pyruvate, tested up to 1 raM, did not have any effect o n the activities of the purified enzymes of S. cerevisiae and C. utilis. Fructose 2,6-bisphosphate was found to inhibit the enzymes competitively, but only at high concentrations (2-5 mM) which are not to be expected in vivo [20,21]. The affinity of the enzyme of b o t h organisms for pyruvate was determined at three
TABLE V
Effect of phosphate on the Km of pyruvate decarboxylase for pyruvate The value of K m at 0 mM Pi was calculated by linear extrapolation of K m values vs. Pl concentration.
[Pl] a (raM) S. cercvisiae C. utilis
0 b 3.0 3.6
10 7.7 4.5
50 25.0 7.0
100 48.0 ll.0
a Pl, inorganic phosphate.
different p h o s p h a t e concentrations. T h e results (Table V a n d Fig. 4) s h o w that the pyruvate decarboxylase o f b o t h yeasts was inhibited b y p h o s p h a t e . I n h i b i t i o n b y
p h o s p h a t e has also b e e n reported for the e n z y m e o f Saccharomyces carlsbergensis [10]. T h e inhibition was competitive with p y r u v a t e (Fig. 4) a n d was stronger for the 2;. cerevisiae enzyme.
Effect o f the in'racellular phosphate concentration on al- coholic fermentation
A possible physiological significance o f the observed inhibition o f p y r u v a t e decarboxylase b y p h o s p h a t e was studied via d e t e r m i n a t i o n o f t h e intracellular c o n c e n t r a - tion o f p h o s p h a t e d u r i n g transition o f the yeasts f r o m glucose limitation to glucose excess. O n l y the cytosofie p h o s p h a t e c o n c e n t r a t i o n is a significant p a r a m e t e r in this respect, since p y r u v a t e decarboxylase is a cytosofic e n z y m e (Table I). Via 3~p-NMR the p h o s p h a t e c o n - c e n t r a t i o n in a single subcellular c o m p a r t m e n t c a n b e .G.. u.LLL~
(act Ivlty)-I l
( m g / U ) o.~o o,os - : o o -Ioo [pyruvatq] -! (M .1)(activity)'! l
(,.w/U)[py,uv,t,]'* m")
Fig. 4. Lineweaver-Burk plots of pyruvate decarboxylatinn experiments at different phosphate concentrations (e, 10 ml~J; o, !;(I raM; U-~ 100 raM) with partially purified enzymes from (A) C. utilis and (B) S. cereuisiae.
A
]~e '
Pl
(
(mM]
4 S PS,¢erevislee
P. . ,, .Hall
I
* "
time (mini
,,, e C_.u,_~., .2o [ .m rp]... ~
12 (raM)
0
"
o
~ 4~ ~
2b 2:, 2~ ,,. time (.~..:::.}Fig. 5. Results of JI P-NMR experiments with (A) S. cerevisiae anti iB) C. utilis. Glucose was added at zero time. Pi, inorganic phosphate; SP, sugar phosphates.
established [15,16]. After addition of glucose to cells, pre-cultured under glucose limitation in a chemostat, the concentration of inorganic phosphate in the cyto- plasm decreased rapidly (within 1 min), whereas the sugar phosphate concentration increased (Fig. 5). From the chemical shift of the intracelhilar phosphate peak and from the shift of the sugar phosphate peak in the NMR-spectra, the intracellular pH can be calculated [~6]. In order to obtain evidence that the intracelhilar phosphate peak represents the eytosolic phosphate, it should be estabfished that the pH calculated from the cytosolic sugar phosphate peak is equal to the pH calculated from the phosphate peak [15]. This proved to be the case. Because the extraeellular phosphate con- centration was high under our conditions, no clear signal could be detected for the vacuolar phosphate peak.
The results shown in Fig. 5 are qualitatively similar to those obtained with batch-grov~.'n cells of C. utilis and
S. cerevisiae [15,17]. In S. cereoisiae, the cytoplasmic phosphate concentration decreased from 13 to 3 mM, whereas in C. utilis it decreased from 6 to 1 mM. However, as pointed out in Materials and Methods, care has to be taken with respect to the physiological interpretation of the N M R data. As a result of the dense suspensions used in these experiments, oxygen limitation will occur. It is therefore possible that the decrease in cytosolic phosphate may also be contributed to a shortage of oxygen. However, if this decrease also occurs under fuRRy aerobic conditions it can be con- cluded that a glucose pulse will stimulate alcoholic fermentation via a release of pyruvate decarboxylase from the inhibition by inorganic phosphate (Table V).
Discussion
During glucose-limited growth in chemostat cultures, the intracellular pyruvate concentration of both yeasts is below the K m of pyruvate decarboxylase for pyru- rate. This can be concluded from low extracellular pyruvate concentrations as reported by van Urk et al. [5]. Assuming that the extracellular undissociated form of pyruvic acid is in equilibrium with the intracellular concentration, the intracellular pyruvate concentration can be calculated as follows:
[pyruvateli,t = ([H + hxl/[H + ]i,0 x [pyruvateh~,
in which ' i n t ' and 'ext' stand for the intracellular and extracellular concentrations, respectively. Because the extracellular and intracellular pH (Fig. 5) and the ex- tracellular pyruvate concentration (20-30 /~M for S.
cerevisiae, see [5]) are known, the intracellular pyruvate concentration can be calculated to be 1-2 mM. This is below the K m values of the enzyme pyruvate decar- boxylase ~or pyruvate at the high phosphate concentra-
tions that are present in the cytosol (Fig. 5, Table V). Therefore, under glucose limitation these conditions are unfavourable for pyrnvate decarboxylase. Nevertheless, despite these unfavourable conditions, the contribution of pyruvate decarboxylase to pyruvate metabolism in glucose-limited cells is not negligible. On the basis of the levels and the kinetic properties of pyruvate decar- boxylase and pyruvate dehydrogenase complex, and a cytosolic pyruvate concentration in glucose-limited cells of 1 - 2 mM, it can be calculated that under steady-state conditions about 50% of the pyruvate from glycolysis in
S. cerevisiae is converted in the cytosol to acetaldehyde by pyruvate decarhoxylase. Thus, under glucose limita- tion in S. cerevisiae, and to a smaller extent also in C
utilis, oxidation of pyruvate may be accomplished not only via the mitochondrial pyruvate dehydrogenase complex but also via pyruvate decarboxylase with acetaldehyde as an intermediate (Fig. 1). The mecha- nism of acetaldehyde metabolism in yeasts is presently unknown. Acetaldehyde may be oxidized to acetate and converted in the cytosol to acetyl-CoA, which then must be transported into the mitochondria. Alternatively, fur- ther metabolism of acetaldehyde may occur in the mito- chondria.
Although the relief of phosphate inhibition on the enzyme pyruvate decarboxylase plays a role, a major factor in the onset of aerobic alcoholic fermentation in
S. cerevisiae after a transition to glucose excess will be intraceRRular accumulation of pymvate. This is likely to occur, since, after a glucose pulse, cells excreted pyru- rate [5]. Apart from this, also the decrease in cytosolic phosphate concentration contributes to the activation of pyruvate decarboxylase after a glucose pulse, since the enzyme was inhibited by inorganic phosphate at physio- logical concentrations (Table V). The acetaldehyde thus produced may either be oxidized via acetate to acetyl- CoA or reduced to ethanol (Fig. 1). Both processes may, in fact, occur simultaneously after a glucose pulse in S.
cerevisiae, since cells excrete ethanol and acetate under these conditions [4,5].
In C. utilis alcoholic fermentation does not occur immediately after transfer of cells to glucose excess [5]. It was therefore relevant to make a comparison between
S. cerevisiae and C. utilis with respect to parameters that affect the division of the flow of pyruvate between respiration and fermentation. The results clearly indi- cate that the different behaviour of S. cerevisiae and C.
utilis does not reside in the potential for pyruvate oxidation in these yeasts, since the pyruvate dehydro- genase activities and the kinetics for mitochondrial pyruvate oxidation were similar in both organisms ('Fa- ble I and Fig. 3).
In contrast to results reported by Evans et al. [9] aad Wills and Melham [71 the e ~ y m e pyruvate carboxylase was found to be localized i~ the cytosol in bodl S.
w o u l d result f r o m leakage o f t h e e n z y m e f r o m t h e m i t o c h o n d r i a l m a t r i x . U n f o r t u n a t e l y , n o s u i t a b l e m a r k e r e n z y m e s a r e available for this c o m p a r t m e n t in yeasts. However, t h e m i t o c h o n d r i a were i n t a c t o n t h e b a s i s o f v a r i o u s criteria a n d , f u r t h e r m o r e , t h e e n z y m e w a s n o t d e t e c t a b l e in t h e m i t o c h o n d r i a l fraction. A cytosolic localization for p y r u v a t e c a r b o x y l a s e in S. cerevisiae
h a s also b e e n r e p o r t e d b y H a a r a s i h a a n d T a s k i n e n [6] a n d Lira et al. [8]. F u r t h e r m o r e , n o s i g n i f i c a n t dif- ferences were f o u n d w i t h r e s p e c t t o t h e kinetics o f m a l a t e o x i d a t i o n b y isolated m i t o c h o n d r i a . F r o m t h e s e results it c a n b e c o n c l u d e d t h a t r e p l e n i s h m e n t o f t h e tricarboxyfic acid cycle i n t e r m e d i a t e s will b e e q u a l l y effective in S. cerevisiae and C. utilis a n d will p l a y n o role o f i m p o r t a n c e in t h e m e c h a n i s m o f t h e s h o r t - t e r m C r a b t r e e effect. T h e o n l y p a r a m e t e r s t h a t were f o u n d to b e different in t h e t w o y e a s t s were t h e level o f p y r u v a t e d e c a r b o x y l a s e ( T a b l e I) a n d t h e sensitivity o f this e n - z y m e for i n o r g a n i c p h o s p h a t e ( T a b l e V).
S u m m a r i z i n g , we s u g g e s t t h a t t h e o b s e r v e d dif- f e r e n c e s b t w e e n S. cerevisiae and C. utilis with r e s p e c t 1o aerobic e t h a n o l f o r m a t i o n u p o n t r a n s i t i o n f r o m glu- c o s e l i m i t a t i o n t o g l u c o s e excess, t h e so-called s h o r t - t e r m C r a b t r e e effect [4,5], a r e m a i n l y d u e to d i f f e r e n c e s i n t h e levels o f p y r u v a t e d e c a r b o x y l a s e . T h e a c t i v a t i o n o f p y r u v a t ¢ d e c . ~ b o x y l a s e a f t e r a g l u c o s e p u l s e is c a u s e d b y d e c r e a s e d levels o f c y t o s o ~ c p h o s p h a t e a n d i n c r e a s e d levels o f p y r u v a t e , c a u s e d b y a n i n c r e a s e d glycolyfi¢ activity [5], t h u s e n a b l i n g p y r u v a t e d e c a r b o x y l a s e to effectively c o m p e t e w i t h t h e m i t o c h o n d r i a a s w a s first o u t l i n e d b y H o l z e r [2]. A c k n o w l e d g e m e n t W e t h a n k P r o f e s s o r J.G. K u e n e n for m a n y v a l u a b l e d i s c u s s i o n s . References
1 Ruiz-Amil, M., De Torrontegui, (3., Palachh~ E., Catalina, L. and Losada, M. (1965) J. Biol. Chem. 240, 3485-3492.
2 Holzer, H. (1961) Cold Spring Harbor Syrup. Quant. Biol. 26, 277-288.
3 Keech, D.B. and Wallace, J.C. (1985) Pyruvate Carboxylase pp. 23-27, CRC Press, Boca Raton, FL.
4 Petrik, M., K~ppeli, O. and Fiechter, A. (1983) J. Gen. MicrobioL 129, 43-49.
5 Van Urk, H., Mak, P.R., Scheffers, W.A. and Van Dijkea, J.P. (1988) Yeast 4, 283-291.
6 Haarasilta, S. and Taskinen, L. (1977) Arch. Microbiol. 113, 159-161.
7 Wills, C. and Melham, T. (1985) Arch. Biochem. Biophys. 236, 782-791.
8 Lim, F., Rohde, M., Morris, C.P. and Wallace, J.C. (1987) Arch. Biochem. Biophys. 258, 259-264.
9 Evans, C.T., Scragg, A.H. and Ratledge, C. (i983) Eur. J. Bio- chem. 130,195-204.
10 Boiteux, A. and Hess, B. (1970) FEBS Lett. 9, 293-296. 11 Bruinenberg, P.M, Van Dijken, J.P., Kuenen, J.G. and Scheffers,
W.A. (1985) J. Gen. Microbiol. 131,1035-1042.
12 Flury, U. (1973) Isoenzyme der Malat-Dehydrogenase in Schizo- saccharomyces pombe. Ph.D. Thesis, ETH Zfirich.
13 Martin, B.R. and Denton, R.M. (1970) Biochem. J. 117, 861-877. 14 Kresze, G.B. and Ronft, H. (1981) Eur. J. Biochem. 119, 573-579. 15 Nicolay, IC, Scheffers, W.A., Bruinenberg, P.M. and Kaptein, R.
(1982) Arch. Microbiol. 133, 83-89.
16 Gillies, R.J., Alger, J.R., Den Hollander, J.A. and Shulman, R.G. (1982) in Intracellular pH: its Measurement, Regulation and Utili- zation in Cellular Functions (Nuccitelli, IL and Deamer, D.W., eds.), pp. 79-104, Alan R. Liss, New York.
i7 Den Hollander, J.A., Ugurbil, K., Brown, T.R. and Shulman, R.G. (1981) Biochemistry 20, 587!-5880.
18 LaNo, le, K.F. and Schoolwerth, A.C. (1979) AJ~nu. Rev. Biochera. 48, 871-922.
19 Bra;l~¢ord, M.A., Thomspon, A.G., Kaderbhai, N. and Beechey, R.B. (1986) Biochem. J. 239, 355-361.
20 Lederer, B., Vissers, S., Van Schaftingen, E. and Hers, H.G. (1981) Biochem. Biophys. Commun. 103,1281-1287.
21 Reibstein, D., Den Hollander, J.A., Pilkis, S.J. and Shulman, R.G. (1986) Biochemistry 25, 219-227.
