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I~I. J. Biochem.Vol. 24, No. 7, pp. I I I I-I 116, 1992 Printed in Great Britain. All rights reserved

,!?-OXIDATION AS CHANNELED REACTION LINKED TO CITRIC ACID CYCLE: EVIDENCE FROM MEASUREMENTS OF MITOCHONDRIAL PYRUVATE OXIDATION DURING FATTY ACID DEGRADATION MALTE E. C. F~RSTER and WOLFGANG STAIB Institut fiir Physiologische Chemie II, Heinrich-Heine-Universitat Diisseldorf, Mooren-str 5, 4000 Diisseldorf, Fed. Rep. Germany (Received II October 1991) Abstract-I. The kinetics of mitochondrial mammalian pyruvate dehydrogenase multienzyme complex (PDHC) is studied by the formation of CO, using tracer amounts of [l-‘4C]pyruvate. It is found that the Hill plot results in a (pseudo-)cooperativity with a transition of n-l +3 at a pyruvate concentration about K,. 2. Addition of L-carnitine, octanoate, palmitoyl-CoA or palmitate + L-carnitine + fatty acid-binding protein results in a Hill coefficient of n = 2 following the kinetics of pyruvate oxidation. 3. Addition of fatty acid-binding protein to an assay system oxidizing palmitate in presence of L-carnitine alters the pattern of the kinetics in the Hill plot so that an apparently lower level of L-carnitine is necessary for the reaction course of p-degradation. 4. It is concluded that P-degradation is a coordinated, multienzyme-complex based mechanism tightly linked to citric acid cycle and it is proposed that L-carnitine is actively involved into the reaction and not only functioning as carrier-molecule for transmembrane transport.

INTRODUCTION In a recent paper we reported the kinetics of pyruvate oxidation by the mammalian pyruvate dehydrogenase multienzyme complex (PDHC) (F6rster and

Staib, 1990). This isolated enzyme complex is composed of pyruvate dehydrogenase (E,, EC 1.2.4.1), dihydrolipoyl transacetylase (E2, EC 2.3.1.12), and dihydrolipoyl dehydrogenase (E3, EC 1J.4.3) (reviewed by Hucho, 1975; Reed and Pettit, 1981; Wieland, 1983). We reported the results of measurements of pyruvate oxidation followed by NADH formation (Linn et al., 1972) as well as the coupled arylamine-acetyltransferase (EC 2.3.1.5)/p-nitroaniline assay (Coore et al., 1971; Wieland et al., 1972). We observed a transition of the Hill coefficient from n = 1-+ 3 at a pyruvate concentration of about K,. A third method of following pyruvate oxidation is given by the use of labeled pyruvate, following COZ formation (Hagg et al., 1976; FGrster et af., 1984). This paper describes experiments assay isolated liver mito-

PDHC, pyruvate dehydrogenase multienzyme complex [E, pyruvate dehydrogenase (EC 1.2.4.1)+ E, dihydrolipoyl-transacetylase (EC 2.3.1.12) + E, dihydrolipoyl dehydrogenase (EC 1.6.4.3)]; BCA, bicinchoninic acid; FA, fatty acid (s); L-FABPc, fatty acid-binding protein(s) from liver cytosol; glu, glutamate; b-HMGCoA, /3-hydroxy-methylglutaryl-CoA; mal, malate; PAGE, polyacrylamide gel electrophoresis; TCC, tricarboxylic acid cycle (citric acid cycle).

chondria. Furthermore, the kinetics were studied under conditions favouring B-oxidation follow downregulation of pyruvate oxidation. The results confirm the formulated model for the reaction-mechanistic implications of the three binding sites previously described (Fiirster and Staib, 1990). From the kinetics of the inhibition studies a fresh look at the role of L-carnitine as a carrier substrate becomes possible (carnitine reviewed by Bieber, 1988). Thus further evidence is given for the understanding that /Ioxidation as a channeled reaction (e.g. Garland et al., 1965; Greville and Tubbs, 1968; Sumegi et al., 1991).

MATERIALS AND

METHODS

Chemicals were purchased from Merck (Darmstadt) and Serva (Heidelberg), biochemicals from Boehringer (Mannheim), and Sigma (Deisenhofen), bicinchoninic acid (BCA) protein assay reagent from Pierce (Weiskirchen). [l-‘4C]Pyruvate was from Amersham Buchler (Braunschweig), Hydroxide of Hyamine and Permafluor V from Canberra-Packard (Frankfurt). Bovine serum albumine (Cohn fraction V) was from Serva (Heidelberg), fatty acidbinding protein from liver cytosol (L-FABPc) prepared according to the procedure given by Glatz et al. (1985), purity proven by SDS-PAGE (Weber and Osborn, 1969). Liver mitochondria (Q = 6.2, P/O with glu/mal = 2.5) were prepared from fed male Han: Wistar rats (200-250 g) according to a standard procedure (Klingenberg and Slenczka, 1959) as described (Fiirster and Staib, 1990).

1111

MALTE

E. C. F~RSTERand WOLFGANG STA~B JtJwJLTS

Pyruvate kinetics The kinetics of COz formation from pyruvate resembles the NAD-assay as described (FGrster and Staib, 1990): the Hill coefficient n exhibits a transition from n = 1-+ 3 exceeding a pyruvate concentration of about KS (Fig. 1, + ). Pyruvate ki~etjc~ under c~~diti~~~fuvo~ring fl-oxidative 100

103 +JM pqwate

IO2

10'

Fig. 1. Hill plot of mitochondrial pyruvate turnover followed by CO, formation. Values of the original data ( + ) > rC, were recalculated after subtraction of an appropriate offset and replotted (0). This operation results in n = 1.2 (r =0.98). (The assay system contained in a final volume of 3 ml of 10, 20, 35, 50 and 75pM pyruvate, OS kBq [l-i4~]-pyruvate~10 PM pyruvate.)

Activity of PDHC was followed with an equipment consisting of a 20 ml Super Poly Vial and a Pica Plastic Vial (Canberra-Packard) as described (Schadewaldt et al., 1983). The assay system contained in a final volume of 3 ml (25°C) 50mM sucrose, 50mM phosphate buffer, pH 7.4, 5 mM MgCI,, 15mM KCL and 5 mg mitochrondrial protein. ‘In addition, depending on the experiments, L-carnitine, L-carnitine + palmitoylcarnitine and octanoate, paimitate f fatty acid binding protein was present (see figure legends for details). Three identical assays were pipetted and the reaction started by addition of the appropriate amounts of pyruvate containing tracer amounts of [I-‘4C]pyruvate. The reaction was stopped by addition of 1 mf 2 M citric acid after 1. 1.5 and 2.5 min, respectively.

Addition of L-carnitine to the assay system results in Hill plots as depicted in Fig 2. The effect observed is the appearance of a growing n = 2 part at the cost of the n = 1 and n = 3 part in the pure pyruvate kinetics, finally resulting in n = 2 kinetics. The same behavior is observed in the presence of octanoate (Fig. 3) and palmitoylcarnitine (Fig. 4) In the last series of experiments L-carnitine, paimitate and fatty acid binding protein was added. The corresponding Hill plots are shown in Fig. 5: here, too, the transition of the Hill coefficient from n = 1- 3 kinetics to II = 2 is the result. Additional L-FABPc changes the pattern of pyruvate kinetics in the Hill plot with the effect that growing amounts of L-FABPc reduce the apparent carnitine concentration utilized for pdegradation to kinetics which are seen with lower levels (Fig. 7), without altering pyruvate turnover significantly (Fig. 6). This effect is accompanied by a return to n = 1-+ 3 from n = 2 kinetics in the Hill plot (Fig. 7). DISCUSSION

The deliberated CO, was trapped with 500~1 Hydroxide of Hyamine ( > 12hr) and radioactivity counted after addition of 5 ml scintillation cocktail. From these values the initial velocity of pyruvate oxidation was calculated. If not stated differently, values shown are mean of N = 3 exper-

M~malian pyruvate dehydrogenase multienzyme complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate and the transfer of the resulting acetyl-moiety to coenzyme A. The regulation of this enzyme complex depends on a variety of parameters, above all end-product inhibition, namely NADH and acetyl-CoA, and interconversion, moduiated by a deand phosphorylating phosphatase and kinase system

iments. Protein content was estimated with the BCA protein assay (Smith et al., 1985) using bovine serum albumine as standard.

Y

,

Vmax

-



100

101

102

103pm pr)ruvare

Fig. 2. Hill plot of mitochondrial pyruvate turnover followed by CO2 formation in presence of 0 ( f 1, 20 ( x ). 60 (0) and 100 (m) #M L-camitine. (The assay system contained in final volume of 3 ml of 10, 25 and 50 FM pyruvate, 0.5 kBq Al-“C~-pyruvatei IO PM pyruvate.)

/I-Oxidation linked to citric acid cycle ”

I

Vmax

-

1113

Y

1~ 4

!Ol

100

+

.

100

10’

102

103pn

ply-uvare

Fig. 3. Hill plot of mitochondrial pyruvate turnover followed by CO, formation in presence of 0 ( + ). 20 ( x ), and 40 (0) p M octanoate. (The assay system contained in a final volume of 3 ml of 5, 15, 30 and 50 PM pyruvate, 0.5 kBq [i-‘4CJ-pyruvate/10 PM pyruvate.)

Fig. 4. Hill plot mitochondrial pyruvate turnover followed by CO, formation in presence of 0 ( + ), 10 ( x ) and 30 (0) /tM palmitoylcarntine. (The assay system contained in a final volume of 3 ml of 5, 10, 30 and 50 /.IM pyruvate, 0.5 kBq ]I-“C]-pyruvate/lOpM pyruvate.)

(see Hucho, 1975; Reed and Pettit, 1981; Wieland, 1983). In a previous paper we studied the kinetics of pyruvate turnover through isolated PDHC by NAD and the coupled arylamine-acetyltransferase/p-nitroaniline assay systems (Fdrster and Staib, 1990). The analysis of kinetics by Hill plots resulted in a complex turnover pattern. From this we derived the interpretation that three binding sites can come into different interaction leading to various reaction mechanisms, resulting in different products. In this paper we wanted to (i) establish the results of the two assay systems used hitherto by a third method, following the end-product CO,, (ii) expand the experiments on mitochondria to study PDHC in more physiological surroundings and (iii) study on that background in detail the kinetics of the regulation of PDHC by using conditions favoring /3-oxidation, since PDHC activity should be down-regulated by acetyl-CoA as an end-product inhibitor. The results of our experiments are discussed under three aspects: Firstly, it has to be discussed if the kinetics observed are due to cooperativity or pseudo-

cooperativity. Though the Hill coefficient n is usually used to discuss changes of binding constants by interacting subunits (Monod ea al., 1965) the possibility should be taken into account that the Hill plot discussed solely this way, may lead to misinterpretation. Depending on experimental conditions a formal cooperativity may result but be reduced to variations in the reaction course or availability of substrate (e.g. Pauly and McMillin, 1988; Richards et al., 1991), in this way resulting in pseudo-cooperativity. The inspection of our data led to the following recalculation: values of the pyruvate concentration about KS and higher were recalculated after subtracting an appropriate offset and replotted resulting in a Hill coefficient of n = 1. This new plot is equivalent to the n = 3 part of the original set of data (Fig. 1, 0). It is stated, therefore, that pseudo-cooperativity is followed. From this we state that binding sites resulting from Hill coefficients may be used to derive a stoichiometry as proposed in our previous report (Forster and Staib, 1990).

Fig. 5. Hill plot of mitochondrial pyruvate turnover followed by CO, formation in presence of 0, 10, 25, 35, 50 and 75 PM L-carnitine (The assay system contained in a final volume of 3 ml of 10,20,35, 50 and 75 nm pyruvate, 0.5 kBq [I-“Cl-pyruvate, 50 nM palmitate and 100 pg L-PABPc.)

MALTEE. C. F~RSTERand WOLFGANG STAIB

0.5

0

E 0

,

50

LOO prl

/

pyrwate

Fig. 6. Michaelis-Menten plot of mitochondrial pyruvate turnover followed by CO, formation ( + ) in presence of 50 PM palmitate, 50 PM L-carnitine in absence ( x ) and presence of 100 (0) and 200 pg (m) L-FABPc. (The assay system contained in a final volume of 3 ml of 10, 20, 35, 50 and 75wM pyruvate, 0.5 kBq [I-“‘Cl-pyruvate/lO PM pyruvate.)

Secondly, it was interesting to follow mitochondrial kinetics of CO, formation from pyruvate and to compare it with the results obtained with the different assay systems used measuring kinetics of the isolated enzyme complex (Forster and Staib, 1990). In contrast to our expectation based on the assumption that an intact mitochondrion should exhibit an un-

hindered channeled flux of pyruvate into citric acid degradation, we did not observe a pure n = 1 or n = 3 coefficient in the Hill plot. The pattern observed (see Fig. 1, + ) resembles the NAD-assay system where a side reaction of pyruvate oxidation, namely acetoin formation (Jagannathan and Schweet, 1952; Alkonyi et al., 1976), was discussed as the reason for kinetics with pyruvate concentrations lower than KS. This result, however, is in absolute agreement with the findings of Baggetto and Lehninger (1987a,b), who reported acetoin formation with intact mitochondria and stated that this reaction became physiologically relevant during metabolic changes as shown with Ehrlich and AS30-D tumor mitochondria. Thirdly, though this observation was not expected but readily understood, we were suprised by the kinetics resulting from experiments with carnitine and fatty acids. Fatty acids are thought to inhibit pyruvate oxidation by the end-product acetyl-CoA

,m,.,

,.,.,

,

..,,..,

100

.

,mTy

10'

.

“-_

.,..,,,

IO2

.

103l.m

pyruvate

Fig. 7. Hill plot of mitochondrial pyruvate turnover followed by CO, formation in prescene of 50/.~M palmhate, 50 PM L-carnitine in absence ( x ) and presence of 100 (0) and 200 pg (m) L-FABPc. (The assay system contained in a final volume of 3 ml of 10,20,35,50 and 75 PM pyruvate, 0.5 kBq [I-WI-pyruvate /IO FM pyruvate.)

and this, indeed was the effect shown by Michaelis-Menten diagrams (Fig. 6). The Hill plots, however, did not result in a n = 1 diagram as expected from the mechanistic scheme given (see Fiirster and Staib, 1990: scheme 1). This scheme was based on the understanding of the feed-back inhibition of pyruvate oxidation at the PDHC. In contrast, a pure n = 2 coefficient resulted with increasing amounts of carnitine (Fig. 2), octanoate (Fig. 3) or fatty acid supply (Fig 4 and 5). We conclude, therefore, that one binding site is occupied by camitine, the remaining ones still accepting pyruvate and catalyzing its turnover, and it is stated, that the kinetics observed mirror the handling of acetyl-moieties. Well aware of other possibilities for an interpretation of the data we make the following proposals with respect to the lowered V,,,,Xof pyruvate oxidation as seen in the Michaelis-Menten plots (Fig. 6): (a) Camitine occupies one binding site serving as an acetyl donor thus feeding C2 into TCC by degradation, simultaneously causing: (i) one molecule of pyruvate to be carboxylated another is oxidatively decarboxylated, (ii) two molecules of pyruvate to be condensed (tail to tail condensation, scheme 1A), feeding Cq, in this way, into a leaky TCC

(A)

OH I

CH2 =

c -

CH2 -

c //O ‘OH

Scheme 1. Formation of C4 molecules from enol-acetic acid by (A) “head to tail” and (B) “tail to tail” condensation (redrawn according to Fiirster, 1988: Scheme 5).

1115

/?-Oxidation linked to citric acid cycle of pyruvate to be condensed to acetoin (a known side reaction of PDC);

(iii) two molecules

or (b) Carnitine occupies one binding site serving as an acetyl donor thus enabling /?-HMG-CoA synthesis, simultaneously causing: (iv) two molecules of pyruvate to be oxidatively decarboxylated and condensed (head to tail condensation, Scheme 1B) to acetoacetylCoA. This interpretation is confirmed by the support of fatty acids, so that p-degradation becomes a strong source for the acetyl-moiety: the kinetics plotted according to Hill are almost identical. Since it is known that mitochondrial p-degradation of fatty acids with a chain length > N = 8 is carnitine dependent, two different conditions have to be considered: (a) N > 8: acetyl-CoA from /?-degradation occupies one binding site or carnitine itself occupies one binding site actively feeding acetyl-CoA from B-degradation into TCC; (b) N < 8: acetyl-CoA from p-degradation occupies one binding site. Summarizing this interpretation, it is concluded that, as long as the chain length of fatty-acids is N > 8, carnitine becomes actively involved in /3degradation and is not only participating as a transmembrane carrier for fatty acids as hitherto formulated. Further confirmation for this statement is derived from two facts: (i) doubling the amount of L-FABPc (Fig. 7, l vs n ) in an assay containing pyruvate, carnitine and fatty-acid ( x ) results in a Hill plot pattern that equals an assay with a lower level of carnitine (compare the kinetics obtained with 50pM carnitine and 10pM carnitine in Fig. 5) without affecting pyruvate turnover significantly. This apparently reduced need of camitine may be due to the formation of a tight cluster by enhanced protein-protein interaction resulting in a channeled reaction course favouring transmembrane-cycling or, (ii) if mechanistically involved, regeneration of carnitine. This second mechanism, furthermore, is in agreement with the experiments and interpretation for the role of FABP described by Foumier and Richard (1990) stating that FABP is an active link between the cytoplasm and the mitochondrion.

CONCLUSION

The data reported are in agreement with the inhibition of pyruvate oxidation under conditions favoring B-degradation of fatty acids. The kinetics derived from the Hill plots, however, suggest that the binding sites mirror the handling of activated acetyl-moieties and thus confirm the growing evidence for an organized B-degradation mechanism based, at least, on the formation of an enzyme complex of the enzymes

involved (Garland et al., 1965). Furthermore, the growing evidence for an active role of carnitine during b-degradation is confirmed (see the discussion “free palmitoyl-CoA vs external acyl-carnitine” in Stanley and Tubbs, 1975). The formulation of an underlying reaction mechanism based on the concept of a metabolon (Srere, 1985) and supramolecularorganization (Fiirster, 1988) as proposed for TCC is published elsewhere (Fiirster, 1992). With the mechanism given the discussion of /I-oxidation enzymes existing as a multienzyme complex to which intermediates are tightly bound and the fact that intermediates of B-degradation are present at very low concentrations, can be reduced to an organizational aspect being in agreement with other discussions dealing with this subject (Watmough et al., 1989; Sumegi et al., 1991). REFERENCES

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Beta-oxidation as channeled reaction linked to citric acid cycle: evidence from measurements of mitochondrial pyruvate oxidation during fatty acid degradation.

1. The kinetics of mitochondrial mammalian pyruvate dehydrogenase multienzyme complex (PDHC) is studied by the formation of CO2 using tracer amounts o...
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