Biochimica et Biophysicu Actu 919 (1987) 171-174

Elsevier

171

BBA 52532

CoA-persulphide: a possible in vivo inhibitor of mammalian short-chain acyl-CoA dehydrogenase

Lee Shaw and Paul C. Engel

Department of Biochemistry, Universi@ of Sheffie& Sheffield (U.K.)

(Received 15 January 1987)

Key words: CoA-persulfide; Acyl-CoA dehydrogenase; Beta-oxidation

The characteristic green colour of native short-chain acyl-CoA dehydrogenases (EC 1.3.99.2) results from a charge transfer complex between the FAD prosthetic group and a tightly bound molecule of CoA-per- sulphide. The native enzyme from ox liver mitochondria was found to have about 60% of its FAD cofactor liganded with CoA-persulphide. When artificially fully liganded with CoA-perstdphide, this enzyme was inhibited by 90% in comparison to unliganded enzyme. Enzymic activity could be slowly restored by displacing the CoA-persulphide with high concentrations of butyryl-CoA, the enzyme’s physiological substrate. The results show that CoA-persulphide is a potent inhibitor of short-chain acyl-CoA dehydro- genase and may have a physiological role in the regulation of /3-oxidation.

Introduction

Acyl-CoA dehydrogenases are a group of solu- ble FAD-linked enzymes that catalyse the forma- tion of cu-,/3-enoyl-CoA products from saturated acyl-CoA substrates during mammalian mitochon- drial /&oxidation [l]. In common with most simple flavoproteins, the general and long-chain acyl-CoA dehydrogenases are isolated as yellow enzymes with ultraviolet/visible absorption spectra similar to that of free FAD [2,3]. However, the absorption spectra of the native short-chain acyl (or butyryl) -CoA dehydrogenases from a variety of mam- malian sources [4-61 and at least one bacterial source [7,8] possess an additional band centred at about 700 nm which gives these enzymes a char- acteristic green colour. The true cause of this green colour eluded various investigators [5,8,9] until, with the bacterial enzyme, it was finally

Correspondence @resent address): L. Shaw, Biochemisches Institut, Christian-Albrechts-UniversitZt, 2300 Kiel 1, F.R.G.

found to be the result of a charge-transfer interac- tion between the flavin cofactor and a tightly bound molecule of CoA-persulphide [lO,ll]. The same ligand is evidently responsible for the green colour of the mammalian enzymes, since it also restored the long wavelength absorbance band of chemically de-greened ox liver short-chain acyl- CoA dehydrogenase [12].

The presence of this ligand has little apparent effect on the activity of the bacterial enzyme un- der the conditions of a normal catalytic assay, presumably because the CoA-persulphide is dis- placed by the large excess of substrate [8]. How- ever, the native green enzyme isolated from pig liver was reported to be half as active as the de-greened form [5], suggesting that this ligand is an inhibitor of mammalian short-chain acyl-CoA dehydrogenases and as such, may be of signifi- cance in vivo.

The recent identification and synthesis of CoA-persulphide now permit a more rigorous in- vestigation into these observations.

In this paper, we describe the inhibitory effects

0005-2760/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

172

of CoA-persulphide on ox liver short-chain acyl-

CoA dehydrogenase and speculate on its possible

significance in vivo.

Materials and Methods

Enzyme purification and assay. Short-chain acyl- CoA dehydrogenase was purified from ox liver as

previously described [12] and, unless otherwise

stated, was used in 50 mM p.otassium phosphate

buffer (pH 7.6).

Enzymic activity was measured at 25’C with a

dye-reduction assay employing 0.6 mM phenazine

ethosulphate, 0.001% dichlorophenolindophenol

(w/v) (Em = 21000 1. mol-’ . cm-‘) and 50 PM butyryl-CoA in 0.12 M potassium phosphate (pH

7.1). The reaction was initiated by addition of the

enzyme instead of phenazine ethosulphate [12]. Production of enzyme ful& liganded with CoA-

persulphide and degradation of enzyme-bound CoA- persulphide. CoA-persulphide was generated by in- cubating a 30-fold molar excess of Na,S vvith 1

mg CoA in 1 ml 50 mM potassium phosphate (pH 7.6) for 3 h at room temperature [lo]. To produce

fully green enzyme, aliquots of the CoA-per-

sulphide incubation mixture were added to a soht-

tion of enzyme until no further absorbance changes

at 685 nm occurred. A ratio A,,,/A,,, (the ab-

sorbance ratio of the long-wavelength charge-

transfer band and the blue-shifted main flavin

band) of 0.29 was indicative of fully-liganded en-

zyme [12]. Excess unbound CoA-persulphide was removed by dialysis, during which no change in

the ratio A685/A430 occurred. To ‘de-green’ the native or artificially liganded enzyme, the bound

CoA-persulphide was chemically degraded at 4“ C by an overnight anaerobic dialysis with 10 mM sodium dithionite in the enzyme buffer, followed

by extensive dialysis against the buffer alone. Slight turbidity resulting from this process was removed by centrifugation prior to spectral measurements.

Rt?Sldt.S

Spectral characteristics of native short-chain acyl- CoA dehydrogenase from ox liver

The chromatofocusing step during enzyme purification resulted in some de-greening of the enzyme [12]. However, the clear maxima at 685

15-

I,0 -

: 2

io.5- 9

f

I L 04 I

300 400 500 600 700 800 Wavelength (nm)

Fig. 1. Absorption spectrum of ox liver short-chain acyl-CoA dehydrogenase before final purification by chromatofocusing.

nm, 435 rnn and 366 nm in the absorption spec-

trum of the enzyme prior to this final purification step (Fig. l), suggest that the preparation at this

,’ ,’

,’ ,’ P / /

/

T Aabs=O-02

t I I , I

4 2 0 Time (mm)

Fig. 2. Demonstration of the inhibition of ox liver short-chain acyl-CoA dehydrogenase due to bound CoA-perstdphide. The solid line represents the progress curve of dichlorophenolin- dophenol reduction (Aa) recorded immediately on addition

of 61 pmol of fully green enzyme to 1 ml 0.001% dichlorophe- nolindophenol in 120 mM potassium phosphate (pH 7.1) con- taining 50 pM butyryl-CoA and 0.6 mM phenazine etho- sulphate at 25 o C. The tangential dashed line (- - - - - -) indi-

cates the estimated initial rate of dichlorophenolindophenol reduction. The steeper broken line (. -. - .) indicates the rate of

dye-reduction using yellow enzyme under conditions identical to those above.

173

stage was relatively free of ~n~a~g chromo- phores. The ratio A,,,/A,3S (0.19) therefore indi- cated that this partially pure enzyme was about 60% liganded with CoA-persulphide. Hence, the enzyme as extracted from mitochondria must have been at least 60% liganded and, because of the two previous purification and dialysis steps, may have been more extensively liganded.

Effect of bound CoA-persulphide on enzyme activity Because CoA-persulphide is unstable in free

solution [lo], inventions steady-state ~bition studies were not feasible. The studies reported here are therefore restricted to the influence of bound CoA-persulphide on enzymic activity.

When tested in the standard assay, the initial activity of the enzyme saturated with CoA-per- sulpbide was only 10% of that of the unliganded enzyme (Fig. 2) and apparently underwent a slow reactivation during the course of the assay, as seen by a gradual steepening in the reaction trace for dic~oropheno~dophenol reduction. Indeed, the true initial rate may have been smaller due to an unavoidable mixing time of a few seconds.

The apparent reactivation of the enzyme was further studied by incubating enzyme, maximally liganded with CoA-persulphide, with a large ex- cess of butyryl-CoA, the enzyme’s optimal sub-

l&Or

04 , J

0 25 50 Time (min.1

Fig. 3. Reactivation of green short-chain acyK!oA dehydro- genase by butyryl-CoA. 8.2 gM fuIly green enzyme (by flavin)

was incubated with 410 CM butyryEoA at 25*C. Aliquots were removed after known time intervals and assayed im- mediately for initial enzymic activity.

OL, 1 I I , J 300 100 ml 600 700 em

Waveierqthlnml

Fig. 4. Spectral changes showing displacement of CoA-per-

sulphide from green short-chain acyLCoA dehdyrogenase by

butyryKoA. 8.2 yM fully green enzyme @y fiavin) (spectxum

1) was incubated at 25 o C with 410 mM butyryl-CoA. Spectra

were recorded after 5 and 75 min (spectra 2 and 3, respec-

tively).

strate [12]. Aliquots were removed at timed inter- vals and assayed immediately for initial activity. The results (Fig. 3) show that a reactivation of the enzyme does indeed occur and happens over a period of about 25 min to give 93% restoration of the initial enzymic rate (the expected full rate is 150 min-r). This incomplete reactivation prob- ably results from a residual equilibrium eoncentra- tion of CoA-persulphideliganded enzyme. Moni- toring of the spectral changes occurring during the reactivation by excess butyryl-CoA revealed that the green CoA-persulphide-liganded form was being slowly converted to a species spectrally identical to the normal active enzyme-butyryl-CoA complex [12] (Fig. 4). This suggests that reactiva- tion occurs by slow dissociation of CoA-per- sulpbide from the active site, followed by binding of the substrate. The 50-fold excess of substrate in this incubation and also in the activity assays ensures that the enzyme remains in the active butyryl-CoA liganded state.

174

Discussion

In this work it has been shown that CoA-per- sulphide, the so-called ‘greening ligand’, associates very avidly with the substrate binding site of ox liver short-chain acyl-CoA dehydrogenase, causing marked (at least 90%) inhibition of its enzymic activity.

Reactivation of enzyme, maximally liganded with CoA-pers~p~de, could be achieved by chemical destruction of the ligand with sodium dithionite or by its replacement using high con- centrations (0.41 mM) of butyryl-CoA, the en- zyme’s natural substrate. Although the concentra- tion of butyryl-CoA within mitochondria is un- known (as far as the authors are aware), the enzyme’s low K, for this substrate (2.7 PM) would suggest that the concentration required to re- activate the enzyme in vitro is unphysiologically high and that inhibition in vivo may be more pronounced. Since mammalian short-chain acyl- CoA dehydrogenases are usuaI.ly isolated together with bound CoA-persulphide, this ligand could be of significance in vivo in the control of P-oxida- tion. It has previously been suggested that short- chain acyl-CoA dehydrogenase catalyses one of the regulatory steps in P-oxidation [13], although the proposed regulatory compounds were CoA- containing F-oxidation intermediates.

The origin of CoA-persulphide bound to native short-chain acyl-CoA dehydrogenases is unknown. If this ligand were of physiological relevance, its production, presumably from sulphur incorpora- tion into free CoA would be expected to be under the control of a specific, possibly enzymic, pro- cess.

Rhodanese, a mitochondrial enzyme, catalysing the transfer of sulphur to a variety of compounds [14,15], does not generate CoA-persulphide (Wil- liamson, G., personal communication) but another sulphur transferase may be involved. The as yet unexplored possibility, that CoA-persulphide is generated in situ by the dehydrogenase itself IlO] is also an attractive hypothesis, especially in view

of the instability of the free persulphide in solu- tion.

In the context of this discussion, it is also worthy of note that covalent sulphur incorpora- tion reactions may be involved in the control of other metabolic processes [16].

Clearly, it is not yet understood why CoA-per- sulphide occurs bound to short-chain acyl-CoA dehydrogenase. However, the demonstration that this unusual CoA-cont~~g species can potently inhibit a potentially regulatory enzyme in a central pathway of energy metabolism is sufficiently im- portant to warrant further investigation.

Acknowledgements

This work was supported by an SERC student- ship to L.S.

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