Biochimica et Biophysica Acta, 706 (1982) 245-248 Elsevier Biomedical Press

BBA Report

245

BBA 30027

A CONVENIENT AND RAPID METHOD FOR THE COMPLETE REMOVAL OF CoA FROM BUTYRYL-CoA DEHYDROGENASE

GARY WILLIAMSON and PAUL C. ENGEL

Department of Biochemistry, University of Sheffield, Sheffield SIO 2TN (U.K.)

(Received May 4th, 1982)

Key words: Butyryl-CoA dehydrogenase; Ligand removal," Thiopropyl-Sepharose 6B; CoA removal

The commercially available gel, 2-pyridyl disulphide hydroxypropyl ether-Sepharose (thiopropyl-Sepharose 6B), can be used to remove bound ligand completely from butyryI-CoA dehydrogenase (EC 1.3.99.2) in two simple operations. The resultant enzyme forms normal complexes with acetoacetyl-CoA and CoA per- sulphide, contains no bound CoA as determined by the enzymatic assay for CoA, and retains full catalytic activity.

Butyryl-CoA dehydrogenase (butyryl-CoA: (acceptor) oxidoreductase, EC 1.3.99.2) from the obligate anaerobe Megasphaera elsdenii is isolated in a green form containing CoA persulphide in a charge-transfer interaction with the enzyme-bound FAD [1-3]. The tightly bound CoA persulphide cannot be removed by gel filtration or extensive dialysis [2]. Acetoacetyl-CoA, an inhibitor of butyryl-CoA dehydrogenase [4,5], has a dissocia- tion constant of less than 0.5 #M (Williamson, G., unpublished results) and is also sometimes present as a ligand on the purified enzyme [6]. The tight binding of substrates and inhibitors has seriously hindered the study of the mechanism and active site of the short-chain acyl-CoA dehydrogenases. For useful studies of substrate binding, chemical modification, rapid reaction kinetics, etc., and bi- nding studies, it is essential that the enzyme be homogeneously unliganded.

The mammalian butyryl-CoA dehydrogenases are similar in many properties to the bacterial enzyme, and are also isolated in a green form [7-9]. Steyn-Parv~ and Beinert [10] reported that

Abbreviations: Thiopropyl-Sepharose 6B, 2-pyridyl disulphide hydroxypropyl ether-Sepharose.

added acyl-CoA compounds could only be re- moved by denaturation, although they could be readily replaced by the addition of an excess of further (labelled) substrate. Engel and Massey [4] showed that extensive anaerobic dialysis of the bacterial enzyme against the reducing agent, sodium dithionite, decreased the CoA content sub- stantially. However, this method was time-con- suming, with an associated risk of some flavin release occurring during the long dialysis, and most importantly the removal of the CoA-con- taining ligand was incomplete [2]. If the enzyme was reoxidised without anaerobic dialysis after treatment with sodium dithionite, it showed no long-wavelength absorption band [1,10], but never- theless could be shown to retain the CoA moiety [2,4], probably as bound CoASH [2]. In this paper it is shown that 2-pyridyl disulphide hydroxypro- pyl ether-Sepharose, which reacts with sulphydryl groups by thiol-disulphide interchange, can be used to remove bound CoA completely from butyryl- CoA dehydrogenase.

Green butyryl-CoA dehydrogenase was pre- pared as described by Engel [11] and was then dialysed overnight against 10 mM sodium di- thionite in 0.1 M potassium phosphate, pH 7, and

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for a further 2 h against the same buffer alone. These dialyses ensure that any bound acyl-CoA is converted to CoASH, and that the dithionite and its oxidised products are partially removed, allow- ing the enzyme-bound FAD to reoxidise. The concentration of yellow enzyme thus produced was measured spectrophotometrically, using E450 ---- 12.6 mM -I • cm -1 [1].

Thiopropyl-Sepharose 6B was purchased from Pharmacia Fine Chemicals. When supplied this material is ready for use, with the immobilised sulphydryl group protected in a disulphide linkage with a 2-pyridyl group (Pharmacia product infor- mation). For use, the column was equilibrated in buffer A (0.1 M potassium phosphate (pH 7)/1 mM EDTA). The gel has a high content of 'active' groups - - 20 /~mol 2-thiopyridyl g r o u p s p e r ml swollen gel and hence may be used up to four times before recycling. The following recycling procedure was performed batchwise: (i) 2 × 5 vol. 10 mM cysteine in buffer A; (ii) 150 vol. buffer A; (iii) 2 × 10 vol. 1.5 mM 2,2'-dithiopyridyl dis- ulphide ('Aldrithiol-2' from Aldrich Chemical Co.) in buffer A for 60 min each with stirring; (iv) 150 vol. buffer A. The gel was then repacked into a 20 × 1.5 cm column.

CoA was assayed by the 'catalytic' procedure of Michal and Bergmeyer [12] using phosphotransa- cetylase, citrate synthase and malate dehydro- genase and measuring the production of NADH.

Enzyme samples (10 nmol) in buffer A were freeze-dried and then prepared for the CoA assay by denaturation in 0.8 ml 5% trichloroacetic acid for 5 min with vigorous mixing, followed by neu- tralisation with 0.16 ml 1 M K2CO 3. The mixture was then centrifuged and the original concentra- tion of enzyme was determined from the ab- sorbance of the supernatant at 450 nm due to released FAD (c at 450 n m = 11.3 mM -n . cm-~). An aliquot, usually 100/~1, was withdrawn for the CoA assay. A standard curve for CoA (from Sigma Chemical Co. Ltd.) was constructed for 0-700 pmol CoA, similarly treated with 5% trichloro- acetic acid and 1 M K2CO 3.

In routine use of the column, the yellow form of the enzyme, prepared as described above and con- taining CoASH, was loaded onto the gel and then eluted with buffer A. A flow rate of about 0.1 ml per min was suitable for the 20 × 1.5 cm column. 2-ml fractions were collected; butyryl-CoA dehy- drogenase could be readily identified by its yellow

colour and by its absorption maximum at 450 nm. The assay for CoA, performed on butyryl-CoA

dehydrogenase, prior to treatment on thiopropyl- Sepharose 6B, showed that the CoA content varied between 0.2 and 0.5 mol CoA/mol butyryl-CoA dehydrogenase. The enzyme eluting from the col- umn, however, contained less than 0.02 mol CoA/mol butyryl-CoA dehydrogenase. The reac- tion of sulphydryl groups, such as that of CoASH, with the thiopropyl-Sepharose 6B stoichiometri- cally releases 2-thiopyridone, which is an excellent leaving group (Pharmacia product information), and has an absorption maximum at 343 nm (~ at 343 nm = 8.08 m M - 1. cm - t, [ 13]). Quantitation of the number of sulphydryls reacted by the routine method is not possible, however, since residual dithionite and its breakdown product, sulphite [ 14], will reduce disulphide bonds and release extra, non-stoichiometric amounts of 2-thiopyridone. In order to determine the amount of 2-thiopyridone released by the reaction of the enzyme, the sample must be extensively dialysed against phosphate buffer prior to loading on the thiopropyl-Sep- harose 6B column, to ensure complete removal of residual sulphite. Fig. 1 shows the elution profile of a 2 ml sample of enzyme, dialysed consecutively against the following: (i) 10 mM sodium dithionite

30

z0

10

0 v - 10 20 30 ~0 - 50 60

ml e tut ecl

Fig . 1. E l u t i o n p ro f i l e f r o m t h i o p r o p y l - S e p h a r o s e 6B. T w o ex-

p e r i m e n t s were separately performed, and in both 120 nmol of butyryl-CoA dehydrogenase containing CoA were loaded onto the gel. 1. With the gel protected by 2-thiopyridyl groups, enzyme elution (0) is followed by release 2-thiopyridone (A). 2. With the gel in the free sulphydryl form, the elution position of butyryl-CoA dehydrogenase (O) is earlier.

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in buffer A; (ii) 3 × 500 vol. buffer A for 3 h each; (iii) 500 vol. buffer A for 16 h.

Integration under the enzyme peak and 2-thio- pyridone peak eluted from the column showed that for 120 nmol of enzyme loaded, 110 nmol was recovered under a peak centred at 38 ml, followed by 42 nmol of 2-thiopyridone centred at 49 ml. This indicates that approx. 0.39 mol sulphydryl have reacted per mol butyryl-CoA dehydrogenase. The enzyme before treatment on the thiopropyl- Sepharose 6B column contained 0.47 mol CoA per mol enzyme by the CoA assay, in reasonable agreement with the release of 2-thiopyridone. En- zyme eluting from the column contained no detec- table CoA. We believe, therefore, that on the column there is a thiol-disulphide interchange be- tween the enzyme-bound CoASH and the im- mobilised 2-pyridyl disulphide hydroxypropyl ether moiety (Scheme I).

ECoAS. C . : . C.2 S 3 I

OH

- O - C H 2 - C H - C H 2 -S-S-CoA.E

'1l OH

2-thiopyridone

~ - O - C H 2 - C H - C H 2 - S - S - C o A + E I CoA-free

OH enzyme

If the thiopropyl-Sepharose 6B column is treated with 0.5M 2-mercaptoethanol [15] then the 2- thiopyridyl protecting groups are removed. In this form, the gel does not bind sulphydryl groups.

Fig. 1 shows that under these conditions the en- zyme elutes earlier, peaking at 29 nil. Thus the reaction of bound CoASH with the gel appears to retard the elution of the enzyme.

Although butyryl-CoA dehydrogenase has a very high affinity for the CoA moiety [4], steric factors presumably severely weaken the binding of the enzyme, (E, above) to the support-linked CoA. This is further supported by the observation that butyryl-CoA dehydrogenase will not bind to a solid matrix containing CoA immobilised via the sulphur atom, but will bind to immobilised CoA if the SH- group is free (Williamson, G., unpub- lished results).

The CoA-free enzyme is fully active in the dye-coupled catalytic assay [12], and gives the normal charge-transfer complex [3], with an ab- sorption band centred at 580 nm, on addition of acetoacetyl-CoA. Butyryl-CoA dehydrogenase as isolated with bound CoA persulphide has absorp- tion peaks centred at 266 nm, 365 nm, 430 nm and 710 nm, with a A266:A43 o ratio of 7.3 [1]. CoA-free enzyme exhibits no 710 nm band, and a shift in the other peaks to 269, 375 and 451 nm, respec- tively. The shift in the ultraviolet peak from 266 nm to 269 nm is consistent with the removal of bound CoA, which has an absorption peak at 260 nm. Furthermore, there is a decrease in the A266 : A430 ratio to less than 5.5 (cf. reported value of 7.3 [1]). When stored, the CoA-free enzyme is less table than enzyme either with bound CoA or bound CoA persulphide (Table I).

Incubation of 1 mM sodium sulphide with butyryl-CoA dehydrogenase containing CoA pro- duces the characteristic absorption band centred at 710 nm [2]. A yellow sample of butyryl-CoA

TABLE I

STABILITY OF BUTYRYL-CoA DEHYDROGENASE

Enzyme samples were stored at 4°C in 0.1 M potassium phosphate pH 7 containing 0.02% sodium azide. Aliquots were removed for assay at the times indicated and the activities expressed as percentages.

Time of storage (h)

0 24 48 72 96

CoA-free butyryl-CoA dehydrogenase 100 Butyryl-CoA dehydrogenase before thiopropyl-Sepharose 6B 100 Butyryl-CoA dehydrogenase containing CoA persulphide 100

100 93 83 75 - - 1 0 0 92 84 - - - - - - 1 0 0

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dehydrogenase prior to treatment on the thiopro- pyl-Sepharose 6B column contained 0.47 mol CoA/mol enzyme by the CoA assay. When this sample was incubated for 16 h with 1 mM Na2S, the A710:A430 ratio increased to 0.17, indicating 0.31 mol CoA persulphide per mol enzyme. By contrast, when the thiopropyl-Sepharose 6B- treated enzyme was incubated with 1 mM Na2S, there was no increase in the absorbance at 710 nm, indicating that no CoA persulphide was formed. When, however, free CoA was added to the in- cubation mixture, the expected increase in .4710 [2] was seen.

We believe that the mild method described here will be generaUy useful in the preparation of butyryl-CoA dehydrogenase from various sources in a state suitable for mechanistic studies.

We are grateful to the Science and Engineering Research Council for the award of a studentship for Training in Research Methods to G.W.

References

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Massey, V. (1982) J. Biol. Chem. 257, 4314-4320

3 Williamson, G., Engel, P.C., Nishina, Y. and Shiga, K. (1982) FEBS Letts. 138, 29-32

4 Engel, P.C. and Masscy, V. (1971) Biochem. J. 125, 889-902 5 Davidson, B. and Schulz, H. (1982) Arch. Biochem. Bio-

phys. 213, 155-162 6 Engel, P.C. (1980) in Flavins and Flavoproteins (Yagi, K.

and Yamano, T., exls.), pp. 423-430, Japan Scientific Socie- ties Press, Tokyo

7 Mahler, H.R. (1954) J. Biol. Chem. 206, 13-26 8 Steyn-Parv6, E.P. and Beinert, H. (1958) J. Biol. Chem. 223,

843-852 9 Hoskins, D.D. (1966) J. Biol. Chem. 241, 4472-4479

10 Steyn-Parv6, E.P. and Beinert, H. (1958) J. Biol. Chem. 223, 853-861

11 Engel, P.C. (1981) Methods Enzymol. 71,359-366 12 Michal, G. and Bergmeyer, H.U. (1974) in Methods of

Enzymatic Analysis, 2nd Edn. (Bcrgmeyer, H.U., ed.), Vol. 4, p. 1967, Verlag Chemie, Academic Press Inc., New York

13 Stuchbury, T., Shipton, M., Norris, R., Malthous¢, J.P.G., Brocklehurst, K., Herbert, J.A.L. and Suschitzky, H. (1975) Biochem. J. 151,417-432

14 Mayhew, S.G. (1978) Eur. J. Biochem. 85, 535-547 15 Thiopropyl-Scpharose 6B immobilised thiol reagent, p. 14

(1977) Pharmacia Fine Chemicals