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Conditions for the Self-Catalysed Inactivation of Carnitine Acetyltransferase a NOVEL FORM of ENZYME INHIBITION

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Conditions for the Self-Catalysed Inactivation of Carnitine Acetyltransferase a NOVEL FORM of ENZYME INHIBITION Biochem. J. (1969) 111, 225 225 Printed in Great Britain Conditions for the Self-Catalysed Inactivation of Carnitine Acetyltransferase A NOVEL FORM OF ENZYME INHIBITION By J. F. A. CHASE AND P. K. TUBBS Department of Biochemi8try, University of (Jambridge (Received 2 Augw8t 1968) 1. Carnitine acetyltransferase is very rapidly inhibited in the presence of bromoacetyl-(-)-carnitine plus CoA or of bromoacetyl-CoA plus (-)-carnitine. 2. Under appropriate conditions, the enzyme may be titrated with either bromo- acetyl substrate analogue; in each case about lmole of inhibitor is required to inactivate completely 1 mole of enzyme of molecular weight 58 000 + 3000. 3. Inhibition by bromoacetyl-CoA plus (-)-carnitine results in the formation of an inactive enzyme species, containing stoicheiometric amounts of bound adenine nucleotide and (-)-carnitine in a form that is not removed by gel filtration. This is shown to be S-carboxymethyl-CoA (-)-carnitine ester. 4. The inhibited enzyme recovers activity slowly on prolonged standing at 4°. 5. Incubation with S-car- boxymethyl-CoA (-)-carnitine ester causes a slow inhibition of carnitine acetyl- transferase. 6. The formation of bound S-carboxymethyl-CoA (-)-carnitine ester by the enzyme is discussed. Presumably the resulting inhibition reflects binding of the ester to both the CoA- and carnitine-binding sites on the enzyme and its consequent very slow dissociation. These observations confirm that carnitine acetyltransferase can form ternary enzyme-substrate complexes; this also appears to be the case with carnitine palmitoyltransferase and choline acetyltransferase. The enzyme carnitine acetyltransferase [acetyl- Preliminary reports of part of this work have CoA-(-)-carnitine O-acetyltransferase, EC 2.3.1.7] been published (Chase & Tubbs, 1966b, 1967). is widespread and active in animal tissues (Beenakkers & Klingenberg, 1964), but as yet no MATERIALS precise physiological role can be ascribed to it (Fritz, 1965). It might be expected, however, that Enzymes. Pigeon breast muscle carnitine acetyltransferase a study of the metabolic disturbances caused by was obtained from Boehringer Corp. (London) Ltd. (Lon- specific inhibitors of the enzyme would yield in- don, W. 5) and recrystallized twice to a specific activity of formation relevant to this problem. 120-130 units/mg. (Chase, Pearson & Tubbs, 1965). The effects of bromoacetyl derivatives of CoA Citrate synthase (EC 4.1.3.7) was prepared from pig and carnitine on isolated preparations of carnitine heart by the method of Srere & Kosicki (1961). Chemical&. CoA was bought from Boehringer Corp. acetyltransferase have been investigated. It was (London) Ltd.; desulpho-CoA was prepared from it by the hoped that such substrate analogues, after being method of Chase, Middleton & Tubbs (1966). Chloroacetyl specifically bound to the enzyme, would alkylate chloride, bromoacetyl bromide, bromoacetic acid, thio- susceptible amino acid residues at or near the active phenol and ,B-alanine were the reagent grade of British centre. Similar active-site-directed inhibitors have Drug Houses Ltd. (Poole, Dorset). S-Carboxymethyl-2- been designed for a number of enzymes (Baker, mercaptoethylamine was prepared from 2-mercaptoethyl- 1967). Although it appears that bromoacetyl-(-)- amine hydrochloride (Koch-Light Laboratories Ltd., carnitine at least may be used to inhibit the enzyme Colnbrook, Bucks.) by the method of De Marco, Riva & and irreversibly and probably in the manner envisaged Dupre (1964) and recrystallized twice. (+)-, (-)- it (±)-Carnitine hydrochlorides were obtained from Koch- above, has also become clear that under certain Light Laboratories Ltd.; samples of the two optically active can cause a more conditions either analogue much isomers from British Drug Houses Ltd. were also used. rapid inhibition, which is slowly reversible. It is (+±)-[Me-14C]-Carnitine hydrochloride, nominally 11-65/Lc/ the somewhat novel nature of this rapid inhibition ,umole, was a gift from Dr P. B. Garland. Before use it that is reported here. was chromatographed on a column of Zeo-Karb 225 8 Bioch. 1969, 111 226 J. F. A. CHASE AND P. K. TUBBS 1969 (SRC 16) cation-exchange resin (British Drug Houses Ltd.) Preparation and assay of bromoacetylcarnitine. The by the procedure of Friedman, McFarlane, Bhattacharyya bromoacetyl derivatives of (+)-, (-) and (±)-carnitine & Fraenkel (1960). An emergent peak with 95% of the were prepared by treating the appropriate carnitine hydro- radioactivity of the starting material proved to contain all chloride, dissolved in bromoacetic acid, with bromoacetyl the carnitine. This material was pooled and stored frozen. bromide. The procedure was otherwise identical with that 5,-5'-Dithiobis-(2-nitrobenzoic acid) was obtained from described by Fraenkel & Friedman (1957) for acetyl- the Aldrich Chemical Co. Inc. (Milwaukee, Wis., U.S.A.). carnitine. Bromoacetylcarnitine was recrystallized twice Tris (Trizma base; Sigma Chemical Co., St Louis, Mo., from acetic acid-acetone (Chase & Tubbs, 1966a) and stored U.S.A.) was neutralized with HCI. Phosphate buffers were in a desiccator away from light. prepared from KH2PO4 and KOH. No specific assay for bromoacetylcarnitine is available, Other reagents were of analytical grade and glass- as this compound strongly inhibits carnitine acetyltrans- distilled water was used throughout. ferase. Two indirect methods have therefore been used. In the first, bromoacetylcarnitine was hydrolysed by incu- bation at room temperature for 10min. in the presence of a METHODS 0-1 M concentration excess of KOH. After neutralization Preparation of chloroacetyl-CoA and bromoacetyl-CoA. In the (-)-carnitine released was determined enzymically the early stages ofthis work these compounds were prepared (Pearson & Tubbs, 1964). This procedure can only be used by treating CoA solutions with the appropriate haloacetyl with derivatives of (-)- or (±)-carnitine. Bromoacetyl halide (Seubert, 1960). However, as this procedure gives esters of all carnitine enantiomers were also estimated rather poor yield (20-30%) of impure haloacetyl-CoA, a chemically by a hydroxamate method (Friedman & milder technique has been developed based on transhalo- Fraenkel, 1955). The first method measures both free and acetylation between thiophenol and CoA. alkali-labile carnitine in the sample, whereas the second (i) Preparation of S-bromoacetylthiophenol. A 2-Oml. measures all esters of carnitine, so that the reported con- portion (19-2m-moles) of thiophenol was added to 2-4ml. centrations of bromoacetylcarnitine are maximum values. (26-4m-moles) of freshly redistilled bromoacetyl bromide Sub8trate8 of carnitine acetyltransferase. Solutions of in a Thunberg tube. HBr was removed as formed by means CoA, acetyl-CoA, (-)-carnitine and acetyl-(-)-carnitine of a water pump. After about 60min. at room temperature were prepared, stored and assayed as described by Chase no further gas was evolved and saturated KHCO3 solution & Tubbs (1966a). was added to bring the pH of the mixture to about 8. This Measurement8 of carnitine acetyltran8ferase activity. precipitated bromoacetylthiophenol as colourless crystals. Except where specific conditions are mentioned, carnitine The mixture was extracted twice with ether, which dissolved acetyltransferase activities were compared by adding the crystals, and the aqueous supernatant was washed enzyme samples to a system containing tris-HCl buffer, twice with this solvent. The ethereal solution was washed pH7-8 (100mm), acetyl-CoA (0-1mM) and (-)-carnitine three times with dilute aqueous KHCO3 to remove traces hydrochloride (1-25mM) in a final volume of 2-Oml. The of bromoacetic acid and dried with Na2SO4. Removal of initial rate of decrease of E232, due to the deacetylation of the ether in a stream of N2 left a pale-yellow solid, which CoA, was observed by using a Beckman DK-2 recording was recrystallized from aqueous ethanol. A yield of 2-7g. spectrophotometer with the cell housing maintained at (57% of theory) of colourless bromoacetylthiophenol (m.p. 30+0-30. 36.3-37.3°, uncorr.) was obtained. On storage at 40 in Molecular-weight determination8. A single determination vacuo in the dark, bromoacetylthiophenol develops a yellow of the molecular weight of carnitine acetyltransferase was colour, and it was freshly recrystallized before use. made by using the Archibald technique as modified by (ii) Bromoacetylation of CoA. A 0-5g. portion (2-Om- Ehrenberg (1957). Observations on the meniscus at the moles) of bromoacetylthiophenol was dissolved in 3ml. of air/solvent interface were carried out on a solution con- acetone-0-1M-KHCO3 (5:2, v/v) and mixed with 20mg. taining 9-85mg. of protein/ml. in 0- M-phosphate buffer, (approx. 20,umoles) of CoA dissolved in the same solvent. pH7-5, by using a Spinco model E analytical ultracentrifuge This system was usually monophasic; if it was not, drops at 7950 rev./min. and 170. The subsequent artificial- ofacetone or KHCO3 were added as required. After 45min. boundary experiment was conducted at 16000 rev./min. at room temperature, the solution was brought to pH 1-2 Calculated values for the molecular weight after 48, 64 and with conc. HCI and the acetone removed with a stream of 80min. were 59300, 58900 and 57500 respectively. A value N2. The aqueous slurry that remained was extracted ten of 0-74 was taken for the partial specific volume of the times with ether to remove thiophenol and acylthiophenol, enzyme. This was obtained from the amino acid com- and the last traces of ether
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