Joiirnai (?i ,A"worhunzisrry. 1976. Vol. 21. pp. 873-881. Pergarnon Preys Printed i n Great Britam

CHOLINE ACETYLTRANSFERASE-EVIDENCE FOR ACETYL TRANSFER BY A HISTIDINE RESIDUE D. MALTHE-SORENSSEN Norwegian Defence Research Establishment. Division for Toxicology, PO Box 25 - N-2007 Kjeller. Norway ( R t ~ r i i w d2 Fehriror), 1976. Accepfrd I2 March 1976)

Abstractxholine acetyltransferase from bovine brain has been extensively purified to a specific activity of 2.5 pmol ACh/min mg protein. Attempts to isolate an acetyl enzyme intermediate after incubation of the enzyme with [l-"T]acetyl-CoA were unsuccessful. Such an intermediate could only be isolatcd using a 30-fold less purified enzyme preparation. The protein. binding "C in this preparation. did not correspond to choline acetyltransferase as shown by disc-electrophoresis. The highly purified enzyme could. however, be labelled when choline acetyltransferase was immobilized on a mercuribenzoate sephrtrose gel and incubated with [l-'4C]acetyl-CoA. Subsequently. the immobilized labelled enzyme or the labelled enzyme which had been released by cysteine from the gel. formed ACh after incubation with choline. The labelling and the following formation of [14C]ACh was pH dependent. Masking histidine residues of the enzyme with diethylpyrocarbonate almost abolished the labelling of the immobilized enzyme and completely abolished the formation of ['4C]ACh. EnLyme inhibited with S.S'dithiobis(2-nitrobenzoate) was partially reactivated when the thionitrobenzoatederivative was cleaved by KCN treatment to a thiocyanatederivative. A reaction mechanism for ChAT is proposed based o n the present data

CHOLINE acetyltransferase (ChAT. EC 2.3.1.6) catalyses the biosynthesis of acetylcholine after the following stoichiometry

MATERIALS AND METHODS Chernirals

[1-'4C]Acetyl-CoA, 59 mCi/mmol was obtained from New England Nuclear Corp. Unlabelled acetyl-CoA was purchased from Schwarz-Mann. New York. U S A . Although the reaction mechanism of partially purified Sephadex gels and Sepharose 4B were obtained from Pharenzymes (POTTERet al., 1968; MORRISet a]., 1971; macia fine chemicals. Uppsala. Sweden. p-ChloromercuriHENIERSON& RAMASASTRY,1972 a n d GLOVER & benzoate sodium salt and BrCN were obtained from Koch POTTER, 1971) from different species has been studied Light Lab., Ltd., Bucks., U.K. and diethylpyrocarbonate for several years a great many questions regarding was obtained from Eastman Kodak. l-Ethyl-3(3-dimethylits mechanism remain unsolved. Kinetic evidence has aminopropy1)carbodiimideHCI and Coomassie Blue 3 O G were obtained through Sigma. 5'5-Dithiobis-(2-nitrobenbeen presented that the reaction involves a Theorellzoate) was purchased from Aldrich. All other chemicals Chance reaction mechanism (WHITE& CAVALLITO. were analytical grade and provided by Merck or BDH. 1970; MORRIS rt al., 1971; RAMA SASTRY & HENDERMercurisepharose 4 8 SON. 1972; EMSONet al., 1974). Inhibition studies sugSepharose 4B was activated by cyanogen bromide gest the involvements of both a thiol group (POTTER (250 mg/ml gel). aminoalkylated by diaminoethane folet a/.. 1968; MANNERVIK & SORBO.1970; ROs1 gelfiltration. 15Opg of protein w a s applied to each gel. 2.5 rnA was used per gel and the electrophoresis \\.as r u n for 1 h. The gels were either stained b! Coomassie blue 250G or sliced in Imm pieces and tested for ChAT activit) and [I

"1.

stability was observed when the labelled enzyme. which was eluted directlq with cysteine (Scheme 1). was allowed to stand overnight. Approximately 70", of the total label was removed from the protein. This was determined by measuring the amount of radioactivity which could be precipitated together with the enzyme by 60", ethanol (v/v). Preincubation of the enzyme with diethylpyrocarbonate ( 5 x lo-" M). masking histidine residues before binding it to mercurisepharose. abolished the formation of [ "CIACh. The non transferable ['"C] labelling of protein. however. still occurred (Fig. 4b). The enzyme eluted from the column by qsteine a a s partially reactivated and could be fully reactivated by treatment with hydroxylamine. The reactivation achieved by the immobiliiation and elution was dependent on the pH used during the gel chromatography. Less reactivation was obtained at pH 6.0 (2") than at pH 7.2 (69""). Enzyme inhibited by DTNB (Table 2) could be partially reactivated by treatment with KCN (28""). There was no rractivation of the enzyme when CN was omitted. Thiocyanate could be detected in the reactivated preparation.

DISCUSSION The generally accepted reaction mechanism for ChAT IS the Theorell-Chance mechanism (CLtLAio.

The mechanism predicts that thc concentration of any ternary complex (intermediate) formed during the reaction is low. Despite this it has recently been claimed that ChAT from bovine brain forms a stable acetyl thioester intermediate in the acetyl transfer reaction (ROSKOSKI. 1973). According t o the present investigation a sulphydryl mediated acetyl transfer reaction, seems unlikely. This conclusion is based on several results. Attempts to isolate an acctyl thioester intermediate using highly purified bovine brain ChAT were unsuccessful (Fig. ?a). Similar results were obtained by CURRIER& MAL~TNER (1974) using highly purified ChAT from squid head ganglia. However, it was possible to isolate such an acetyl labelled intermediate when a less purified enzyme (preparation B) such a s used by ROSKOSKI(1973). was used for the acetylation experiments (Fig. 2b). As demonstrated by disc electrophoresis the labelled protein. This preparation did not correspond to ChAT (Fig. 3). Although the protein isolated by disc electrophoresis contained SO",, of the labelling compared to the protein fraction isolated by gelfiltration (Fig. 2b), this label could not be transferred to ACh. Since approx. 40:, of the label bound to the proteins after gelfiltration could be transferred to ACh. this provides evidence for the existence of at least two components in the preparation able to bind acetyl-CoA. One of them is in some way able to transfer the acetylgroup to choline. but none of them corresponded to ChAT which has previously been claimed (WHITE & CAVALLITO, 1970; ROSKOSKI.1973. 1974). Although no acetyl thioester intermediate of soluble enzyme could be isolated using the highly purified preparation one cannot exclude the possibility that a thiol group is essential to the catalytic function of ChAT a s proposed by several groups (BFRMANREISBERG. 1957: POTTCR et 01.. 1968; MANNLRVIE & SORBO.1970: ROSKOSKI,1974). Thiol reagents such as p-chloromercuribenzoate, N-ethylmaleimide, iodoacetate and DTNB inhibit the enzyme. These claims are complicated by considerable species diffcrcnccs in the sensitivity of ChAT to such reagents (EMSONet ul., 1974). by the uncertainty as to whether some of these reagents are truly thiol specific (GLAZER, 1970; WHITE & CAVALLITO. 1970). and by the possibility that the action of the thiol reagents may be more peripheral in nature. causing conformational changes and dena-

Mechanism of choline acetyltransferase

879

Fractions FIG.4. Acetylation of immobilized ChAT and formation of ACh by the immobilized enzyme. 0.5 ml of enzyme preparation was applied to a column (1 x 1 cm) of mercurisepharose previously equilibrated by buffer A at pH 6.0 containing SO mM-NaC1. Unabsorbed protein was washed out before the immobilized enzyme was incubated for 1 h at 5°C with 0.5 ml 60 p~-[l-'"C]acetyl-CoA. Excess [1-'"Clacetyl-CoA was washed out by the same buffer and the column was further incubated under the same conditions M-physostigmine and washed with buffer A and finally with with 0.5 ml 25 mM-choline containing 20 mwcysteine in buffer A and pH 6.0. Fractions of 1 ml were collected at a flow rate of 4.0 ml/h. Each incubation is marked with an arrow on the diagram and the compound used for the incubation. (a) Untreated enzyme, (b) Enzyme preinhibited by diethylpyrocarbonate (5 x ~O-'M). Ezso, A----A ['"C].

turation (WHITE& CAVALLITO, 1970; M A N N ~ R V&I K %KBO, 1970). Highly purified ChAT was inhibited both by p-chloromercuribenzoate and DTNB and acctyl-CoA protected against this inhibition, demonstrating the presence of a thiol group in or near by the active site. The formation of a partially functional thiocyanate derivative of ChAT suggests that the thiol group is not necessary for the enzyme reaction. The formation of thiocyanates has previously been used to demonstrate the existence of non functional thiol groups in the active site of several enzymes (CHUNG, 1972; VANAMAN & STARK,1972 and MORRISON& HEYDE,1972). The low activity of the thiocyanate derivative of ChAT could either be explained by steric hindrance by the cyanide ion introduced, or partial irreversible denaturation of the enzyme inhibited by

DTNB caused by conformational changes (MANNER& SORBO, 1970). Since enzyme preincubated with acetyl-CoA was not bound to the mercurisepharose gel, it is reasonable to believe that the binding of the enzyme to the gel occurs at the thiolgroup in the active site. The acetylation of the immobilized enzyme and formation of ACh by the same enzyme (Fig. 4a) provides further evidence for a non-functional thiol group in the active site and for a non-thiol mediated transfer of the acetyl group from acetyl-CoA to choline. On the other hand. no acetylation of soluble enzyme inhibited by p-chloromerucirbenzoate took place. This seems to be inconsistent with the acetylation taking place on the mercurisepharose column. But, the effect of binding the enzyme to p-chloromercuribenzoateVIK

880

D. MALTHE-S(JREYSSES

FIG.5. Proposed reaction mechanism for ChAT. (-)

Indicate the binding site of choline.

sepharose could be explained by conformational formation of ACh by non-enzymatic catalysis where changes introduced and less steric hindrance achieved imidazole was proposed to act as a nucleophile. In by IJ-chloromrrcuribenzoate under these conditions. Fig. 5 is shown a possible reaction mechanism of making the enzyme susceptible to acetylation. I t is ChAT. based on the data from the present investiganot reasonable to believe that the number and type tion. with a nucleophilic attack of histidine on acetylCoA with a concomitant formation of acetylated of different residues involved in the reaction mechanism changes. but rather that the reaction rates histidine and transfer of acetyl to choline. The acetyof the different reaction steps change. It has been lated intermediate could only be demonstrated by means of the immobilized enzyme. The concentration demonstrated for several enzymes that immobilizing enzymes to different types of supports changes their of any acetylated intermediate of the soluble enzyme afinities for substrates. their pH optima (GOLIXTEIS must be very low, which is in agreement with the kinetics of the enzyme. The rate limiting step in the i’i a/.. 1964; HOMBY c’i d..1968) and their reaction reaction would be the nucleophilic attack of histidine t’i d.. 197 I ) . kinetics (KASCHI.of ( 7 1 . . 1971 : GOLDMAS The formation of a ,V-carbethoxqhistidine residue on acetyl-CoA with a fast reaction with choline 1959). A similar mechanism of acetyl transfer (Fig. 1) and the protective effect of acetyl-CoA against (BRUICE, diethylpyrocarbonate provides evidence for a func- has been suggested by CHASE& TUBRS(1970) for tional histidine residue in the active site. Previously carnitine acetyltransferase containing a histidine resiit has been demonstrated b) different techniques that due in the active center directly involved in the catalysis of the acyl transfer reaction by this enzyme. A a histidine residue may play an important role in the catal!tic function of ChAT WHIT^ & CAVALLITO.nucleophilic catalysis of this type requires a highly 1970: CURRIER & MAUTNER. 1974: ROSKOSKI.1974). activated acyl derivative, represented in this case by acetyl-CoA. Histidine has previously only been shown and carnitine acetyltransferase. an enzyme catalysing an analogous reaction (CHASE& TLBBS.1970). to act as a nucleophile in enzymes which catalyse phosphate transfers (JENCKS, 1969). Further evidence for a functional histidine residue is provided by the fact that diethylpyrocarbonate preskilled work of Ms H r L r N SHAW vented the acetylation of the immobilized enzyme and .4(krio\r/~,.r~grriirJir-The the formation of ACh To determine whether or not IS gratelitlly acknowledged. acetyl-CoA merely binds very strongly to the enzyme without any further reaction. further investigation is REFERENCES necessary. But. since ethylformylation and acetylation R . (1957) J . b i d . Mrd., Yale 29. 403-435. of the immobilized enzyme were dependent on pH BERMAS-REISBERG BKL 1ct T. C. (1959) J . Ant. chrm. Soc. 81. 5444-5449. to the same extent, indicating that ethylformylation B C R T A. M . & SILVFR A . (1973) Nurttrr. Lotid. 243. and acetylation take place on the same residue of 157-159. the enzyme. this strengthened an assumption of an CHASEJ. F. A. & TC’BBS P. K. (1970) Biocheni. J . 116. active acetylation of a histidine residue on the 7 I 3-720. enzyme. The low stability of the acetylated interme- CHWGA. E. (1977) A r c h . B i o c h i . Biophys. 152. 125-135. diate of ChAT. either in the immobilized form or after CLELAND W. W. (1963) Biockim. biophys. Acra 67. 104137. elution by cysteine from the column. could be CLATRECASAS P. (IY70) J . hiol. Chum. 245. 3059-3065. S . F. & MALTNERH. (1974) Proc. tiam. Acad. explained in terms of an unstable acetyl-histidine deri- CLRRIFR Sci. U.S.A. 71. 3355-3358. vative. Such a derivative is known to undergo sponDAVIS B. J. (1964) Ann. N.Y. Acad. Sci. 121. 404-427. taneous hydrolysis under the conditions used and D. & FONNUM F. (1974). more easily at pH 7.1 than at pH 6.0 (JENCKS,1969). EMSON P. C.. MALTHE-SORENSSEN J . Neurochem. 22. 1089-1098. According to the results, acetylation of choline seems FOUNUM F. (1966) Biochrnt. J . 100. 479-484. to proceed through an acetylation of histidine. sug- FOSSLMF. (1969) Bioclirni. J . 113. 291-298. gesting that the reaction occurs by a nucleophilic FONSLMF. (1975) J . Nrurochmi. 24. 407409. attack on acetyl-CoA by histidine. Previous results GLAZER A. N . (1970) A n n . Rrr. Biuchern. 39. 101-130. by BURT& SILVER (1973) and HERRe l a / . (1975) sup- GI.OVI R V. A . S. & POTTFR L. T. (1971) J . Nrurochcwl. 18. 571-5YO. port this assumption. They have demonstrated the

Mechanism of choline acetyltransferase

R., KtDtRN 0. & KATSHALSKI E. (1971) Bio10. 165-1 72. GOLIXTEIN L.. LEVINY. & KATSHALSKI E. (1964) Bioc7hrrTtisrr.y. Eu.rtott 3. 1913-1919. H r m C., MANNS. P. & MEADJ. (1975) Biochrm. Pharmac. 24. 1007-1011. HENDERSON G . I. & RAMASASTRY B. V. (1972) Fedit. Proc. Fedrr. hi.Socs. e s p . B i d . 31. 516. HOMBY W. E.. LILLYM. D. & CROOKE. M. (1968) Bioi . 1 w i t . J . 107. 669- 670. JMCKS W. P. (1969) in Curalysis in Chrmisrry and Enzymology pp. 42-111. McGraw-Hill, New York. KASCHF. V.. LUNDQClST H. & BERGMAN R. (1971) Biochem. hiop/lJx Res. Conimurt. 45. 615-621. LOWRI' 0. H., ROSEBROLGHN. J.. FARRA. L., RANDALL R . J. (1951) J . bid. Chrrn. 193. 265-275. MANNERVIK B. & SGRBO B. (1970) Biocketn. Phtrrtnuc. 19. 2509-25 16. MILESE. W. & KUMAGAI H. (1974) J . hid. Chetn. 249. 2843 ~2851. MORKlS D. & GRLWAAL D. s. (1971) Eur. J . Biochem. 22. GOLDMAN

~ / t c , ~ t t i ~ r Enstort rl~,

563 512.

88 I

MOKKIS D.. HEBBC. & BULLG. (1966) Nature. Lord. 209. 91 4-91 5. MORRISD., M A N E C K JA. E ~& HEBBC. (1971) BioCIiPttt. J 125. 857-866. MORRISON J. F. & HEYDEE. (1972) .4. Rev. Biocherii. 41. 29-54. POTTERL. T. & MURPHYW. (1967) Biocheni. P/lur,rtuc. 16. 13861388. POTTERL. T.. GLOVERV. A. S.. SALLENS J . K. (1968) J. hid. Clirm. 243. 3864-3870. RAMA SASTRYB. V. & HENDERSON G. I. (1972) ~ i 0 C h C ~ Phannuc. 21. 787-802. ROSKOSKIR. JR.(1973) Biochemisrry, Easrort 12. 3709-.3714. R., JR. (1974) J. biol. Cltem. 249. 2156-2159. ROSKOSKI SCHNEIDER J. F. & WESTLEY J. (1969) J . hiol. Chevt. 244. 5735-5744. T. C. & STARKC. R. (1970) J. h i d Chriir. 245. VANAMAN 3565-3573. WHIT^ H. L. & CAVALLITO C. J . (1970) Biochim. hioph!,r Acru 206. 343-358. WHlTF H . L. & WL F. c.(1973) J . N e u r o c k m . 20. 297 307.

l .

Choline acetyltransferase--evidence for acetyl transfer by a histidine residue.

Joiirnai (?i ,A"worhunzisrry. 1976. Vol. 21. pp. 873-881. Pergarnon Preys Printed i n Great Britam CHOLINE ACETYLTRANSFERASE-EVIDENCE FOR ACETYL TRAN...
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