Journal of Neurochemistry. 1977. Vol. 29, pp. 433-437. Pergamon Press. Printed in Great Britain.

THE SYNTHESIS OF ACETYLCHOLINE FROM ACETYL-COA, ACETYL-DEPHOSPHO-COA AND ACETYLPANTETHEINE PHOSPHATE BY CHOLINE ACETYLTRANSFERASE HEATHER BANNS,CATHERINE HEBBand S. P. MAN" A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT (Received 14 December 1976. Revised 23 February 1977. Accepted 4 March 1977)

Abstract-The synthesis of ACh by choline acetyltransferase (ChAc) has been examined using acetylCoA, acetyl-dephospho-CoA and acetylpantetheine phosphate. At pH 7.5 K, values of 25.7 PM for acetyl-CoA, 54.8 PM for acetyl-dephospho-CoA and 382 PM for acetylpantetheine phosphate were obtained and are similar to those at pH 6.0. This indicates that the 3-phosphate may not be required for binding the substrate to the enzyme unlike carnitine acetyltransferase. for CoA, dephospho-CoA and pantetheine phosphate were also measured Inhibitor constants (Ki) and when considered with the K , values obtained for the acetyl derivatives it is concluded that acetyldephospho-CoA could be a successful acetyl donor in the synthesis of ACh. Acetyl-dephospho-CoA was found to be less satisfactory as a substrate for citrate synthase.

THESUBSTRATE specificity of the enzyme choline acetyltransferase for choline analogues has been repeatedly examined. However, there is less information on the specificity for analogues of the other substrate acetyl-coenzyme A. Preliminary work in this laboratory has indicated that the acetyl-derivatives of two

ana kindly donated the pantetheine phosphate. All other materials were of the highest purity obtainable. Methods

Assay of choline acetyltransferase. The synthesis of ACh by choline acetyltransferase was measured radiometrically CoA precursors, dephospho-CoA and pantetheine using the method previously described by HEBB et a!. phosphate, may be suitable substrates for the acetyl- (1975). The incubation medium consisted of (final concentrations) 33 mM-Na phosphate buffer pH 7.5 or 6.4,0.5 mMtransfer reaction (unpublished results). EDTA, 0.13 mM-eserine sulphate, 20 mM-NaC1 (Na' final In the present work the velocity constants of the concentration approx. 100 mM); the incubation was conacetyl derivatives and the inhibitor constants of the tinued at 39°C for 10 min. Choline concentration was norprecursors have been determined. Similarly these con- mally saturating (12.5 mM) while the concentrations of stants have been measured for a second acetyltrans- [l-'4C]acetyl-CoA, [1-'4C]acetyl-dephospho-CoA and phosphate were varied as ferring enzyme, citrate synthase. The results obtained [1-'4C]acetylpantetheine are compared with those obtained by CHASE(1967) required. The enzyme used was prepared from acetone powders for carnitine acetyltransferase. The significance of the results reported here in elu- of rat brain as described by MANN& HEBB(1975); the equivalent of 1 mg dry powder was used in each incubacidating the substrate binding mechanism and the tion. Control samples contained either no enzyme or possibility that alternative substrates for acetyl- enzyme that had been inactivated by boiling. transfer reactions may exist in uiuo are discussed. Separation of labelled ACh from the labelled substrate was achieved on columns of Zerolit FFIP resin (10 x 0.63cm) as described by HEBB et al. (1975). The MATERIALS AND METHODS amount of label appearing in the blank when acetylMaterials dephospho-CoA or acetylpantetheine phosphate was used, Eserine sulphate and choline chloride were obtained was greater than with acetyl-CoA, but even with acetylpanfrom British Drug Houses, Poole, U.K.; bovine plasma tetheine phosphate, which has the smallest negative charge albumin was a product of the Armour Pharmaceutical Co., of the three substrates, the number of counts in the control Eastbourne, U.K., and Zerolit FFIP resin SRA63 was sup- samples was less than 0.7% of the counts added to the plied by Permutit Co., 652 London Road, Isleworth, U.K.; columns. DTNB, cis-oxaloacetic acid and Tris were products of Preparation of [1-14C]acetyl derivatives of dephosphoSigma London Chemical Co. Ltd., Kingston-upon- CoA and pantetheine phosphate. Labelled acetyl derivatives Thames. The citrate synthase (from pig heart) was obtained of dephospho-CoA and pantetheine phosphate were preas a crystalline product suspended (2 mg/ml) in a solution pared using the original method described by SIMON& of ammonium sulphate from Boehringer, CoA and dephos- SHEMIN(1953). A small quantity of the compound to be pho-CoA were also Boehringer products. Dr. H. G . Khor- acetylated was reacted with [l-'4C]acetic anhydride and then with cold acetic anhydride, the resulting acetylated derivative was purified on a DEAE-cellulose column as To whom correspondence should be addressed. 433

HEATHER BANNS. CATHERINE HEBBand S . P. MANN

434

tent for some experiments the final Na' concentration was adjusted to l O O r n ~ by adding 20 mM-NaC1 for all routine observations.

I80

Determination of K, for acetyl-CoA, ucetyl-dephosphoC o A and acrtylpuntetheine phosphate with ChAc I50

Michaelis constants were obtained in two ways: first, initial velocities were measured for varying substrate concentrations. The values obtained were plotted using the double reciprocal plots of LINEWEAVER & BURK (1934) and gave intersecting lines characteristic of enzymes with sequential mechanisms (Fig. 2 a and b). Michaelis constants were then obtained by replotting the values for V,,,, obtained from the Lineweaver-Burk plots, against l/S thus obtaining the K , at saturating values of the second substrate

f

m

E

p

120

0)

. 2

E

U

3

90

0

g

f

-0

60

s

I a Ace t y I-dephospho - CoA 30

1 100

I 200

I 300

I

I

400

500

(NaCO rnM FIG.1. The effect of Na ' concentration on choline acetyltransferase activity. ChAc activity was measured in the presence of increasing NaCl concentration at saturating substrate concentrations; 0 acetyl-CoA, acetyl-dephospho-CoA. A acetylpantetheine phosphate. previously described for acetyl-CoA (Hebb et al. 1975). The elution pattern of acetyl-dephospho-CoAand of acetylpantetheine phosphate was different from that observed with acetyl-CoA. Acetyl-dephospho-CoA eluted earlier than acetyl-CoA while acetylpantetheine phosphate eluted before the acetyl-dephospho-CoA.This was expected since these two compounds are less negatively charged than acetyl-COA. Assay of citratr synthase. The citrate synthase was assayed using the spectrophotometric method described by CHASE (1967). In this DTNB is used to react with the CoA released when the enzyme transfers an acetyl group from acetyl-CoA to oxaloacetic acid to form citrate. The reaction of the -SH- group with DTNB causes an increase in absorbance at 412 nm which is measured on a recording spectrophotometer at 39°C. The final incubation mixture of 3ml contained (final concentration) 100 mM-Tris-HC1 buffer pH 7.8, 1 mM-DTNB, 1 mhi-oxaloacetic acid and 25-250 ng of citrate synthase.

I

(acetyl- dephospho-Co A )

I

mM-'

(b) Acetylpontetheine phosphate

/.

RESULTS

Optimal Na' concentration for ChAc The addition of NaCl to the incubation medium increased the activity of ChAc with all three substrates (see Fig. 1). However, in the presence of 12.5 mM-chohe the concentration at which activity decreased due to the competition between Na' and choline for the choline binding site (see MANN & HEBB, 1975) was lower with acetyl-dephospho-CoA and acetylpantetheine phosphate as substrates. Because there was a need to reduce the choline con-

I

(ocetylpontetheine phosphate)

rn M -1

FIG. 2. Lineweaver-Burk plots for choline acetyltransferase. Enzyme activity was measured at variable substrate concentrations. Choline concentrations were as follows :0 0.0625 mM; 0.125 mM; A 0.25 mM; 0 0.5 mM

Synthesis of ACh by choline acetyltransferase

435

t K,,, 25.7 700

A

-

/

A

2.5

3.0

Vrnaa799

500

E

300-

Vmoa

2

756

0

0.5

1.5

1.0

I

2.0 pM-1

( Acetyl-Con)

FIG. 4. Inhibition of choline acetyltransferase by dephospho-CoA. ChAc activity was measured at saturating choline and varying acetyl-CoA concentrations in the presence of dephospho-CoA at 2 3 0 p ~0, 115 p~ A, 5 7 p W, ~ and

'A I 0

100

I 200

I 300

I 400

(Substrate) ,UM

2 9 p ~0. 3. Michaelis constants for choline acetyltransferase. Michaelis constants and maximal velocities for acetyl-CoA Determination of product inhibition by CoA, dephos0, acetyl-dephospho-CoA W, and acetylpantetheine phosphate A were calculated according to BLISS(1970) at satu- pho-CoA and pantetheine phosphate rating choline concentration. All three compounds were inhibitors of ChAc and were competitive with acetyl-CoA; Pig. 4 shows the (for an example see MORRISet a!., 1971). Constants inhibition of ChAc by dephospho-CoA. Inhibitor conwere also calculated from data obtained by incubat- stants, Ki, for the three compounds were determined ing a wide range of substrate concentrations in the graphically by the method of Dixon (DIXON& WEBB, presence of a saturating concentration of choline 1964) (Fig. 5), the values obtained are given in Table (12.5 RIM). The figures when plotted gave the rectan- lb. There is no difference between CoA and dephosgular hyperbolas shown in Fig. 3. By computing the pho-CoA but pantetheine phosphate is a much less potent inhibitor. This agrees well with the K , values data according to BLISS(1970) values for K , and V,,, obtained for the acetyl derivatives of these comwith standard errors were obtained. The constants obtained in this way for the three pounds. substrates at pH 7.5 and 6.4 are given in Table la. Determination of K, for acetyl-CoA and acetyl-dephosThere is no measurable difference between the values pho-CoA with citrate synthase obtained at these two differing pH values. The K , Data obtained by the spectrophotometric method for acetyl-dephospho-CoA was slightly greater than for acetyl-CoA as was the V,,,. The K , for acetylpan- were processed according to the method of BLISS tetheine phosphate was much greater than for the (1970). The values obtained are shown in Table 2; other two substrates although the V,, remained the The K , for acetyl-dephospho-CoA is 50 times greater same as that for acetyl-CoA. There was no difference than that for acetyl-CoA and although the V,, is between the values obtained for V, at pH 6.4 and slightly greater with dephospho-CoA the change is small compared with the difference in K,. It was not those obtained at p H 7.5. FIG.

TABLE1.

VELOCITY AND INHIBITOR CONSTANTS FOR CHOLINE ACETYLTRANSFERASE

(a) Velocity constants Acetyl-CoA Acetyl-dephospho-CoA Acetylpantetheine phosphate (b) Inhibitor constants at pH 7.5 and acetyl-CoA as substrate

pH 7.5

K*

25.7 _+ 2.3 54.8 f 7.8 382.0 46.1

* 1 ml enzyme contains 50mg acetone powder.

V,,, (nmol/ml enzyme h - ')*

pH 6.4

pH 7.5

pH 6.4

22.0 f 1.0 61.9 f 8.6 380.6 5 53.8

799 f 15.0 1073 5 41.6 756 4.2

785 & 6.5 1185 46.5 738 k 60.8

CoA

dephospho-CoA

80 pM

88 pM

pantetheine phosphate 416 p~

HEATHER BANNS.CATHERINE HEBBand S. P. MANN

436

lor

2t

1; 4 u

1

0

I

125

[Pantetheine phosphate]

I 250

I

I

375

500

mY

FIG.5. Dixon plot of choline acetyltransferase inhibition by pantetheine phosphate. ChAc activity was measured at 16.5 LIM 0, 33 p~ A, and 100 W M 0 acetyl-CoA in the pres-

ence of

increasing concentrations of phosphate.

pantetheine

possible, by the present assay method, to measure any activity with acetylpantetheine phosphate. DISCUSSION

The transfer of acetyl groups from acetyl-dephospho-CoA has already been examined by CHASE(1967) who observed that the maximal velocity of the reaction in which acetylcarnitine is formed by carnitine acetyltransferase was the same when acetyl-dephospho-CoA was substituted for acetyl-CoA. The K , of acetyl-CoA, however, was 3 4 p ~as opposed to 1300 PM for acetyl-dephospho-CoA. Chase also observed that although K , for acetyl-dephospho-CoA remained constant from pH 6.1 to 7.4 the K , for acetyl-CoA changed as the pH approached 6.4 which is the ionizing pH of the 3-phosphate group on CoA. From this he concluded it was likely that this was responsible for binding to one site in the active centre. It can be seen from Table la, however, that there is no significant difference between the values for K , obtained at pH 7.5 and at 6.4 for ChAc. This must indicate that the 3-phosphate group, which is present in CoA but absent in dephospho-CoA and pantetheine phosphate. is not an active part of the process which binds the substrate to the enzyme. In the present work acetyl-dephospho-CoA and acetylpantetheine phosphate have been shown to be substrates for ChAc with maximal velocities equal to that observed with acetyl-CoA. A comparison between acetyl-CoA, and acetyl-dephospho-CoA

shows that the K , for the former is approximately half that for the latter. This difference may simply reflect a change in the K , caused by the increased V,,, observed with acetyl-dephospho-CoA. This is borne out by the inhibitor studies where the K i for CoA is the same as that for dephospho-CoA. The considerable difference in K , between acetylCoA and acetyl-pantetheine phosphate also indicates that the adenosine part of the CoA or dephosphoCoA molecule must play an important part in the binding of the substrate to the enzyme in addition to the binding of the pantetheine residue. It is interesting that the difference between the constants obtained for the first substrate and its product (i.e. K , acetyl-CoA 25-50 PM and K , CoA 8&88 p ~ ) is small when compared with those obtained for the second substrate and its product ( K , choline 290 p~ and K i ACh 2 2 . 5 m ~ ;MANS & HEBB.1975). This may indicate that the acetyl group of acetyl-CoA and the -SHgroup of CoA have little or no binding effect compared with the --OH of choline. From these observations it can be seen that acetyldephospho-CoA could be a suitable substrate for the synthesis of ACh in viuo. The results with citrate synthase, however. indicate that little acetyl-dephosphoCoA would be formed by this enzyme either inside or outside the mitochondrion unless the concentration of dephospho-CoA was greatly in excess of that of the CoA. There is little evidence to suggest that large quantities of dephospho-CoA are present in tissues, although some will be present since dephospho-CoA is the precursor of CoA. Dephospho-CoA is phosphorylated to become CoA by the enzyme dephospho-CoA kinase (EC 2.7.1.24) as the last step in the biological synthesis of CoA (HOAGLAND& NOVELLI, 1954). Carnitine acetyltransferase like citrate synthase is a mitochondria1 enzyme and both would appear to need the 3-phosphate group of CoA to bind the substrate to the enzyme. Choline acetyltransferase is an extramitochondrial enzyme and although many workers have examined the source of the acetyl group in the ACh molecule (see HEBB, 1972; TUCEK& CHENG,1974) the means by which the active acetylgroups are transferred from the mitochondria to the cytoplasm for the synthesis of ACh remains unresolved. Acknowledgements-The authors wish to express their gratitude to Dr. H. G . KHORANA for the gift of pantetheine phosphate which was referred to i n the text. and to Dr.

TABLE2. VELOCITYCONSTANTS FOR

CITRATE SYNTHASE

K,pH 7.8 (PM)

Acetyl-CoA Acetyl-dephospho-CoA Acetylpantetheine phosphate

2.0 110.8

0.52

+ 9.42

V,,,. PH 7.8 (pmoi min-' mg enzyme-') 40 3.24 54 f 3.43

No detectable activity

(NB Citrate synthase has an activity of approx. 110 pmol/mg at 25°C. Present estimations like those for ChAc were carried out at 39°C).

Synthesis of ACh by choline acetyltransferase J. PATERWN for advice on the statistical treatment of some of the data. The work was partly funded by a grant to C.O.H. from the Wellcome Trust, whose help she gratefully acknowledges.

REFERENCES BLISSC. I. (1970) Statistics in Biology. McGraw-Hill, New York. CHASE.I. F. (1967) Biochem. J. 104, 503-509. DIXONM. & WEBBE. C. (1964) Enzymes, 2nd Ed. Longmans. London.

431

HEBBC. (1972) Physiol. Rev. 52, 918-957. HEBBC., MANNS. P. & MEADJ. (1975) Biochem. Pharmac. 24, 1007-1011. HOAGLAND M. B. & NOVELLIG. D. (1954) J. b i d . Chem. 261, 767-773. LINEWEAVER H. & BURKD. (1934) J . Am. chem. Soc. 56, 658- 666. MANNS. P. & HEBBC. (1975) Biochem. Pharmnc. 24, 1013- 1017. A. & HEBBC. (1971) Biochem. J . MORRISD., MANECKJEE 125. 857-863. SlMON E. J. & SHEMIND. (1953) J. Am. chem. Soc. 75, 2250. TUEEKs. & CHENG S.-C. (1974) J . Neurochem. 22, 893-914.