P h e n y l a c e t y l C o e n z y m e A H y d r o l a s e
Phenylaeetyl coenzyme A + H~O--~ phenylaeetie acid + eoenzyme A An enzyme catalyzing this reaction has been recognized in extracts of a member (Wis 51-20 F~) of the "Wisconsin Family" of high penicillinyielding mutants of Penicillium chrysogenum and in a number of related mutants?
Assay M e t h o d
Principle. [1-~4C]Phenylacetyl coenzyme A is incubated at 37 ° with P. chrysogenum extract as well as buffer, EDTA, and a sulfhydryl compound. The reaction is stopped by adding an excess of citrate buffer, pH 5.0, and the mixture is chromatographed on paper. The radioactivity of the spot corresponding to [1-14C]phenylacetic acid is counted by liquid scintillation and quantitated by reference to the radioactivity of standard [ 1-~4C] phenylacetyl CoA.
Reagents [1-14C]Phenylacetyl coenzyme A is prepared by the mixed anhydride method of Stadtman. 2 To 50 ~Ci of [1-14C]phenylacetic acid (39 mCi/mmole, The Radiochemical Centre, Amersham) is added 8.9 mg of phenylacetic acid, and the mixture is dissolved in 400 ~l of ether. After addition of 6,1 ~l of pyridine the mixture is cooled in ice and 7 ~l of ethylchloroformate is added slowly with agitation. During standing for 1 hr in ice, the tube is occasionally twirled by hand, causing the precipitated pyridinium chloride to stick to the sides of the vessel. The clear supernatant is pipetted off and added to a solution of 33.8 mg coenzyme A. (Sigma Chemical Co. Grade 1) in 1 ml of water, which has been adjusted to pH 7.6 with solid potassium bicarbonate. The tube containing the mixture is gassed with nitrogen and shaken for 30 rain. The solution is then adjusted to pH 2.0 with HC1 and extracted 4 times with 1.5 ml ether. The remaining ether is evaporated off by a stream of nitrogen. The solution is adjusted to pH 7.0 with solid lB. Spencer and Chit Maung, Biochem. J. 118, 29P (1970). E.R. Stadtman, this series, Vol. 3, p. 931.
PHENYLACETYL COENZYME A HYDROLASE
potassium carbonate and distributed in 100-t~l portions in small tubes for storage at --20 ° . Radioactivity is counted by liquid scintillation and thiol ester content is assayed by the DTNB method of Elhnan 3 scaled down to deal with 40 t~l of diluted (1/9 v/v) sample. The yield is 1 ml of 35 mM [1-14C]phenylacetyl coenzyme A, and 10~1 spotted on paper give 450,000 epm measured as described below. Potassium phosphate buffer, 0.1 M, pH 7.5, containing 1 mM EDTA and 10 mM glutathione or N-acetylcysteine Citrate buffer, pH 5.0, 10% (w/v) Ethanol~n-butanol~28% (w/v) ammonium sulfate (2:1:1 by volume) 4 Whatman No. 1 chromatography paper that has been wetted with potassium phosphate buffer, pH 6.0 (75 g/liter of KH2P04, 25 g/liter of K~HPO~), blotted to remove excess buffer and dried 5 2,5-Diphenyloxazole (PP0), 5 g, and of 2,2-p-phenylenebis(5phenyloxazole) (POPOP), 0.3 g, dissolved in 1 liter of toluene
Assay of the Hydrolase. The enzyme preparation, 20 td, is incubated with l0 t~l of 24 mM [l-14C]phenylacetyl CoA and 10 t~l of 0.1 M potassimn phosphate buffer, pH 7.5 containing 1 mM EDTA and 10 mM glutathione. In controls the enzyme preparation nmst be replaced by the buffer-sulfhydryl mixture in which the enzyme sample is contained and the controls must be incubated along with the enzyme reaction tubes. After incubation for 15 rain, the reaction is stopped by adding 20 ~1 of 10% (v/v) citrate buffer, pH 5.0. Portions (10 ul) of the treated reaction mixtures including controls are chromatographed for 5-10 hr by descending chromatography on treated Whatman No. 1 paper using the ethanol/ n-butanol/28% ammonium sulfate solvent. The spot at RI 0.65, corresponding to ['~CIphenylacetic acid, is cut out, placed in a glass vial with 10-12 ml of the scintillation fluid and counted for sufficient time (usually 2-5 rain) in a Packard Tri-Carb liquid scintillation spectrometer Model 3375 to obtain a count rate with an SE. of less than 1%. The count rate is quantitated by reference to the radioactivity of standard amounts of [1-~C]phenylacetyl CoA which are spotted onto a piece of chromatography paper. The assay is linear with enzyme concentration and with time only over 20 rain. C. Ellman, Arch. Biochem. Biophys. 82, 70 (1959). 4 p. L. Tardrev and M. J. Johnson, J. Bacteriol. 76, 400 (1958). D. L. Pruess and M. J. Johnson, g. Bacteriol. 94, 1502 (1967).
A unit of phenylacetyl CoA hydrolase is defined in the standard manner as that amount which hydrolyzes 1 ~mole of the substrate per minute.
Production and Purification
Culture on Agar Slopes. Penicillium chrysoqenum Wis 51-20F3 is maintained as a spore suspension in soil and subcultured on tomato juice agar [canned tomato juice adjusted to pH 6 with 1 N NaOH and diluted 1:1 (v/v) with 2.5% (w/v) agar solution (Difco Bacto) ]. Shake Cultures. Inoculations are carried out with fresh spore suspension prepared by washing spores from a tomato-agar slope with 5 ml of sterile deionized water containing 0.1% (v/v) Tween 80. Five milliliters of this spore suspension are used to inoculate 250-ml Erlenmeyer flasks containing 40 ml of the following sterile glucose-lactate salts medium (in g/liter): D-glucose, 40.0; ammonium lactate, 21.0; KH:PO~, 3.0; Na2SO~, 0.74; magnesium acetate, 0.25; ZnCl~, 0.02; CaCO3, 13.0; MnCl~. 4H20, 0.02; FeCl~ • 6H20, 0.02; and CuCl~ 2H~_O,0.005. The glucose/ lactate is sterilized separately from the salts solution. The inoculated flasks are shaken for 2 days at 25 ° and 250 rpm on a gyratory shaker. The preculture (40 ml) is used to inoculate l-liter Erlenmeyer flasks containing 400 ml of sterilized fermentation medium composed of (g/l) : lactose, 50; corn liquor, 50; Na2SO~, 1. Growth is carried out at 28 ° on a gyratory shaker (250 rpm). At 24 hr, 1 ml of lard oil containing 5% v / v Tween 80 is added to prevent foaming. After 3 days growth, the mycelium is harvested by filtering through a double layer of cheesecloth and washed thoroughly with cold water. The mycelium is then pressed between filter papers to remove excess water and is used either immediately or after storage at --20 °. A yield of 15-25 g of "pressed-dry" mycelium per flask is obtained.
Step 1. Ten grams of press-dried mycelium is suspended in 40 ml of ice-cold 50 mM potassium phosphate buffer, pH 7.6 (containing 5 mM dithiothreitol and 1 mM EDTA), and ground lightly in a cooled mortar for about 2 min. The mixture is then extruded twice in a cooled French press at 5000-7000 psi. The pH of the extract is adjusted to pH 7.6 and mycelial debris is removed by centrifugation. The resulting supernatant, 38-40 ml, has a protein concentration of 15-20 mg/ml.
PHENYLACETYL COENZYME A HYDROLASE
Alternatively, the mycelium can be ground with buffer and sand until a thin paste is given and then centrifuged. The volume of supernatant is less and the protein concentration about half that achieved using the French press. Step 2. Solid ammonium sulfate is added to a supernatant solution to a concentration of 209 g/liter (35% saturation). The precipitate is removed by centrifugation, and further ammonium sulfate is added to a final concentration of 313 g/liter (50% saturation). The precipitate is collected by centrifugation and dissolved in 2 ml of 50 mM phosphate buffer, pH 7.5, containing 1 mM D T T and 1 mM EDTA. The enzyme at this stage is stable for several weeks at --20% Step 3. The 35-50% ammonium sulfate fraction (2.5 ml) is desalted on a Sephadex G-25 (coarse) column which has been equilibrated with 50 mM phosphate buffer pH 7.5 containing 1 mM D T T and 1 mM EDTA, the elution being carried out with the same buffer. A DEAE-cellulose column (0.9 }( 12.5 cm) is equilibrated with 50 mM phosphate buffer pH 7.5 containing 1 mM D T T and 1 mM EDTA. The desalted extract (12 ml) is absorbed onto the colmnn which is then washed with 20 ml of the equilibrating buffer. The enzyme is eluted from the column using the equilibrating buffer containing 0.1 M KC1. After discarding the void volume, the next 6 ml are collected. Step 4. A Sephadex G-100 column (1.2 X 75 cm), Vo 34 ml, flow rate 20-25 ml/hr, is first equilibrated with 50 mM phosphate buffer pH 7.5 containing 1 mM DTT and 1 mM EDTA. The DEAE fraction (6 ml) is applied to the column and eluted with the same buffer. Two-milliliter fractions are collected, and the most active fractions (fraction numbers 27-31) are pooled (10 ml). The pooled fractions from step 4 represent a 130-fold purification from step 1 with recovery of 25% of the activity. The specific activity is 0.15 unit per milligram of protein.
Properties of the Purified Enzyme
Specificity. It has been suggested 1 that phenylacetyl CoA hydrolase is only one of four activities attributable to a single thiol-dependent enzyme whose action involves a Ping-Pong Bi Bi mechanism with an alternate hydrolytic step. The other activities are penicillin acyltransferase, 6-aminopenicillanic acid acyltransferase, and penicillin acylase. The evidence is based on the constant ratio between the activities during various fractionation and purification procedures, inhibition, and pH and temperature inactivation. In the crude extract (step 1) the ratio of phenylacetylCoA hydrolase to the other activities is higher than in the purified ex-
tracts, suggesting the presence of nonspecific enzymes at this stage which can hydrolyze phenylacetyl-CoA. The enzyme is approximately 5.0 times as active against phenoxyacetyl-CoA as compared to phenylacetyl-CoA, 6 and this compares to a similar ratio between the acylase activity of the preparation in hydrolyzing penicillins V and G. The purified preparation also hydrolyzes p-nitrophenyl acetate and phenoxyacetyl glycine at about the same rate as the hydrolysis of phenylacety]-CoA. The ratio of these various activities is the same in steps 3 and 4, but there is no other indication to confirm that the same enzyme is responsible. Other Properties. The hydrolase activity shows a pH optimum of 7.6-7.8 and a Km of 3.95 raM. The molecular weight by Sephadex gel filtration 7 is 25,000. The enzyme is sensitive to sulfhydryl inhibitors and is completely inhibited by pretreatment wth 2 mM N-ethylmaleimide, 2 mM dithiobis(2-nitrobenzoic acid), and 2 mM p-mercuribenzoate. The inhibition is reversible by subsequent addition of excess thiol compounds. The enzyme is sensitive to oxidation during the purification procedure, and it is necessary to keep the enzyme in a reduced state by the presence of thiol compounds. The oxidized enzyme fractionates differently on DEAE-cellulose. Free thiol compounds are not involved in the mechanism of action, and when they are removed by passing the enzyme preparation through Sephadex G-25 and thiols are omitted from the assay mixture, some activity is still observed. However, thiol compounds need to be present during the assay for maximum activity. Despite the presence of 1 mM D T T and 1 mM EDTA the purified preparation is not stable, and about 50% of the activity is lost during storage at 0 ° for 24 hr. Comments on the Assay Procedure. The necessity for free thiol during assay introduces complications due to a rapid S -~ S intermolecular acyl migration between phenylacetic acid and the thiol that occurs at pH above 6.5 and which leads to the elimination of free CoA. When the added thiol does not contain NH.. or OH groups proximal to the SH group (e.g., N-acetyl cysteine, thioglycolic acid, glutathione), the acyl group simply equilibrates between CoA and the added thiol, the rate of equilibration increasing with the pH. When proximal NH2 or OH groups are present in the thiol (e.g., DTT, DTE, cysteine, cysteamine, mercaptoethanol), the S-> S acyl migration proceeds to completion owing to subsequent S--> N and S--> O intramolecular acyl transfer. Further reactions can Chit Maung, Ph.D. Thesis, Dublin University, Ireland, 1970. 7p. Andrews, Biochem. J. 91, 22"2(1964).
ERYTHROMYCIN C 0-METHYLTRANSFERASE
include elimination of free [14C]phenylacetic acid and the formation of cyclic compounds t h a t contain [1-14C]phenylacetic acid. These reactions not only alter the concentration of substrate during the reaction, but nonenzymieally liberated [1-1~C]phenylacetic acid and other products, which chromatograph at the same R~, can lead to spurious high results. By avoiding those thiols t h a t can carry out intramoleeular S --> 0 and S ~ N aeyl migrations, interference during the assay can be limited, even at its fullest extent, to the equilibrimn position governed by the amounts of phenylacetyl-CoA and thiol used. This interference can be further limited by keeping the ratio, phenylaeetyl CoA:thiol, high and the p H and assay time low. The assay conditions recomn~en(ted take these points into consideration. In the preparation of the enzyme it is convenient to use buffer containing D T T , and the residual amounts of this compound will produce some nonenzymieally liberated [~4C~]phenylacetie acid. I t is therefore necessary for the control to contain the same buffer as t h a t in which the enzyme is dissolved and for the control to be incubated.
 S-Adenosylmethionine: Erythromycin C O-Methyltransferase By JOHN W. CORCORAN E r y t h r o m y c i n s A, B, and C, 1 the macrolide antibiotics elaborated by Streptomyces erythreus have the structures shown in Fig. 1. I t has been demonstrated t h a t the methyl groups attached to the C-3"-,3"-0, and N atoms of the sugars of the erythromycins are derived from L-methionine. -°,3 The O-methylation of the L-mycarose moiety of erythromycin C by a partially purified enzyme obtained from extracts of S. erythreus is described here. The reaction catalyzed is shown in Fig. 1Abbreviations : Ea, the lactone of erythromycin A, also called erythronolide A; D, D-desosaminyl group; M, L-mycarosyl group; C, b-cladinosyl group; EaDM, erythromycin C; EaDC, erythromycin A; EDTA, ethylenediaminetetracetic acid, disodium salt; DTT, dithiothreitol; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homoeysteine; REV, relative elution volume, defined as a ratio of the elution volume (Ve) over the bed volume (Vt). 2 j. W. Corcoran, J. Biol. Chem. 236, PC 27 (1961). 3j. Majer, M. Puza, L. Dole~ilov£, and Z. Vanek, Chem. Ind. (London) 1961, p. 669 (1961).