ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1979, p. 719-723
Vol. 16, No. 6
0066-4804/79/12-0719/05$02.00/0
Kinetic Studies on Enzymatic Acetylation of Chloramphenicol in Streptococcus faecalis YOJI NAKAGAWA, YOSHIYUKI NITAHARA, AND SADAO MIYAMURA* Department of Bacteriology, Niigata University School of Medicine, Niigata, Japan 951 Received for publication 1 October 1979
The l2inetics of chloramphenicol (CP) acetylation by CP acetyltransferase from Streptococcus faecalis was studied. CP was shown to be acetylated enzymatically to its 3-0-acetyl derivative (3-AcCP) in the presence of acetyl coenzyme A, after which 3-AcCP was converted nonenzymatically to its 1-O-acetyl isomer, 1-0acetyl CP (1-AcCP). At equilibrium, the 1-AcCP and 3-AcCP were present in a 1:4 ratio. Subsequently the diacetylated product, 1,3-0-0-diacetyl CP [1,3(Ac)2CP], was enzymatically produced from 1-AcCP by the same enzyme. Theoretical calculation of rate constants (k1, k2, k3) for each successive reaction is as follows: k3 ki k2 CP-*3-AcCP--* 1-AcCP-_ 1,3-(Ac)2CP This calculation gave k, = 0.4 min-', k2 = 0.002 min-', and k3 = 0.016 min-'. Experimental results agreed closely with these calculated values.
Miyamura noted chloramphenicol (CP) inactivation in cultures of CP-resistant bacteria carrying R factor and suggested that the phenomenon was related to the mechanism of CP resistance (3, 4). Suzuki (12) and Shaw and coworkers (8-10) reported that CP inactivation was due to enzymatic acetylation of CP by chloramphenical acetyltransferase (CAT) in the presence of acetyl-CoA. However, specific reactions involved in acetylation have not been elucidated. In this connection, it was interesting to study the correlation between 3-O-acetyl CP (3-AcCP) and 1-0-acetyl CP (1-AcCP) and the mechanisms for formation of diacetylated CP from monoacetylated CP. The present studies were undertaken to clarify the mechanism of the acetylation of CP by an enzyme isolated from Streptococcus faecalis (5). MATERIALS AND METHODS Bacterial strain. S. faecalis N-117, isolated from the urine of a cystitis patient in the Niigata University Hospital, was used as the source of enzyme (CAT). The minimal inhibitory concentration of CP for this
strain was 125 pg/mI. Extraction and purification of the inactivating enzyme. S. faecalis N-117 was grown for 24 h at 370C with shaking in heart infusion broth containing 1% glucose and 10 Ag of CP per ml. Bacterial cells were harvested by centrifugation at 8,000 rpm for 20 min, washed twice with 0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.8), resuspended in the same buffer, and disrupted by a cell homogenizer (Braun Co.) for 10 min. Five milliliters of cell-free protein extract was added to an affinity
chromatography column, and the column was washed at 40C with 50 mM Tris-hydrochloride buffer (buffer A) containing 0.5 M NaCl, followed by 1 M NaCl, until no further ultraviolet-absorbing material was eluted. The enzyme was then eluted with buffer A containing 1 mg of CP per ml. The flow rate was 4 ml/h; 2-ml fractions were collected. CAT activity was determined by the spectrophotometric assay, which utilizes the reduction of 5,5'-dithiobis-2-nitrobenzoic acid by reduced acetyl coenzyme A (acetyl-CoA) (1, 5, 11). The enzyme was separated from CP by passing it through a Sephadex G-25 column. Fractions containing CAT were pooled and stored at -20°C. The protein content of the enzyme preparation used in these studies was 152 ,ug/ml as determined by the method of Lowry et al. (2). The specific activity of the electrophoretically homogeneous preparation was 74.5 units per,g of protein. This enzyme preparation was used in the following experiments unless otherwise specified. Radioisotope assay of CP acetylation. CP and its derivatives were extracted from the reaction mixture by ethyl acetate as described by Shaw (9). After three successive extractions, the total recovery of both CP and its derivatives was about 90%. A portion of the extract was then spotted on a silica gel thin-layer plate (Merck Silica Gel 60 Sheet) and submitted to chromatography with the solvent described below. CP and its products were located by an ultraviolet lamp and extracted three times with ethyl acetate by scraping from the appropriate area. The radioactivity was measured quantitatively with a liquid scintillation counter. The yield of CP and its derivatives was calculated from the known specific activity of the starting material. Thin-layer chromatography of silica gel. The thin-layer plate used was a Merck Silica Gel 60 sheet (silica gel with fluorescent indicator). The plate was 719
720
ANTimICROB. -AGENTS CHEMOTHER.
NAKAGAWA, NITAHARA, AND MIYAMURA
activated by heating it at 80°C for 15 min, before the extract from the reaction mixture was spotted on it. Development was carried out with chloroform-meth-anol (95:5) for about 60 min. Radioactivity measurement with a liquid scintillation counter. The solvent consisted of 6 g of 2,5diphenyloxazole, 0.3 g of 1, 4-bis[2-(5-phenyloxazolyl)] benzene, 450 ml of methanol, and 550 nl of toluene. The sample was dissolved in 8 ml of solvent and counted with a liquid scintillation counter (ALOKA, Liquid Scintillation Spectrometer LSC-602). Procedure for preparation of affinity chromatography column. Activated CH-Sepharose 4B swollen in 1 mM hydrochloric acid for 10 min was filtered by suction and washed with 1 mM hydrochloric acid on a glass filter. The filtered gel was treated in 0.1 M sodium bicarbonate solution containing 2 - amino-1(p-nitrophenyl) - 1,3-dihydroxypropane (7); the mixture was shaken gently at 220C for 28 h, after which the gel was filtered and washed with 50 mM Trshydrochloride buffer (pH 7.8). The gel was washed successively with 50 mM Tris-hydrochloride buffer (pH 8.0) containing 0.5 M NaCl and with 50 mM formic acid buffer (pH 4.0) containing 0.5 M NaCl. Before use the gel was equilibrated with buffer A. Enzymatic inactivation of CP. The complete reaction mixture contained 25 pxnol of Tris-hydrochloride buffer (pH 7.8) or Tris-maleate buffer (pH 7.0) and various amounts of ['4C]CP (4.87 uCi/,umol), acetyl-CoA, and enzyme in a total volume of 0.5 ml. The ratio of CP to acetyl-CoA was 1:4. The mixture was incubated at 30 or 370C for an appropriate time. A 0.1ml sample was pipetted into 1 ml of precooled ethyl acetate to terminate the reaction, and the inactivated products of CP were extracted. Chemicals. ['4C]CP (D-threo[dichloro-acetyl-1,2'4C]CP; 4.87 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. CH-Sepharose 4B and activated CH-Sepharose 4B were purchased from Pharmacia Fine Chemicals. Acetyl-CoA was purchased from P-L Biochemicals Inc. The acetyl derivatives of CP and the nonacetyl derivative, 2-
amino-1-(p-nitrophenyl)-1,3-dihydroxypropane, were synthesized in our laboratory.
RESULTS Time course of acetylation of CP and its derivatives. The acetylating enzyme from S. faecalis N-117 cells produced two monoacetyl derivatives and one diacetyl derivative of CP when incubated with CP and acetyl-CoA. In the complete system, the time course of product formation could be followed by thin-layer chromatography as shown in Fig. 1. CP was acetylated to 3-AcCP within the first minute of incubation; 3-AcCP rose sharply to a maxinum at 1 min and then declined slowly, whereas 1-AcCP increased slowly from 1 to 30 min and thereafter decreased gradually. The formation of 1,3(Ac)2CP increased markedly after the first 5 min of incubation. The data in Fig. 1 suggested the following sequence of reactions: CP -- 3-AcCP -* 1-AcCP -. 1,3-(Ac)2CP
24[ 18 (A
w
-J
0 12 B [ 0 z 4
z
6
60 01 5 15 30 MINUTES
90
FIG. 1. Kinetic studies on the formation of monoacetyl and diacetyl derivatives of CP. The complete system consisted of 50 punol of Tris-hydrochloride buffer (pH 7.8), l.(XI) umol of acetyl-CoA, 0.24 pmol of [4C]CP (4.87 yICi/Mmol), and 100 gd of enzyme protein solution in a final volume of I ml. Incubation was at 37°C. In each case, the reaction was terminated by the addition of ethyl acetate and the products were extracted as described in the text. Symbols: (O) CP; (-) 3-AcCP; (A) 1-AcCP; (A) 1.3-(AC)2CP.
Relationship between 3-AcCP and 1AcCP. Suzuki and Okanoto (12) and Shaw (9) suggested that 1-AcCP was derived from 3-AcCP nonenzymatically. To confirm this suggestion, 3AcCP separated with thin-layer chromatography was incubated in Tris-maleate buffer (pH 7.0) under conditions which minimized hydrolysis as much as possible. It is clearly shown in Fig. 2 that the 1-AcCP increased gradually while 3-AcCP decreased, and the equilibrium resulted. Diacetylation of CP. Diacetylated CP was derived from monoacetylated CP. To determine whether 1-AcCP or 3-AcCP is the direct precursor of 1,3-(Ac)2CP, the relationship of the relative concentration of both monoacetylated CPs to the formation of 1,3-(Ac)2CP was investigated. As shown in Fig. 3, the amount of 3-AcCP decreased with time, whereas 1-AcCP diminished rapidly and simultaneously 1,3-(Ac)2CP increased at the same rate for the first 20 min. When the concentration of 1-AcCP was high, a pronounced increase in diacetylation was observed. Kinetics. From the experimental data obtained the acetylation reaction of CP could be described as follows: k k3 k2 -.1,3-(Ac)2CP CP 43-AcCP -1-AcCP where k1, k2, and, k3 are rate constants. We assumed that the acetylation of CP was a successive reaction. If the iiiltial concentration of CP is a, and the concentrations of CP, 3-AcCP, I-AcCP, and 1,3-(Ac)2CP after t min are (A),
721
ACETYLATION OF CHLORAMPHENICOL IN S. FAECALIS
VOL. 16, 1979
(B), (C), and (D), respectively, the following
fornulas were derived. (a
0 0 z
5
(A) ae-k'
(1)
3
(B) ak, (e-kit -e-k2t k2- k)
(2)
.4
2
1
as
0
2
1
(Ck,
3
R(2- k,)(k
-
k,)
HOURS
FIG. 2. The conversion from 3-AcCP to 1-AcCP. 3AcCP obtained under the conditions described in Fig. 1 was dissolved in I ml of Tris-maleate buffer (pH 7.0). At the indicated time, 0.1-mi samples were taken for assay. In each case, the reaction was terminated by adding the sample to ethyl acetate, and the extracts were fractionated by thin-layer chromatography. 3-AcCP (0) and 1-AcCP (A) were quantitatively deternined by measuring their "4C activities with a scintillation counter.
+
+
a)(
(3)
(ki - k2)(k3- k2) e (k2- k3)(k, -k3)
(k3- k,)(k, - k2) +
kAk,ek2 (ki- k2)(k2- k3)
(4)
kik2e-k +
(k2 -k3)(k3 -k)
The measured values of (A), (B), (C), and (D) Lu 0
are 2-
z
O
=
60 0 10 2030 MINUTES
90
FIG. 3. Time course of acetylation reaction from monoacetylated to diacetylated CP. The reaction was stopped by extraction with ethyl acetate. The solvent was evaporated to dryness, and the residue was dissolved in Tris-hydrochloride buffer. The solution was divided into two parts, A (dotted lines) and B (solid lines). Part A was incubated immediately, and B was incubated after remaining for 3 h at 37°C. The solid lines show that the initial concentration of 1-AcCP and 3-AcCP was 4.3:20, and the dotted lines show the ratio was 1:20 after 3 h at 37°C. Symbols: (0) 3AcCP; (A) 1-AcCP; (A) 1,3-(Ac)2CP.
shown in Table 1. The rate constants (k1, k2,
k3) calculated from these values were 0.4, 0.002, and 0.016 min-', respectively. Figure 4 compares the curves of equations 1, 2, 3, and 4 by introducing the calculated rate constants from these equations, with the experimental data. The calculated and observed values of (A) and (B) were completely identical for the first 5 min. Thereafter, they remained essentially identical. Satisfactory agreement between calculated and experimental values of (D) was also observed. In the case of (C) only, a notable discrepancy was observed between experimental and calculated values. Equilibrium between 3-AcCP and 1AcCP. The mutual changes of the two monoacetyl derivatives of CP were observed on the thinlayer chromatograph (Fig. 5). By one-dimensional development (Fig. 5A), 1-AcCP was derived from 3-AcCP; by two-dimensional development (Fig. 5B), part of 1-AcCP changed into 3-AcCP.
TABLE 1. Concentrations of CP, 3-AcCP, I-AcCP, and 1,3-(Ac)2CP over time CP and CP derivative
CP 3-AcCP 1-AcCP
1,3-(Ac)2CP
Concn (nmol) at miin: 1 7.60 3.72 0.16 0.02
2 5.00 6.20 0.28 0.02
3 3.18 7.76 0.54 0.02
5 1.02 9.80 0.66 0.02
10 0.21 10.41 0.86 0.02
20 0.17 10.42 0.88 0.02
40 0.12 10.15 1.13 0.10
60 0.08 9.79 1.39 0.24
90 0.07 0.48 1.33 0.62
120 0.05 9.21 1.24 1.00
150 0.05 8.76 1.14 1.55
8 -w8 0 2 6 0
C
z
4
ANTIMICROB. AGENTS CHIZMOTHER.
NAKAGAWA, NITAHARA, AND MIYAMURA
722
4
C2
IC]01020 40
90 60 MINUTES
120
150
FIG. 4. The comparison of calculated and experimental concentrations of CP [A], 3-AcCP [B], 1AcCP [C], and 1,3-(Ac)2CP [D1. The dotted lines show the calculated values. and the solid lines show the experimental values. The reaction mixture contained 60 pumol of Tris-maleate buffer (pH 7.0), 0.60 pmol of acetyl-CoA, 0.15 ,mol of [4C]CP, and 50 IlI of enzyme in a total volume of 1.2 ml. The reaction mixture was incubated at 30°C, and 0.1-ml samples were taken for assay at the indicated times. The initial concentration of CP was 11.50 nmol. The ethyl acetate extracts were fractionated by thin-layer chromatography. Symbols: (0) CP; (0) 3-AcCP; (A) 1AcCP; (A) 1,3-(Ac)2CP.
DISCUSSION Conclusive evidence has not yet been presented for the whole course of the acetylation of CP to its diacetylated derivative by CAT. However, it may be postulated that 1,3-(Ac)2CP is derived from monoacetyl CP enzymatically. In the present work, it was confirmed that 3-AcCP formed from CP by CAT in the presence of acetyl-CoA is converted non-enzymatically to its isomer, 1-AcCP (Fig. 1 and 2). To account for the observation, one of the two schemes of the following types must be adopted. CP -. 3-ACCP -* 1-ACCP -* 1,3-(Ac)2CP (1) CP -- 3-AcCP -- 1-AcCP (2) I 1,3-(Ac)2CP
The rate of 1,3-(Ac)2CP produced depended mainly on the amount of 1-AcCP (Fig. 3), and the kinetics of product formation was related to 1-AcCP disappearance. These data strongly suggested that 1-AcCP was a direct precursor of 1,3(Ac)2CP, as shown in scheme 1. The essential agreements between calculated and observed values of each compound in scheme 1, with the exception of the values for 1-
AcCP, support this view. The exception seemed to be caused by a reverse reaction from 1-AcCP to 3-AcCP. Spontaneous conversion of 1-AcCP to 3-AcCP and vice versa occurred as shown in Tris-maleate buffer (Fig. 2) and on thin-layer chromatography plates (Fig. 5). It appeared of interest to investigate further the reversible mechanism of this reaction to establish definitive kinetics of acetylation of CP. If several different enzymes participated in the acetylation of CP to C-1 or C-3 acetyl derivatives (Fig. 6), the reaction rates of mono- and diacetylation of CP should be almost equal. However, a large difference between the rates of mono- and diacetylation was observed, indicating the existence of a single enzyme which acetylates the hydroxyl groups attached to C-3 of both CP and 1-AcCP. Some bacterial species having CAT, such as Staphylococcus and Pseudomonas, have been reported to produce no diacetylated CP (6,13). Our experiments suggest that the reported experiments may not have been carried out under optimal conditions. Moreover, the inactivated products, monoand diacetylated CP, are easily hydrolyzed to
B
A
*
3-ACCP
1-AcCP
...
...:
_.
i-AcCP 3-ACCP
FIG. 5. Autoradiographs showing the changes between 3-AcCP and I-AcCP; one development is onedimensionally, and the other is two-dimensionally. After incubation at 37°C for 3 min, the mixture was added to ethyl acetate to terminate the reaction and to extract the inactivated products. Monoacetyl CP was isolated by thin-layer chromatography. The onedimensional development is lengthwise and the twodimensional is sideways. Development was carried out with chloroform-methanol (95:5) for about 60 min. H 02N
NHCOCHC12
1 -2C-3CH2 OH H OH
FIG. 6. Structure of CP.
ACETYLATION OF CHLORAMPHENICOL IN S. FAECALIS
VOL. 16, 1979
723
3-AcCP
CP
NHCOCHC12
H
H
CATI
NHCOCHC12 I
02NC-:O H02NOCH-H -CH2
-
I-
OH
H
OH
CH2
OCOCH3
OH
H
H
NHCOCHC12
A
0
H
02N
NHCOCHC12
C -C
OCOCH3
CAT
-CH2
2-
l l C -C-CH H | OCOCH3
OCOCH3
OH
1 -AcCP
1,3-(Ac) 2CP
FIG. 7. Proposed formula of inactivation of CP.
give CP. This fact indicates that an uncontrolled change in pH could cause hydrolysis and the recovery of antimicrobial activity in the products.
Thus, it is proposed that the CP acetylation is in Fig. 7.
summarized
LITERATURE CITED 1. Alpers, D. H., S. H. Appel, and G. ML Tomkns. 1965. A spectro-photometric asay for thiogalactoside transacetylase. J. Biol. Chem. 240:10-13. 2. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J. Randall. 1965. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 3. Miyamura, S. 1961. Chloramphenicol inactivation by dysentery bacilli, with special reference to chloram-
phenical resistance. (In Japanese.) Jpn. J. Bacteriol. 16: 115-119. 4. Miyamura, S. 1964. Inactivation of chloramphenicol by chloramphenicol-resistant bacteria. J. Pharm. Sci. 53: 604-607. 5. Miyamura, S., H. Ochia, Y. Nitahara, Y. Nakagawa, and M. Terao. 1977. Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcuspneumoniae and Streptococcus faecalis. Microbiol. Immunol. 21:69-76.
6. Okamoto, S., Y. Suzuki, K. Mise, and R. Nakaya. 1967. Occurrence of chloramphenicol-acetylating enzymes in various gram-negative bacilli. J. Bacteriol. 94: 1616-1622. 7. Rebstock, M. C., H. M. Crooks, Jr., J. Controulis and 0. R. Bartz. 1949. Chloramphenicol (chloromycetin). IV. Chemical studies. J. Am. Chem. Soc. 71:2458-2462. 8. Sands, L C., and W. V. Shaw. 1973. Mechanism of chloramphenicol resistance in Staphylococci: characterization and hybridization of variants of chloramphenicol acetyltransferase. Antimicrob. Agents Chemother. 3: 299-305. 9. Shaw, W. V. 1967. The enzymatic acetylation of chloramphenicol by extracts of R factor-resistant Escherichia coli. J. Biol. Chem. 242:687-693. 10. Shaw, W. V. 1975. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 43:737-755. 11. Shaw, W. V., and R. F. Brodsky. 1968. Characterization of chloramphenicol acetyltransferase from chloramphenicol-resistant Staphylococcus aureus. J. Bacteriol.
95:28-36.
12.
Suzuki, Y., and S. Okamato. 1967. The enzymatic ace-
tylation of chloramphenicol by the multiple drug-resistant Escherichia coli carrying R factor. J. Biol. Chem. 242:47224730. 13. Suzuki, Y., S. Okamoto, and M. Kono. 1966. Basis of chloramphenicol resistance in naturaly isolated resistant staphylococci. J. Bacteriol. 92:798-799.
Vol. 16, No. 6
0066-4804/79/12-0719/05$02.00/0
Kinetic Studies on Enzymatic Acetylation of Chloramphenicol in Streptococcus faecalis YOJI NAKAGAWA, YOSHIYUKI NITAHARA, AND SADAO MIYAMURA* Department of Bacteriology, Niigata University School of Medicine, Niigata, Japan 951 Received for publication 1 October 1979
The l2inetics of chloramphenicol (CP) acetylation by CP acetyltransferase from Streptococcus faecalis was studied. CP was shown to be acetylated enzymatically to its 3-0-acetyl derivative (3-AcCP) in the presence of acetyl coenzyme A, after which 3-AcCP was converted nonenzymatically to its 1-O-acetyl isomer, 1-0acetyl CP (1-AcCP). At equilibrium, the 1-AcCP and 3-AcCP were present in a 1:4 ratio. Subsequently the diacetylated product, 1,3-0-0-diacetyl CP [1,3(Ac)2CP], was enzymatically produced from 1-AcCP by the same enzyme. Theoretical calculation of rate constants (k1, k2, k3) for each successive reaction is as follows: k3 ki k2 CP-*3-AcCP--* 1-AcCP-_ 1,3-(Ac)2CP This calculation gave k, = 0.4 min-', k2 = 0.002 min-', and k3 = 0.016 min-'. Experimental results agreed closely with these calculated values.
Miyamura noted chloramphenicol (CP) inactivation in cultures of CP-resistant bacteria carrying R factor and suggested that the phenomenon was related to the mechanism of CP resistance (3, 4). Suzuki (12) and Shaw and coworkers (8-10) reported that CP inactivation was due to enzymatic acetylation of CP by chloramphenical acetyltransferase (CAT) in the presence of acetyl-CoA. However, specific reactions involved in acetylation have not been elucidated. In this connection, it was interesting to study the correlation between 3-O-acetyl CP (3-AcCP) and 1-0-acetyl CP (1-AcCP) and the mechanisms for formation of diacetylated CP from monoacetylated CP. The present studies were undertaken to clarify the mechanism of the acetylation of CP by an enzyme isolated from Streptococcus faecalis (5). MATERIALS AND METHODS Bacterial strain. S. faecalis N-117, isolated from the urine of a cystitis patient in the Niigata University Hospital, was used as the source of enzyme (CAT). The minimal inhibitory concentration of CP for this
strain was 125 pg/mI. Extraction and purification of the inactivating enzyme. S. faecalis N-117 was grown for 24 h at 370C with shaking in heart infusion broth containing 1% glucose and 10 Ag of CP per ml. Bacterial cells were harvested by centrifugation at 8,000 rpm for 20 min, washed twice with 0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.8), resuspended in the same buffer, and disrupted by a cell homogenizer (Braun Co.) for 10 min. Five milliliters of cell-free protein extract was added to an affinity
chromatography column, and the column was washed at 40C with 50 mM Tris-hydrochloride buffer (buffer A) containing 0.5 M NaCl, followed by 1 M NaCl, until no further ultraviolet-absorbing material was eluted. The enzyme was then eluted with buffer A containing 1 mg of CP per ml. The flow rate was 4 ml/h; 2-ml fractions were collected. CAT activity was determined by the spectrophotometric assay, which utilizes the reduction of 5,5'-dithiobis-2-nitrobenzoic acid by reduced acetyl coenzyme A (acetyl-CoA) (1, 5, 11). The enzyme was separated from CP by passing it through a Sephadex G-25 column. Fractions containing CAT were pooled and stored at -20°C. The protein content of the enzyme preparation used in these studies was 152 ,ug/ml as determined by the method of Lowry et al. (2). The specific activity of the electrophoretically homogeneous preparation was 74.5 units per,g of protein. This enzyme preparation was used in the following experiments unless otherwise specified. Radioisotope assay of CP acetylation. CP and its derivatives were extracted from the reaction mixture by ethyl acetate as described by Shaw (9). After three successive extractions, the total recovery of both CP and its derivatives was about 90%. A portion of the extract was then spotted on a silica gel thin-layer plate (Merck Silica Gel 60 Sheet) and submitted to chromatography with the solvent described below. CP and its products were located by an ultraviolet lamp and extracted three times with ethyl acetate by scraping from the appropriate area. The radioactivity was measured quantitatively with a liquid scintillation counter. The yield of CP and its derivatives was calculated from the known specific activity of the starting material. Thin-layer chromatography of silica gel. The thin-layer plate used was a Merck Silica Gel 60 sheet (silica gel with fluorescent indicator). The plate was 719
720
ANTimICROB. -AGENTS CHEMOTHER.
NAKAGAWA, NITAHARA, AND MIYAMURA
activated by heating it at 80°C for 15 min, before the extract from the reaction mixture was spotted on it. Development was carried out with chloroform-meth-anol (95:5) for about 60 min. Radioactivity measurement with a liquid scintillation counter. The solvent consisted of 6 g of 2,5diphenyloxazole, 0.3 g of 1, 4-bis[2-(5-phenyloxazolyl)] benzene, 450 ml of methanol, and 550 nl of toluene. The sample was dissolved in 8 ml of solvent and counted with a liquid scintillation counter (ALOKA, Liquid Scintillation Spectrometer LSC-602). Procedure for preparation of affinity chromatography column. Activated CH-Sepharose 4B swollen in 1 mM hydrochloric acid for 10 min was filtered by suction and washed with 1 mM hydrochloric acid on a glass filter. The filtered gel was treated in 0.1 M sodium bicarbonate solution containing 2 - amino-1(p-nitrophenyl) - 1,3-dihydroxypropane (7); the mixture was shaken gently at 220C for 28 h, after which the gel was filtered and washed with 50 mM Trshydrochloride buffer (pH 7.8). The gel was washed successively with 50 mM Tris-hydrochloride buffer (pH 8.0) containing 0.5 M NaCl and with 50 mM formic acid buffer (pH 4.0) containing 0.5 M NaCl. Before use the gel was equilibrated with buffer A. Enzymatic inactivation of CP. The complete reaction mixture contained 25 pxnol of Tris-hydrochloride buffer (pH 7.8) or Tris-maleate buffer (pH 7.0) and various amounts of ['4C]CP (4.87 uCi/,umol), acetyl-CoA, and enzyme in a total volume of 0.5 ml. The ratio of CP to acetyl-CoA was 1:4. The mixture was incubated at 30 or 370C for an appropriate time. A 0.1ml sample was pipetted into 1 ml of precooled ethyl acetate to terminate the reaction, and the inactivated products of CP were extracted. Chemicals. ['4C]CP (D-threo[dichloro-acetyl-1,2'4C]CP; 4.87 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. CH-Sepharose 4B and activated CH-Sepharose 4B were purchased from Pharmacia Fine Chemicals. Acetyl-CoA was purchased from P-L Biochemicals Inc. The acetyl derivatives of CP and the nonacetyl derivative, 2-
amino-1-(p-nitrophenyl)-1,3-dihydroxypropane, were synthesized in our laboratory.
RESULTS Time course of acetylation of CP and its derivatives. The acetylating enzyme from S. faecalis N-117 cells produced two monoacetyl derivatives and one diacetyl derivative of CP when incubated with CP and acetyl-CoA. In the complete system, the time course of product formation could be followed by thin-layer chromatography as shown in Fig. 1. CP was acetylated to 3-AcCP within the first minute of incubation; 3-AcCP rose sharply to a maxinum at 1 min and then declined slowly, whereas 1-AcCP increased slowly from 1 to 30 min and thereafter decreased gradually. The formation of 1,3(Ac)2CP increased markedly after the first 5 min of incubation. The data in Fig. 1 suggested the following sequence of reactions: CP -- 3-AcCP -* 1-AcCP -. 1,3-(Ac)2CP
24[ 18 (A
w
-J
0 12 B [ 0 z 4
z
6
60 01 5 15 30 MINUTES
90
FIG. 1. Kinetic studies on the formation of monoacetyl and diacetyl derivatives of CP. The complete system consisted of 50 punol of Tris-hydrochloride buffer (pH 7.8), l.(XI) umol of acetyl-CoA, 0.24 pmol of [4C]CP (4.87 yICi/Mmol), and 100 gd of enzyme protein solution in a final volume of I ml. Incubation was at 37°C. In each case, the reaction was terminated by the addition of ethyl acetate and the products were extracted as described in the text. Symbols: (O) CP; (-) 3-AcCP; (A) 1-AcCP; (A) 1.3-(AC)2CP.
Relationship between 3-AcCP and 1AcCP. Suzuki and Okanoto (12) and Shaw (9) suggested that 1-AcCP was derived from 3-AcCP nonenzymatically. To confirm this suggestion, 3AcCP separated with thin-layer chromatography was incubated in Tris-maleate buffer (pH 7.0) under conditions which minimized hydrolysis as much as possible. It is clearly shown in Fig. 2 that the 1-AcCP increased gradually while 3-AcCP decreased, and the equilibrium resulted. Diacetylation of CP. Diacetylated CP was derived from monoacetylated CP. To determine whether 1-AcCP or 3-AcCP is the direct precursor of 1,3-(Ac)2CP, the relationship of the relative concentration of both monoacetylated CPs to the formation of 1,3-(Ac)2CP was investigated. As shown in Fig. 3, the amount of 3-AcCP decreased with time, whereas 1-AcCP diminished rapidly and simultaneously 1,3-(Ac)2CP increased at the same rate for the first 20 min. When the concentration of 1-AcCP was high, a pronounced increase in diacetylation was observed. Kinetics. From the experimental data obtained the acetylation reaction of CP could be described as follows: k k3 k2 -.1,3-(Ac)2CP CP 43-AcCP -1-AcCP where k1, k2, and, k3 are rate constants. We assumed that the acetylation of CP was a successive reaction. If the iiiltial concentration of CP is a, and the concentrations of CP, 3-AcCP, I-AcCP, and 1,3-(Ac)2CP after t min are (A),
721
ACETYLATION OF CHLORAMPHENICOL IN S. FAECALIS
VOL. 16, 1979
(B), (C), and (D), respectively, the following
fornulas were derived. (a
0 0 z
5
(A) ae-k'
(1)
3
(B) ak, (e-kit -e-k2t k2- k)
(2)
.4
2
1
as
0
2
1
(Ck,
3
R(2- k,)(k
-
k,)
HOURS
FIG. 2. The conversion from 3-AcCP to 1-AcCP. 3AcCP obtained under the conditions described in Fig. 1 was dissolved in I ml of Tris-maleate buffer (pH 7.0). At the indicated time, 0.1-mi samples were taken for assay. In each case, the reaction was terminated by adding the sample to ethyl acetate, and the extracts were fractionated by thin-layer chromatography. 3-AcCP (0) and 1-AcCP (A) were quantitatively deternined by measuring their "4C activities with a scintillation counter.
+
+
a)(
(3)
(ki - k2)(k3- k2) e (k2- k3)(k, -k3)
(k3- k,)(k, - k2) +
kAk,ek2 (ki- k2)(k2- k3)
(4)
kik2e-k +
(k2 -k3)(k3 -k)
The measured values of (A), (B), (C), and (D) Lu 0
are 2-
z
O
=
60 0 10 2030 MINUTES
90
FIG. 3. Time course of acetylation reaction from monoacetylated to diacetylated CP. The reaction was stopped by extraction with ethyl acetate. The solvent was evaporated to dryness, and the residue was dissolved in Tris-hydrochloride buffer. The solution was divided into two parts, A (dotted lines) and B (solid lines). Part A was incubated immediately, and B was incubated after remaining for 3 h at 37°C. The solid lines show that the initial concentration of 1-AcCP and 3-AcCP was 4.3:20, and the dotted lines show the ratio was 1:20 after 3 h at 37°C. Symbols: (0) 3AcCP; (A) 1-AcCP; (A) 1,3-(Ac)2CP.
shown in Table 1. The rate constants (k1, k2,
k3) calculated from these values were 0.4, 0.002, and 0.016 min-', respectively. Figure 4 compares the curves of equations 1, 2, 3, and 4 by introducing the calculated rate constants from these equations, with the experimental data. The calculated and observed values of (A) and (B) were completely identical for the first 5 min. Thereafter, they remained essentially identical. Satisfactory agreement between calculated and experimental values of (D) was also observed. In the case of (C) only, a notable discrepancy was observed between experimental and calculated values. Equilibrium between 3-AcCP and 1AcCP. The mutual changes of the two monoacetyl derivatives of CP were observed on the thinlayer chromatograph (Fig. 5). By one-dimensional development (Fig. 5A), 1-AcCP was derived from 3-AcCP; by two-dimensional development (Fig. 5B), part of 1-AcCP changed into 3-AcCP.
TABLE 1. Concentrations of CP, 3-AcCP, I-AcCP, and 1,3-(Ac)2CP over time CP and CP derivative
CP 3-AcCP 1-AcCP
1,3-(Ac)2CP
Concn (nmol) at miin: 1 7.60 3.72 0.16 0.02
2 5.00 6.20 0.28 0.02
3 3.18 7.76 0.54 0.02
5 1.02 9.80 0.66 0.02
10 0.21 10.41 0.86 0.02
20 0.17 10.42 0.88 0.02
40 0.12 10.15 1.13 0.10
60 0.08 9.79 1.39 0.24
90 0.07 0.48 1.33 0.62
120 0.05 9.21 1.24 1.00
150 0.05 8.76 1.14 1.55
8 -w8 0 2 6 0
C
z
4
ANTIMICROB. AGENTS CHIZMOTHER.
NAKAGAWA, NITAHARA, AND MIYAMURA
722
4
C2
IC]01020 40
90 60 MINUTES
120
150
FIG. 4. The comparison of calculated and experimental concentrations of CP [A], 3-AcCP [B], 1AcCP [C], and 1,3-(Ac)2CP [D1. The dotted lines show the calculated values. and the solid lines show the experimental values. The reaction mixture contained 60 pumol of Tris-maleate buffer (pH 7.0), 0.60 pmol of acetyl-CoA, 0.15 ,mol of [4C]CP, and 50 IlI of enzyme in a total volume of 1.2 ml. The reaction mixture was incubated at 30°C, and 0.1-ml samples were taken for assay at the indicated times. The initial concentration of CP was 11.50 nmol. The ethyl acetate extracts were fractionated by thin-layer chromatography. Symbols: (0) CP; (0) 3-AcCP; (A) 1AcCP; (A) 1,3-(Ac)2CP.
DISCUSSION Conclusive evidence has not yet been presented for the whole course of the acetylation of CP to its diacetylated derivative by CAT. However, it may be postulated that 1,3-(Ac)2CP is derived from monoacetyl CP enzymatically. In the present work, it was confirmed that 3-AcCP formed from CP by CAT in the presence of acetyl-CoA is converted non-enzymatically to its isomer, 1-AcCP (Fig. 1 and 2). To account for the observation, one of the two schemes of the following types must be adopted. CP -. 3-ACCP -* 1-ACCP -* 1,3-(Ac)2CP (1) CP -- 3-AcCP -- 1-AcCP (2) I 1,3-(Ac)2CP
The rate of 1,3-(Ac)2CP produced depended mainly on the amount of 1-AcCP (Fig. 3), and the kinetics of product formation was related to 1-AcCP disappearance. These data strongly suggested that 1-AcCP was a direct precursor of 1,3(Ac)2CP, as shown in scheme 1. The essential agreements between calculated and observed values of each compound in scheme 1, with the exception of the values for 1-
AcCP, support this view. The exception seemed to be caused by a reverse reaction from 1-AcCP to 3-AcCP. Spontaneous conversion of 1-AcCP to 3-AcCP and vice versa occurred as shown in Tris-maleate buffer (Fig. 2) and on thin-layer chromatography plates (Fig. 5). It appeared of interest to investigate further the reversible mechanism of this reaction to establish definitive kinetics of acetylation of CP. If several different enzymes participated in the acetylation of CP to C-1 or C-3 acetyl derivatives (Fig. 6), the reaction rates of mono- and diacetylation of CP should be almost equal. However, a large difference between the rates of mono- and diacetylation was observed, indicating the existence of a single enzyme which acetylates the hydroxyl groups attached to C-3 of both CP and 1-AcCP. Some bacterial species having CAT, such as Staphylococcus and Pseudomonas, have been reported to produce no diacetylated CP (6,13). Our experiments suggest that the reported experiments may not have been carried out under optimal conditions. Moreover, the inactivated products, monoand diacetylated CP, are easily hydrolyzed to
B
A
*
3-ACCP
1-AcCP
...
...:
_.
i-AcCP 3-ACCP
FIG. 5. Autoradiographs showing the changes between 3-AcCP and I-AcCP; one development is onedimensionally, and the other is two-dimensionally. After incubation at 37°C for 3 min, the mixture was added to ethyl acetate to terminate the reaction and to extract the inactivated products. Monoacetyl CP was isolated by thin-layer chromatography. The onedimensional development is lengthwise and the twodimensional is sideways. Development was carried out with chloroform-methanol (95:5) for about 60 min. H 02N
NHCOCHC12
1 -2C-3CH2 OH H OH
FIG. 6. Structure of CP.
ACETYLATION OF CHLORAMPHENICOL IN S. FAECALIS
VOL. 16, 1979
723
3-AcCP
CP
NHCOCHC12
H
H
CATI
NHCOCHC12 I
02NC-:O H02NOCH-H -CH2
-
I-
OH
H
OH
CH2
OCOCH3
OH
H
H
NHCOCHC12
A
0
H
02N
NHCOCHC12
C -C
OCOCH3
CAT
-CH2
2-
l l C -C-CH H | OCOCH3
OCOCH3
OH
1 -AcCP
1,3-(Ac) 2CP
FIG. 7. Proposed formula of inactivation of CP.
give CP. This fact indicates that an uncontrolled change in pH could cause hydrolysis and the recovery of antimicrobial activity in the products.
Thus, it is proposed that the CP acetylation is in Fig. 7.
summarized
LITERATURE CITED 1. Alpers, D. H., S. H. Appel, and G. ML Tomkns. 1965. A spectro-photometric asay for thiogalactoside transacetylase. J. Biol. Chem. 240:10-13. 2. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J. Randall. 1965. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 3. Miyamura, S. 1961. Chloramphenicol inactivation by dysentery bacilli, with special reference to chloram-
phenical resistance. (In Japanese.) Jpn. J. Bacteriol. 16: 115-119. 4. Miyamura, S. 1964. Inactivation of chloramphenicol by chloramphenicol-resistant bacteria. J. Pharm. Sci. 53: 604-607. 5. Miyamura, S., H. Ochia, Y. Nitahara, Y. Nakagawa, and M. Terao. 1977. Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcuspneumoniae and Streptococcus faecalis. Microbiol. Immunol. 21:69-76.
6. Okamoto, S., Y. Suzuki, K. Mise, and R. Nakaya. 1967. Occurrence of chloramphenicol-acetylating enzymes in various gram-negative bacilli. J. Bacteriol. 94: 1616-1622. 7. Rebstock, M. C., H. M. Crooks, Jr., J. Controulis and 0. R. Bartz. 1949. Chloramphenicol (chloromycetin). IV. Chemical studies. J. Am. Chem. Soc. 71:2458-2462. 8. Sands, L C., and W. V. Shaw. 1973. Mechanism of chloramphenicol resistance in Staphylococci: characterization and hybridization of variants of chloramphenicol acetyltransferase. Antimicrob. Agents Chemother. 3: 299-305. 9. Shaw, W. V. 1967. The enzymatic acetylation of chloramphenicol by extracts of R factor-resistant Escherichia coli. J. Biol. Chem. 242:687-693. 10. Shaw, W. V. 1975. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 43:737-755. 11. Shaw, W. V., and R. F. Brodsky. 1968. Characterization of chloramphenicol acetyltransferase from chloramphenicol-resistant Staphylococcus aureus. J. Bacteriol.
95:28-36.
12.
Suzuki, Y., and S. Okamato. 1967. The enzymatic ace-
tylation of chloramphenicol by the multiple drug-resistant Escherichia coli carrying R factor. J. Biol. Chem. 242:47224730. 13. Suzuki, Y., S. Okamoto, and M. Kono. 1966. Basis of chloramphenicol resistance in naturaly isolated resistant staphylococci. J. Bacteriol. 92:798-799.
