Biochimica et Biophysica A cta, 1040(1990) 12-18

12

Elsevier BBAPRO 33699

Purification and properties of ampicillin acylase from Pseudomonas melanogenum D e o g J u n g K i m a n d Si M y u n g B y u n Department of Biological Science and Engineerin~ Korea Advanced Institute of Science and Technology (KAIST), Seoul (Korea)

(Received 23 March 1990) Key words: Ampicillinacylase; Purification;(P. melanogenum)

Ampicillin acylase, which is known to have a novel substrate spectrum, was purified to homogeneity from Pseudomonas melanogenum by the crude extract preparation and chromatography with S-Sepharose, hydroxyapatite, CM-cellulose C-52, and CM-Sepharose. The molecular weight of the native enzyme was calculated to he 146 000 by Protein PAK-300 sw HPLC chromatography. SDS-polyacrylamide gel electrophoresis revealed that the enzyme consisted of two identical subunits with a molecular weight of 72 000. The enzyme was a glycoprotein containing 13% total carbohydrate, and its isoelectric point was 7.2. The enzyme catalyzed both synthesis and hydrolysis of ampicillin and hydrolysis of the ester bond of phenylglycinemethylester hydrochioride substrate. The substrate specificity showed that the enzyme required a free amino group on the a-carbon of the acyl group. Chemical modification by diethylpyrocarbonate or N-bromosuccinimide resulted in time-dependent inactivation of the enzyme, and other results suggest the participation of essential histidine residue(s) in the catalytic activity of ampicillin acylase. Substrates of the enzyme, 6-aminopenieiilanic acid a.-td ampiciUin, exhibited protective effects against N-bromosuccinimide inactivation, suggesting that the modification occurred near or at the active site. Introduction Since Sakaguchi and Murao [1] first reported the presence of penicillin acylase (EC 3.5.1.11) in Penicillium chrysogenum, similar enzyme activity has been demonstrated to occur in many kinds of microorganisms [2,3]. Penicillin G, penicillin V, and ampicillin acylase are the three types of acylase classified according to their preferred substrates. Takahashi et al. [4] first described that some bacteria belonging to the family Pseudomonadaceae catalyzed N-acylation of 7-aminocephem compounds with aamino acid esters to synthesize the semi-synthetic cephalosporins. The same bacteria catalyzed N-acylation of 6-aminopenicillanic acid. Because the enzyme involved in the synthetic reactions utilized a-amino acid esters for acyl donors and transferred the acyl groups to

Abbreviations: 6-APA, 6-aminopeniciilanicacid; p-DAB, p-dimethylaminobenzaldehyde;PGM, D-a-phenylglycinemethylester;PG, DC-phenylglycine; NBS, N-bromosuccinimide; DEPC, diethylpyrocarbonate. Correspondence: S.M. Byun, Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, P.O. Box 150, Seoul 131, Korea.

both water and 7-aminocephem compounds or 6aminopenicillanic acid, they named this enzyme a-amino acid ester hydrolase. Among the Pseudomonadaceae, the a-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 [5,6] and Xanthomonas citri IFO 3835 [7,8] have been purified and studied for their substrate specificity, physicochemical properties, and enzyme kinetics. Meanwhile, Nara et al [9] and Okachi et al. [10] reported on ampicillin acylase from Kluyvera citrophila and Pseudomonas melanogenum, which display novel substrate spectra. The ampicillin synthesizing enzyme from P. melanogenum, in particular, is known to be an interesting penicillin acylase which shows activity only for the synthesis of ampicillin and cephalexin, both of which contain a side chain of o-phenylglycine [11]. Although the enzyme from P. melanogenum was referred to as ampicillin acylase on the basis of its preferred substrate, its substrate specificity [12] was very similar to that of a-amino acid ester hydrolase [6,8]. However, little information has been reported concerning the purification and properties of ampicillin acylase from P. melanogenum. In this work, we report the purification and properties of the ampicillin acylase from P. melanogenum and compare its properties with those of the a-amino acid ester hydrolase from X. cirri and A. turbidans.

0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)

13 Materials and Methods

Chemicals 6-Aminopenicillanic acid (6-APA), ampicillin, penicillin G, amoxicillin, cephalosporin C, cephalexin, metal salts, inhibitor compounds, and the other chemical compounds were purchased from Sigma (St. Louis, MO, U.S.A.). o-ct-Phenylglycinemethylester (PGM) hydrochloride was purchased from Wako Pure Chemicals (Osaka, Japan). S-Sepharose, CM-Sepharose, and molecular weight marker proteins were the products of Pharmacia Fine Chemicals AB (Uppsala, Sweden). Phenylisothiocyanate (PITC), 6 M hydrochloric acid, and Amino Acid Standard H were purchased from PIERCE (Rockford, IL, U.S.A.). All other chemicals were of the highest grade commercially available.

Microorganism and cultivation conditions Pseudomonas melanogenum IFO 12020 was grown at 30 °C in a Chemap fermenter containing 200 liters of medium. The growth medium contained 1% polypeptone, 1.0% yeast extract, 0.5% beef extract, 0.5% monosodium glutamate, and 0.25% sodium chloride. Cells were harvested at the late exponential growth phase with a Sharples centrifuge and the cell paste, 2.0 kg wet weight, was stored at - 2 0 °C until use.

Enzyme assays The hydrolytic activity of ampicillin acylase was determined by measured the amount of 6-APA produced by hydrolysis of ampicillin. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 #mol of 6-APA per rain at 37°C (pH 6.0). Enzyme was added to a solution containing 5 mM ampicillin in 10 mM sodium phosphate buffer (pH 6.0), and was incubated at 37°C for 5 rain. The 6-APA formed in the reaction mixture was assayed by the p-dimethylaminobenzaidehyde (p-DAB) method [13]. This reaction depended upon the free amino group of 6-APA and p-DAB to form a Schiff's base which was estimated spectrophotometrically at 415 nm. In the synthesis of ampicillin, the reaction mixture containing enzyme, 20 mM 6-APA, and 60 mM PGM-HC1 in 10 mM sodium phosphate buffer (pH 6.0) was incubated at 37 °C for 5 rain. The ampicillin formed in the reaction mixture was determined colorimetrically at 320 nm according to the method of Smith et al. [14].

Purification of the ampicillin'acylase All steps were carried out in a cold room at 4 ° C, unless otherwise specified. Extraction and ammonium sulfate fractionation. The cells (600 g wet weight) were suspended in 1800 ml of 50 mM sodium phosphate buffer (pH 6.0) with 0.1% Triton X-100 and homogenized by passing the solution through a Manton-Gaulin homogenizer, three times at

500 kg/cm. The eluent was cooled to 4 ° C and centrifuged at 30 000 x g for 30 rain. To the crude enzyme extract, a 30% solution of streptomycin sulfate was added dropwise with continuous stirring to reach 2% final concentration. After further standing for 1 h at 4 ° C, the mixture was centrifuged and the supernatant was obtained. This fraction was brought to 30% (w/v) saturation with solid (NH4)2SO4, stored for 30 min, and centrifuged. The resulting supernatant was then brought to 60% with (NH4)2SO 4 and centrifuged. The precipitate was dissolved in 700 ml of buffer A (10 mM sodium phosphate (pH 6.0)) and dialyzed overnight against the same buffer. S-Sepharose column chromatography. The enzyme solution (1100 ml) was divided into three aliquots and applied to an S-Sepharose column (5.0 x 17 cm) equilibrated with buffer A and washed with 2 bed volumes of the same buffer. The enzyme was eluted with a 1500-ml linear gradient of NaC1 from 0 to 0.5 M in buffer A at a flow rate of 180 ml/h. All active fractions were collected (315 ml) and dialyzed overnight against buffer B (10 mM potassium phosphate (pH 6.0)). Hydroxyapatite column chromatography. The dialyzed enzyme solution was loaded on a hydroxyapatite column (2.6 x 42 cm) previously equilibrated with buffer B. The column was washed with 2 bed volumes of the same buffer and eluted with a 2000-ml buffer gradient of 10-750 mM potassium phosphate (pH 6.0) at a flow rate of 60 ml/h. The pooled fractions with enzyme activity (200 ml) were dialyzed overnight against buffer A. CM-cellulose C-52 column chromatography. The enzyme solution was applied on a CM-cellulose C-52 column (2.6 x 20 cm) equilibrated with buffer A and washed with 2 bed volumes of the same buffer. The enzyme was eluted with 800 ml of salt gradient from 0 to 0.2 M NaC1 in buffer A. The flow rate of the column was 45 m l / h and 7.5 ml fractions were collected. The pooled active fractions (38 ml) were dialyzed overnight against buffer A. CM-Sepharose column chromatography. The enzyme solution was applied to a column (2.6 × 16 cm) with CM-Sepharose, equilibrated with buffer A, and eluted with a 600-ml linear gradient of sodium phosphate buffer from 10 to 100 mM (pH 6.0) at a flow rate of 24 mi/h. The active fractions (55 ml) were pooled and dialyzed against 10 mM sodium phosphate buffer (pH 6.0) and concentrated in a dialysed bag with poly(ethylerie glycol), molecular weight 20 000. This protein solution was used as the purified enzyme in further experiments.

Determination of protein and carbohydrate The protein concentration was determined according to the Bradford assay [15], using bovine y-globulin as a standard. The carbohydrate content was determined by

14 the phenolsulfuric acid method [16], using glucose as standard.

Molecular weight and isoelectric point determination The molecular weight of the native enzyme was estimated by comparing its mobility with standard proteins on a Protein PAK-300 sw HPLC column (7.8 × 30 cm) (Waters, U.S.A.). Standard proteins used for the calibration were ferritin (Mr 440 000), catalase (Mr 232 000), aldolase ( M r 158 000), and bovine serum albumin ( M r 67 000). Subunit analysis was performed by electrophoresis in a 12% polyacrylamide gel containing 0.1% SDS according to the method of Laemmli and Farre [17], with low molecular weight SDS-polyacrylamide gel electrophoresis standards (Pharmacia) as markers. Analytical isoelectric focusing with 1% agarose IEF gel was performed using 6.3% ampholyte (pH 3-10) at 15 W constant power for 1.5 h at 10 o C, according to the method of Rosen [18]. Amino acid analysis The amino acid analysis was performed by reversephase chromatography with phenylisothiocyanate (PITC) precolumn derivatization of the protein hydrolysates. Samples containing about 1.5 /~g protein of purified enzyme were dried under vacuum using a PICO-TAG Workstation and hydrolyzed with 6 M HCI in vacuo at 105 o C for 24 and 72 h. Amino acid analysis was carried out in a Waters amino acid analyzer with a reverse-phase high performance PICO-TAG column [19,20]. Identification of reaction course To determine the reaction course of the purified enzyme, the products from the reaction mixture were analyzed with a Waters HPLC/LBondapak Cls column. The running buffer was 0.2 M sodium acetate (pH 4.0) with a flow rate of 1 ml/min. The retention times for 6-APA, PGM-HC1, PG, and ampicillin were 4.21, 23.03, 3.55, and 50.13 min, respectively.

1

2

3

4

5

6

Fig. 1. Electrophoretic pattern of fractions from each stage of the purification (12% SDS-polyacrylamide gel). Lane 1, crude extract (50 /~g protein); Lane 2, S-Sepharose pool (20 /~g protein); Lane 3, hydroxyapatite pool (10/~g protein); Lane 4, CM-ceilulose C-52 pool (5 t~g protein); Lane 5, CM-Sepharose pool (2/~g protein); and Lane 6, molecular weight standards: phosphorylase b (Mr 97 400), bovine serum albumin ( M T 66 200), ovalbumin (Mr 42 700) and carbonic anhydrase (M r 29 000).

Effects of various inhibitors Chemical modification of the enzyme was carried out at 37 o C in a reaction mixture containing 10 mM sodium phosphate buffer (pH 6.0), the enzyme, and 1-5 mM concentrations of various compounds (Table IV). For studies on protection of the enzyme against chemical modification, the enzyme was preincubated with different concentrations of 6-APA, PGM-HC1, and ampicillin, and modification was done as indicated in the table legend. R~d~

Purification of the ampicillin acylase A summary of the purification steps is given in Table I. The purification protocol resulted in 8847-fold purification, with a final recovery of 12% of the enzymatic

TABLE I

Purification of ampicillin acylase from Pseudomonas melanogenum Purification step

Volume (ml)

Total protein (nag)

Total a activity (units)

Specific activity (units/mg)

Yield (%)

Crude extract

1 600

38 400

4 160

0.108

100

Ammonium sulfate (30-50% fractionation)

1 100

10 400

3 272

0.315

79

S-Sepharose

315

84

2 556

30.4

61

Hydroxyapatite

200

3.3

1 826

553.3

44

CM-cellulose C-52

38

1.25

1 097

877.6

26

CM-Sepharose

55

0.54

516

955.5

12

a Based on the hydrolysis of ampicillin by the enzyme.

15 activity. The relative purity at each step of the purification is shown in Fig. 1.

Molecular properties of the enzyme The molecular weight of the native enzyme was estimated to be 146 000 by gel filtration of Protein PAK-300 sw HPLC with marker proteins (Fig. 2). The subunit size of the purified enzyme was estimated to be 72 000 as determined by SDS-polyacrylamide gel electrophoresis (Fig. 1, lanes 5 and 6). On the basis of these results, this enzyme appears to be a homodimer, consisting of two identical subunits each having a molecular weight of 72 000. Isoelectric focusing of the enzyme showed a single protein band with an isoelectric point of 7.2. Amino acid composition and carbohydrate content The amino acid composition of the ampicillin acylase from P. melanogenum is given in Table II, in comparison with those of the a-amino ester hydrolases from X. citri and A. turbidans. Surprisingly, the three enzymes exhibited similar amino acid composition, despite small differences at the level of cysteine, methionine, and isoleucine. It was seen that the contents of Asx, Glx, and Val were high, while those of His, Ser, and Met were low. Analysis of carbohydrate content by the phenolsulfuric acid method showed that the enzyme is a glycoprotein possessing 13% carbohydrate. Optimal pH, temperature and stability of the enzyme The optimal pH values for synthesis and hydrolysis of ampicillin were the same, around pH 6.0 with 10 mM

7.

6.0

o~

X~ 3

(J

A m i n o acid

mol% P. melanogenum a

X. citri d

A. turbidans e

Asx Thr Ser Glx Pro Gly Ala Val Met lie Leu Tyr Phe Lys His Arg Cys Trp

9.25 4.61 3.82 9.29 5.65 4.07 6.71 8.07 b 2.63 5.27 b 6.58 4.09 6.34 4.96 2.86 6.11 N.D. c N.D. c

10.45 4.55 3.55 8.70 5.87 3.79 4.90 6.39 3.18 2.55 6.54 5.32 5.85 4.75 2.64 7.03 0.00 4.01

10.68 4.52 3.69 8.48 6.80 4.01 5.64 6.26 8.18 2.58 5.90 5.60 5.11 3.90 3.71 6.61 3.06 4.88

a Values based on a molecular weight of 72 000. Average of two hydrolysates at 24 h, unless otherwise indicated. b Average of two hydrolysates at 72 h. e N.D., not determined. d Data of Takahashi et al. [7]. e Recalculated from the data of R y u et al. [5].

of sodium phosphate buffer, and most of the enzyme activity was retained at pH 5.5-6.5 after 2 h incubation at 30 o C. However, after preincubation at a pH below 4.0 or above 8.0 the enzyme was mostly inactivated. The optimal temperature for synthesis and hydrolysis of ampicillin was 37 ° C. For the test of thermal stability, the enzyme was incubated for 2 h at different temperatures and the residual activity was estimated. Thermal inactivation occurred above 37 °C and led to complete loss of activity at 50 o C.

Metal ion effects The effects of various metal ions on the hydrolytic activity of ampicillin were examined. Several divalent cations (Cu 2÷, Hg 2÷, Fe 2÷) decreased the enzyme activity to 50% at 1 mM. A chelating agent, EDTA, did not inhibit the enzyme activity, so it appears that metal ions are not absolutely necessary for the activity.

5.5 )

o

T A B L E II Amino acid composition of ampicillin acylase

5.0 01 0

4.5 !

I

I

I

0.2

0.3

0.4

0.5

Kav Fig. 2. Determination of the molecular weight of native ampiciUin acylasc on a Protein PAK-300 sw H P L C column (7.8 × 30 cm). T h e molecular weight markers used were: 1, fcrritin (M r 440000); 2, catalase (M r 232 000); 3, aldolase (M r 158 000); and 4, bovine serum albumin (M r 67 000). The arrow indicates the Kay value of purified ampicillin acylase.

Substrate specificity The substrate specificity of this enzyme was investigated in the hydrolysis of several fl-lactam antibiotics. The results are shown in Table III. 6-APA was produced only from the fl-lactam antibiotics containing a free amino group located on the a-carbon atom of the acyl group. This result indicates that a free amino group in the acyl group is essential for enzyme activity. It was also noted that cephalexin and cephaloglycin containing

16 TABLE III SUBSTRATE SPECIFICITY OF AMPICILLIN ACYLASE Substrate

Hydrolysis a

fl-Lactam antibiotics

N-acyl residue

Penicillin G

O

CH 2 - C O -

Penicillin V

O

OCH2 - C O -

Amoxicillin

H O - ~

Kinetic constants K m (mM)

Vm~ (retool/rain)

+

4.34

0.45

CH - C O NH 2

NH 2

Cephalosporin C

H 2N x HO2C ~.CH(CH 2) 3- CO -

Cephalothin

~

Cephapirin

O

SCH2- C O -

Cephaloglycin

O

ICH- C O NH2

+

1.96

0.22

Cephradine

O

CH - CO -

+

1.79

0.24

+

1.61

0.32

CH 2 - CO--

NH2 Cephalexin

~

CH - CO NH 2

a Hydrolysis of fl-lactam antibiotics. 6-APA, 7-ACA, 7-ADCA: +, produced; and -, not produced.

the cephem ring were better substrates than ampiciUin containing the penam ring. This difference in reactivity of the acyl acceptor implies a difference in the binding force of the acyl acceptor to the binding site of the enzyme through hydrophobic interaction. Reaction courses of the enzyme The reaction compounds of the purified enzyme were analyzed by means of HPLC (data not shown), and it was confirmed that the enzyme catalyzed synthesis of ampicillin by transfer of the acyl group from PGM-HC1 to 6-APA, hydrolysis of the acid amide bond of ampieillin, and hydrolysis of the ester bond of PGM-HC1. Inhibitor studies Preliminary chemical modification with various amino acid inhibitors showed that N-bromosuceinitnide (NBS) and diethylpyrocarbonate (DEPC) caused a significant decrease in activity, but modification with other inhibitors resulted in little loss of activity (Table IV).

When the enzyme was incubated with various concentrations of NBS and DEPC, a time-dependent inactivation was observed (data not shown). Both the hydrolytic and the synthetic activity of this enzyme were simultaneously inactivated by NBS and DEPC. The protective effects of the three kinds of enzyme substrate were next assayed to examine whether the modified residues were located near or at the active site. Because the DEPC inhibitor reacted with 6-APA, PGM-HC1, and ampicillin, whereas NBS did not react with these substrates, the protective effect of the substrates was tested against NBS inactivation. Table V shows that the substrates 6-APA and ampicillin exhibited protective effects against NBS inactivation, but another substrate, PGM-HC1, displayed no such effect.

Since Okachi et al. [10] reported that ampiciUin acylase from P. melanogenum displays a novel substrate

17 enzyme from X. citri. The optimal pH and temperature values of the three enzymes were very similar, except for the optimal temperature of the enzyme from A. turbidans (P. melanogenum, pH 6.0, 37 o C; X. cirri, pH 6.4, 35 o C; A. turbidans, pH 6.2, 40-45 o C) Studies of their amino acid composition revealed the three enzymes shared a significant degree of similarity (Table II). In the reaction courses, the three enzymes catalyzed both hydrolysis of a-amino acid ester and transfer of the acyl group from the acyl donor to 7-aminocephem compounds or 6-APA. The substrate specificity of these enzymes showed that all required a free amino group on the a-carbon of the donor ester. Even though ampicillin acylase from P. melanogenum was classified and named according to its preferred substrate, previous studies [4,5,7] and the present work have also shown that ampicillin acylase is the same kind of enzyme as a-amino acid ester hydrolase. The unique substrate specificity of these enzymes leads us to the interesting question of why they do not hydrolyze or synthesize natural antibiotics, such as penicillin G, penicillin V, and cephalosporin C, and why they 'do' hydrolyze or synthesize artificially semisynthesized products such as ampicillin, cephalexin, and cephaloglycin. This unusual substrate specificity of the enzyme raises the possibility that this protein may have another physiological function in addition to its known enzyme activity. However, this possibility cannot be investigated at the present time, and further studies will be required to elucidate this point. No studies have been reported to date on the reaction mechanism and active sites of these enzymes. Determination of the essential amino acid residues involved in substrate binding or in catalytic activity is fundamental to an understanding of the nature of the active site of the enzyme. As shown in Table IV, ampicillin acylase was markedly inhibited by two reagents, N-bromosuccinimide (NBS) and diethylpyrocarbonate (DEPC). NBS has been demonstrated to modify to cleave the functional group of tryptophan, tyrosine, cysteine, and histidine [21-23]. It is well known that DEPC reacts with proteins having several amino acid

TABLE IV

Inhibitory effects of various compounds on the hydrolysis of ampicillin by the enzyme The enzyme was preincubated with the compound in the assay buffer for 20 min and dialyzed to remove unreacted compound. Then the enzyme activity on the hydrolysis of ampicillin was assayed. Residual activity is expressed as a percent of the activity in the absence of added compound. Compound

Concentration (mM)

Residual activity (%)

Carbodiimide Pyridoxal 5-phosphate Diethylpyrocarbonate N-Bromosuccinimide 2-Hydroxy-5-nitro benzyl bromide p-Chloromercuribenzoate Dithiothreitol Iodoacetate Phenylmethylsulfonyl fluoride p-Nitrophenyl acetate N-Acetylimidazole

5 5 1 1 1 1 2 5 1 5 5

100 94 10 0 77 94 97 100 75 97 97

spectrum, another report [12] demonstrated partial purification of this enzyme and its substrate specificity. The present report is the first which, deals with the purification to homogeneity and the properties of ampicillin acylase from P. melanogenum. Some properties of the ampicillin acylase from P. rnelanogenum will be discussed in comparison with those reported for the a-amino acid ester hydrolases from X. citri [7,8] and A. turbidans [5,6]. The subunit size of the above three enzymes was approximately similar, at Mr 70 000-72 000. However, the molecular weight and isoelectric point of the native enzyme calculated for ampicillin acylase from P. melanogenum ( M r 146 000, two identical subunits, p I 7.2) was compared to the value of the a-amino acid ester hydrolase from X. citri ( M r 280 000, four identical subunits, p l 7.8) and the enzyme from A. turbidans ( M r 280 000-290 000, four subunits having two subunits each of M r 70 000 and 72 000, p I 5.8). The enzyme from P. melanogenum was a glycoprotein containing 13% carbohydrate. However, carbohydrate moiety of less than 0.4% was detected in the TABLE V

Protective effects of substrates on the inactivation of ampicillin acylase by NBS The enzyme was incubated at 37°C in the assay buffer with 7/~M NBS, in the presence of 70 and 350/LM 6-APA, 70 and 350 #M Amp, and 70 and 350/~M PGM-HC1. At intervals, aliquots were withdrawn and assayed for enzyme activity on the hydrolysis of Amp. Residual activity was corrected with blank value in the absence of Amp substrate and expressed relative to the original enzyme activity. Incubation

Residual activity (Sg)

time (min)

NBS

NBS + 6-APA

NBS + Amp

NBS + PGM-HC1

70/zM

(350 #M)

70 btM

(350 #M)

70 #M

(350/zM)

5

21

70

(86)

57

(93)

26

(26)

10

14

43

(80)

43

(86)

18

(18)

15

14

43

(80)

43

(86)

18

(18)

18

side chains, such as imidazale, phenolate, sulfhydryl, and amino groups [24-26]. However, DEPC has been shown to modify histidine residues in proteins with considerable specificity at a slightly acidic pH of 6.0 [27]. Because other inhibitors, such as 2-hydroxy-5nitrobenzyl bromide, p-nitrophenyl acetate, and pchloromercuribenzoic acid (specific for tryptophan, tyrosine, and cysteine, respectively) did not show a significant activity decrease, these results suggest the participation of essential histidine residue(s) in the catalytic activity of ampicillin acylase. The protective effects of the enzyme substrates 6-APA and ampicillin against NBS inactivation provide definite evidence that the modification occurred near or at the active site. Moreover, the results shown in Table V elucidated the location of the modified residue(s) at the active site of ampicillin acylase. Because the substrates 6-APA and ampicillin, but not PGM-HC1, showed protective effects against NBS inactivation, it is certain that the modified residue is located at the binding site of 6-APA, but not at that of PGM-HC1. However, simultaneous inactivation of the enzyme's hydrolytic and synthetic activity with NBS suggests that the modified residue is located near the binding site of PGM-HC1, close enough to influence the hydrolysis of PGM-HC1. Further investigation of the nature of essential amino acid residues in the active site is necessary for an understanding of the mechanism of the enzymatic reaction, and this investigation is in progress. References 1 Sakaguchi, K. and Murao, S. (1950) J. Agric. Chem. Soc. Jpn. 23, 411-413. 2 Vandamme, E.J. and Voets, J.P. (1974) Adv. Appl. Microbiol. 17, 311-369. 3 Hamilton-Miller, J.M.T. (1966) Bacteriol. Rev. 30, 761-771.

4 Takahashi, T., Yamazaki, Y., Kato, K. and Isono, M. (1972) J. Am. Chem. Soc. 94, 4035-4037. 5 Ryu, Y.W. and Ryu, D.D.Y. (1987) Enzyme Mierobiol. Teehnol. 9, 339-344. 6 Ryu, Y.W. and Ryu, D.D.Y. (1988) Enzyme Microbiol. Technol. 10, 239-245. 7 Kato, K., Kawahara, K., Takahashi, T. and Kakinuma, A. (1980) Agr. Biol. Chem. 44, 1069-1074. 8 Kato, K., Kawahara, K., Takahashi, T. and Kahinuma, A. (1980) Agr. Biol. Chem. 44, 1075-1081. 9 Nara, T., Okachi, R. and Misawa, M. (1971) J. Antibiot. (Tokyo) 24, 321-323. 10 Okachi, R., Kato, F., Miyamura, Y. and Nara, T. (1973) Agr. Biol. Chem. 37, 1953-1957. 11 Shimizu, M., Masuike, T. and Fujita, H. (1975) Agr. Biol. Chem. 39, 1225-1232. 12 Okachi, R. and Nara, T. (1973) Agr. Biol. Chem. 37, 2797-2804. 13 Blasingham, K., Warburton, D., Dunnill, P. and Lilly, M.D. (1972) Biochim. Biophys. Acta 276, 250-256. 14 Smith, J.W.G., Grey, G.E. and Patel, V.J. (1967) Analyst 92, 247-252. 15 Bradford, M. (1976) Anal. Biochem. 72, 248-254. 16 Dubois, M., Gilles, K.A., Hamilton, J.K., Roberts, P.A. and Smith, F. (1956) Anal. Chem. 28, 350-356. 17 Laemmli, U.K. and Farre, M. (1973) J. Mol. Biol. 80, 575-599. 18 Rosen, A. (1980) in Electrophoresis '79 (Radola, B.J., ed.), pp. 105-116, de Gruyter, Berlin. 19 Robert, L.H. and Stephen, C.M. (1984) Anal. Biochem. 136, 65-74. 20 Henning, S. (1985) J. Chromatogr. 350, 453-460. 21 Witkop, B. (1961) Adv. Protein Chem. 16, 221-285. 22 Vallee, B.L. and Riordan, J.F. (1969) Annu. Rev. Biochem. 38, 733-794. 23 Lundblad, R.L. and Noyes, C.M. (1984) in Chemical Reagents for Protein Modification, Vol. II, pp. 47-72, CRC Press, Boca Raton, FL. 24 Miller, E.W. (1977) Methods Ealzymol. 47, 431-442. 25 Muhlrad, A., Heigyi, G. and Toth, G. (1967) Acta Biochim. Biophys. Acad. Sci. Hung. 2, 19-29. 26 Burstein, Y., Walsh, K.A. and Neurath, H. (1974) Biochemistry 13, 205-210. 27 Melchoir, W.B. and Fahrney, D. (1970) Biochemistry 9, 251-258.

Purification and properties of ampicillin acylase from Pseudomonas melanogenum.

Ampicillin acylase, which is known to have a novel substrate spectrum, was purified to homogeneity from Pseudomonas melanogenum by the crude extract p...
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