cephalosporin-sensitive penicillin-binding proteins of

Vol. 254, No. 23, Issue of December 10, pp. 12056-12061, 1979 Printed in U S. A.

Cephalosporin-sensitive Penicillin-binding Proteins of StuphyZococcus aureus and Bacillus subtilis Active in the Conversion of [“C]Penicillin G to [14C]Phenylacetylglycine*

(Received for publication, July 16, 1979)

David J. Waxman and Jack L. Strominger

From The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

Breakdown of the covalent complex formed between [‘“Clpenicillin G and higher molecular weight, cephalosporin-sensitive penicillin-binding proteins was studied using mixtures of the purified proteins isolated from membranes of Staphylococcus aureus and Bacillus subtilis. These penicillin-binding proteins were found to release the bound *T label in a first order process characterized by half-lives of 10 to 300 min at 37°C. Denaturation of the penicilloyl* penicillin-binding protein complex prevented this release, indicating that the process is enzyme-catalyzed. [‘4C]Phenylacetylglycine was identified as the major labeled fragmentation product, indicating that these cephalosporin-sensitive penicillin-binding proteins, for which no in vitro transpeptidase or carboxypeptidase activity has been found, catalyze the same fragmentation of the bound penicilloyl moiety previously described for several penicillin- sensitive D-alanine carboxypeptidases.

Penicillin kills bacteria by inhibiting cross-linking of the cell wall during ‘the final stages of its biosynthesis (1, 2). It was proposed that penicillin acylates the active site of the transpeptidase which catalyzes this reaction by acting as a struc- tural analog of the acyl-D-alanyl-D-alanine terminus of nascent peptidoglycan (2). Consistent with the prediction that a stable, covalent penicilloyl. transpeptidase complex can form, PBPs’ have been detected in bacterial membranes (3, 4). Most re- cently, sequence analysis of active site peptides derived from two of these PBPs has demonstrated that penicillin and substrate do, in fact, both acylate the same active site serine (5, 6), in accordance with the proposed mode of penicillin action (2). In each organism examined thus far, multiple PBPs exist. Genetic, biochemical, and immunological evidence (7- 10) indicate that multiple PBPs are independent proteins, not related by precursor-product relationships. Studies with Esch- erichia coli suggest that the various PBPs may perform different penicillin-sensitive reactions of cell wall biosynthesis (11). Thus, one.PBP may function as a transpeptidase of cell elongation, another as a transpeptidase involved in septation and yet a third may be involved in maintaining cell shape.

The PBP of lowest molecular weight, M, = 40,000 to 50,000, has been purified from membranes of several bacterial species by covalent penicillin affinity chromatography (4, 12-16). These PBPs are penicillin-sensitive enzymes which specifi-

* This work was supported by research grants from the National Institutes of Health (AI 09152) and the National Science Foundation (PCM 78 24129). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: PBP, penicillin-binding protein; SDS, sodium dodecyl sulfate.

tally catalyze hydrolysis of the terminal n-alanine from cell wall-related substrates in uitro. This D-alanine carboxypeptidase activity is lost upon formation of a covalent penicilloyl. enzyme complex. It is not known whether these enzymes function as peptidoglycan transpeptidases in Viva or whether they catalyze a related penicillin-sensitive reaction, e. g. trim- ming terminal D-alanine residues from nascent peptidoglycan strands to limit the extent of cell wall cross-linking (for a more complete discussion, see Ref. 17). Although carboxypeptidases are sensitive to low concentrations of various penicillins, they are generally much less sensitive to cephalosporins and thus it is unlikely that these PBPs are directly involved in the killing effects of cephalosporin antibiotics (18).

Penicillin-inactivated D-alanine carboxypeptidases catalyze breakdown of the covalent penicilloyl. enzyme complex to yield reactivated enzyme and biologically inactive penicillin derivatives. D-Alanine carboxypeptidases from Staphylococ- cus aureus (PBP 4) and E. coli (PBPs 5 and 6) rapidly release the bound penicilloyl moiety as penicilloic acid, i.e. they exhibit /?-lactamase activity, in addition to carboxypeptidase and transpeptidase activity in vitro (14, 19). Early studies indicated that D-alanine carboxypeptidases of Bacillus stearothermophilus and Bacillus subtilis release the bound penicilloyl moiety as a product other than penicilloic acid (20). Subsequently, the released moiety was identified as phenylacetylglycine for these carboxypeptidases (21)’ and for the carboxypeptidases from various species of Streptomyces (22). This unusual fragmentation of the bound penicilloyl moiety also yields dimethylthiazoline carboxylate (23) or its hydrolysis product, N-formyl D-penicillamine (24, 25) from the an- tibiotic’s thiazolidine ring. This fragmentation proceeds at a significantly slower rate (tlp = 80 to 300 min at 37°C) than release of penicilloic acid by the other carboxypeptidases (tl,2 = less than 5 min at 37°C) and involves an enzymatic scission of the C5-C6 bond of the penicillin nucleus. The detailed mechanism of this novel degradation (21, 26, 27) may have a direct bearing on the mechanism of release of the substrate- derived acyl group during a carboxypeptidase or transpeptidase reaction.

High molecular weight PBPs (iV& = 60,000 to 120,000), believed to include penicillin-sensitive transpeptidases, have been purified as mixtures with D-alanine carboxypeptidase from detergent extracts of bacterial membranes (12,14). These high molecular weight PBPs are generally sensitive to both cephalosporins and penicillins, in contrast to the low molecular weight PBPs (carboxypeptidases) which are often much less cephalosporin-sensitive. Release of the penicilloyl moiety bound by these high molecular weight PBPs has been observed using membranes from various bacterial species (2% 30). The presence of carboxypeptidase as a major component

* D. J. Waxman, unpublished results.

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Penicillin-binding Proteins of S. aureus and B. subtilis 12057

in purified mixtures of PBPs has complicated identification of the degraded peniciIloy1 moiety released from the high molecular weight, cephalosporin-sensitive PBPs.

The recent introduction of covalent cephalosporin affinity chromatography (8) has facilitated the isolation of high molecular weight PBPs from B. subtilis (8) and S. aweus free from contaminating carboxypeptidase. Thus far, no in vitro transpeptidase or carboxypeptidase activity has been found for these or for other (30) such PBPs, possibly due to loss of enzymatic activity during purification. It is important to as- certain whether these cephalosporin-sensitive PBPs catalyze the novel fragmentation of the bound penicilIoy1 moiety previously described for the carboxypeptidases. This study pre- sents evidence that several of the higer molecular weight, cephalosporin-sensitive PBPs isolated from S. aureus and B. subtilis do indeed catalyze release of the bound [‘*C]penicilloyl moiety to yield [‘*C]phenylacetylglycine as the major labeled fragmentation product.

MATERIALS AND METHODS

Penicillin G, 6-aminopenicillanic acid, 6-aminohexanoic acid, Tri- ton X-100, Tris base, and SDS were obtained from Sigma. [8-14C]- penicillin G, K’ salt, shown to be greater than 95% radiochemically pure by thin layer chromatography in solvent A (see below) was from Amersham (52 &i/pmol) and was stored at -20°C. Hydroxylamine hydrochloride was from Eastman and solvents used in thin layer chromatography were from Fisher.

A mixture of S. aureus H PBPs 1, 2, 3, and 4 was isolated by covalent penicillin affinity chromatography using 6-aminopenicillanic acid bound to Sepharose 4B-200 (Pharmacia) via a g-carbon spacer, as described (14). A mixture of B. subtilis Porton PBPs 1, 2, and 4 were isolated using 7-amino-cephalosporanic acid-Sepharose, as described (8). Electrophoresis in discontinuous SDS-polyacrylamide slab gels (5% stacking, 7.5% running gel), sample preparation, staining, destaining, fluorography, and densitometry were as previously described (31). Radioactivity was determined by liquid scintillation counting and protein by the Lowry assay in the presence of 1% SDS, using bovine serum albumin as a standard.

Thin layer chromatography was performed on precoated Silica Gel 60 F-254 plates (E. Merck) as described (21) using three solvent systems: Solvent A, acetone/acetic acid, 95:5 (v/v); Solvent B, chloroform/acetone/hexanes, 9:l:l (v/v/v); and Solvent C, ethyl acetate/ hexanes/acetone, 8:2:1 (v/v/v). Radiolabeled compounds were detected by autoradiography using blue-sensitive x-ray film (Kodak, SB-254) and standard compounds with an ultraviolet lamp or by exposure to iodine vapors.

Phenylacetylglycine and its methyl ester were synthesized as described (21). Ethyl acetate extracts of the acidified (pH 2) [‘“Cl- penicillin fragmentation product (1 to 5 ml) were concentrated to 50 to 100 ~1 (gentle stream of Nz) and then methylated with 0.5 ml of diazomethane in ether (10 min on ice, then 20 min at 25°C) prepared from Diazald (Aldrich). [‘4C]Penicilloic acid was prepared by incu- bating [‘4C]penicillin G with penicillinase (Calbiochem).

RESULTS

Isolation of a native [‘*C]Penicilloyl. PBP Complex- [‘*C]Penicillin G was bound to a mixture of S. aureus PBPs 1, 2, 3, and 4 and the [‘*C]penicilloyl.PBP complexes were isolated by gel filtration on Sephadex G-50 (Fig. 1). The peak of radioactivity at the exclusion volume, coincident with the elution position of the applied Triton X-100 micelles, contained all four YBPs, as indicated by SDS-gel electrophoresis (Fig. 2, Lane 1). Although all four PBPs initially bound [‘*C]penicillin G, only PBPs 1, 2, and 3 were labeled after gel filtration (Fig. 2, Lanes 2 and 3), consistent with the finding that PBP 4 rapidly releases the bound penicillin as penicilloic acid (14). Formation of [‘*C]penicilloyl.PBPs 1, 2, and 3 was prevented effectively by mild heat denaturation (5 min at 7O’C) or by treatment with 1% SDS.

3 T. A. O’Brien, D. J. Waxman, and D. M. Lindgren, unpublished results.

9-

4 SALT

I - 6- n h x -04

Fraction Number

FIG. 1. Isolation of S. aurew [‘*C]penicilloyl~PBPs complex by gel filtration. [‘*C]Penicillin G (30 ~1 at 0.7 mg/ml) was incubated with 0.67 mg of a mixture of S. aureus PBPs 1, 2, 3, and 4 in a final volume of 0.5 ml of 0.05 M Tris-Cl, pH 7.5, 0.5 M NaCl, 1% Triton X- 100 for 10 min at 25°C. The reaction mixture was cooled on ice and applied to a column of Sephadex G-50 (superfine) (0.8 x 20 cm) equilibrated and eluted with 0.01 M sodium cacodylate, pH 6.0, at 4°C at a flow rate of 6 ml/h. Aliquots were withdrawn from each fraction (0.38 ml) for determination of radioactivity (5 pl) (--) and for a spectrophotometric determination of Triton X-100 (15 ~1) (- - -) after dilution into 0.5 ml of 0.01 M sodium cacodylate, pH 6.0. Pool of [‘4C]penicilloyl.PBPs (Fractions 7 to 9) yielded 700,000 cpm. VO, column void volume.

4-

FIG. 2. SDS-polyacrylamide gel indicating release of bound [‘*C]penicilloyl moiety from S. aureus PBPs 1,2, and 4. Electro- phoresis and fluorography were as described under “Materials and Methods” with 4 pg of protein applied per lane. Lane 1, Coomassie blue staining of S. aureus PBPs 1, 2, 3, and 4; Lanes 2, 3, and 4, fluorograph of gel. Lane 2, fluorograph corresponding to Lane 1, indicating [‘*C]penicillin G bound to all 4 PBPs. Lane 3, [‘?]penicilloyl.PBPs 1, 2, and 3, as isolated by gel filtration (Fig. 1). Lane 4, same as in Lane 3 after incubation in 0.01 M sodium cacodylate, pH 6.0, 0.2% Triton X-100 for 10 h at 37°C.

Breakdown of the S. aureus [‘*C]Penicilloyl+ PBPs Com- plex-Incubation of S. aureus [ ‘*C]penicilloyl . PBPs 1,2, and 3 (isolated by gel filtration, as in Fig. 1) at 37°C resulted in a time-dependent release of the bound radioactive label. Anal- ysis of the acetone precipitated protein by SDS-gel electrophoresis and fluorography indicated that only PBPs 1 and 2


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12058 Penicillin-binding Proteins of S. aureus and B. subtilis

released the bound [i4C]penicilloyl moiety (Fig. 2, Lane 4). The decrease in the intensity of the radiolabeled band of PBP 3 reflects the total decrease in Coomassie-stained protein after a 10-h incubation at 37°C.

A more careful study of the kinetics of breakdown of S. aureus [i4C]penicilloylePBPs 1, 2, 3, and 4 indicated that breakdown of these covalent complexes proceeded with first order kinetics, characterized by half-lives of approximately 90 min, 40 min, and 1% min for PBPs 1, 2, and 4, respectively, when the incubation was carried out at 37”C, pH 7.5 (Fig. 3). The rates of breakdown of the complexes formed between [‘4C]penicillin G and PBPs in membrane fragments (determined as with the purified components, Fig. 3) were similar to the rates measured with the purified PBPs, indicating that purification of the PBPs by covalent penicillin affinity chromatography did not significantly alter this catalytic activity. Release of the bound penicilloyl moiety resulted in a reacti- vation of the penicillin-binding activity of these PBPs (not shown). Boiled [‘4C]penicilloyl.PBPs 1, 2, and 3 released the bound label from PBPs 1 and 2 at a rate 5 to 8 times slower than that observed with native complex.

Identification of the 14C-Labeled Penicillin Degradation Product-The 14C-labeled penicillin derivative released by S. aureus PBP 4 was identified as penicilloic acid (14). The

PEP 4

I

I I I 1 I I I 25 50

Time (‘m”i ns. o?h”c ) 125 150

FIG. 3. Kinetics of breakdown of S. UUIYIIS [14C]penicilloyl* PBPs. [‘4C]penicillin G (0.35 pg) was bound to a mixture of S. aureza PBPs 1, 2, 3, and 4 (5 pg) in 10 ~1 of 0.02 M Tris-Cl, pH 7.5, 0.2 M NaCl, 0.5% Triton X-100 for 10 min at 25”C, following which 35 pg of unlabeled penicillin G was added in 10 ~1 of H20. Samples were incubated at 37’C for the indicated times, and the release reaction then halted by addition of 4 volumes of cold acetone. Electrophoresis, fluorography, and densitometry were as described under “Materials and Methods”. Plots of log per cent bound uersus time were linear, indicating that breakdown of the [‘4C]penicilloyl. PBP complex was first order with half-lives of 90 min, 40 min, and 1% min for PBPs 1 (A-A), 2 (O---0), and 4 (M), respectively. Penicilloyl. PBP3 (M) did not breakdown.

penicillin degradation product released by the mixture of S. aureus [‘4C]penicilloyl. PBPs 1,2, and 3 was analyzed by thin layer chromatography after acidification to pH 2 and extrac- tion into ethyl acetate. This product was distinguished from penicilloic acid and from penicillin G by chromatography in Solvent A (see “Materials and Methods”) (RF [14C]penicillin degradation product = 0.48; Rr [14C]penicilloic acid = 0 and Rr [‘4C]penicillin G = 0.58). Treatment of the ethyl acetate extract with diazomethane increased the mobility of the major radioactive spot after chromatography in Solvent B from Rr = 0 to RF = 0.43 (Compound A, Fig. 4, Lamss 6 and 7), suggesting that this compound contained a free *carboxy group.

Enzymatically active PBPs catalyzed formation of the major 14C-labeled penicillin degradation product in a time-dependent manner (Fig. 4, Lanes 3 to 5). Boiling the mixture of [‘4C]penicilloyl. PBPs 1,2, and 3 or incubation in the presence of 1% SDS prevented formation of this compound (Lanes 1 and 2). Compounds formed by chemical degradation of the [‘4C]penicilloyl.PBPs 1, 2, and 3 (Lanes 1, 2, and 3) included penicilloic acid, dimethyl ester (Compound B), and other unidentified compounds.

The major 14C-labeled-penicillin degradation product was identified as phenylacetylglycine by co-migration of the radiolabeled compound with synthetic material on silica gel in Solvent A (see “Materials and Methods”). Similarly, the diazomethane derivatized [‘4C]penicillin degradation product co-migrated with synthetic phenylacetylglycine methyl ester on silica gel in Solvents B and C (RF = 0.43,0.58, respectively). These chromatographic systems were also employed to iden- tify the penicillin degradation product of the B. stearothermophilus and B. subtilis D-ahnine carboxypeptidases as phenylacetylglycine (21).2 Further confiiation of this identification was obtained by the co-crystallization of synthetic and radiolabeled methyl ester to constant specific activity (Table I). The possibility that small amounts of products other than phenylacetylglycine (e.g. penicihoic acid) may arise from the enzymatic degradation of the S. aureus penicilloyl. PBPs 1,2, and 3 has not been excluded.

Hydroxylamine-induced Breakdown of the [‘4C/Penicil- 10~1. PBP Complexes-Hydroxylaminolysis of the penicihoyl moiety from [‘4C]penicilloyl a PBP complexes has been shown to be enzyme-catalyzed (32). Although the S. aureus [‘“Cl- penicilloyl. PBP 3 compiex did not fragment spontaneously to yield [ 14C]phenylacetylglycine and reactivated enzyme, this PBP did catalyze hydroxylaminolysis of its bound penicilloyl moiety at a rate similar to that of PBP 2 (Fig. 5). Thus, it is unlikely that the apparent stability of the penicilloyl. PBP 3 complex resulted from complete inactivation of this PBP during purification.

Penicillin Release Catalyzed by B. subtilis PBPs-A mixture of B. subtilis PBPs 1, 2, and 4 was isolated by cephalosporin affinity chromatography as described (8). Breakdown of the B. subtilis [14C]penicilloyle PBPs was monitored by SDS-gel electrophoresis and fluorography (Fig. 6). PBP 1 was seen to catalyze a relatively rapid release of the bound penicilloyl moiety (tip = 10 min at 37°C). PBPs 2, 4, and 5 (the carboxypeptidase) released the bound label more slowly, with half-lives of approximately 300 min, 60 min, and 120 min, respectively, at pH 7.5 and 37°C. This release was significantly slower in the presence of 1% SDS (Lanes 1 and 2). Release of bound radioactivity from these PBPs (principally PBPs 2 and 4) was correlated with formation of [14C]phenylacetylglycine, as identified by thin layer chromatography of the acidified and ethyl acetate-extractable material on silica gel run in Solvent A. Small amounts of [14C]penicilloic acid might also have been formed (not shown). Thus, the higher molecular


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I 2 3 4 5 6 7

TABLE I Co-crystallization of methyl-“C-labeled degradation product and

phenylacetylglycine methyl ester Synthetic phenylacetylglycine methyl ester was mixed with diazo-

methane-treated [‘?]penicillin degradation product, purified by thin layer chromatography (see Fig. 4, Lane 6). Sample was dissolved in warm hexanes/ethyl acetate, 3:l (v/v) (20 al/mg of sample), and then recrystallized at 0°C for 1 h. Crystals were washed once with hexanes/ ethyl acetate, 3:l (v/v), and then dried to constant weight. Sample was dissolved in 1 ml of ethyl acetate, following which duplicate aliquots (50 to 200 ~1) were withdrawn for determination of radioactivity.

Crystallization MS.5 Radioactivity Specific activity

Before 1 2 3

w cPm cpm/mg

27.2 14,580 536 17.7 11,620 656 12.8 8,380 655 10.9 7.020 644

weight, cephalosporin-sensitive PBPs of B. subtilis also catalyze breakdown of the bound [‘?I!]penicilloyl moiety to yield [14C]phenylacetylglycine as the major degradation product.

DISCUSSION

Bacterial D-alanine carboxypeptidases bind penicillin to form covalent penicilloyl a enzyme complexes with concomi- tant loss of carboxypeptidase activity. Penicilloyl. carboxypeptidase complexes are known to reactivate with release of penicilloic acid (14, 19) or via a fragmentation pathway whereby the penicilloyl moiety is degraded to phenylacetylglycine and dirnethylthiazoline carboxylate, or its hydrolysis product, IV-forrnyl n-penicillamine (20-25). This paper dem- onstrates that PBPs other than n-alanine carboxypeptidases catalyze this enzymatic fragmentation.

Fragmentation of the penicilloyl moiety bound by cephalosporin-sensitive, higher molecular weight PBPs was studied in S. aureus, where the lowest molecular weight PBP, PBP4, is a carboxypeptidase-transpeptidase which rapidly releases

-A

-B

-C

FIG. 4. Analysis of [%]penicillin degradation product by autoradiography after thin layer chromatography. A mixture of [Wlpenicilloyl. PBPs 1,2, and 3 was prepared as in Fig. 1. Samples (3700 cpm) were incubated in 0.01 M sodium cacodylate, pH 6.0, 0.2% Triton X-100 for 0 to 10 h at 37”C, acidified, extracted into ethyl acetate, and treated with diazomethane (samples in Lanes 1 to 6) as described under “Mate- rials and Methods”. Samples were spot- ted on silica gel plates which were then developed with Solvent B (see “Materi- als and Methods”) and exposed to x-ray fii to detect radioactive compounds. Lane 1, sample boiled 3 min prior to lo- h incubation at 37°C. Lane 2, incubation for 10 h at 37’C in the presence of 1% SDS. Lanes 3 to 5 incubation for 0 h, 4 h, and 10 h, respectively, at 37°C. Lane 6, the major 14C labeled penicillin degradation product (Compound A) eluted from the silica gel with chloroform/ethyl acetate, 1:l (v/v) (2 X 1 ml), and rechro- matographed. Lane 7, sample as in Lane 5, in absence of diazomethane treatment. The mobility of Compound B corre- sponded to that of penicilloic acid dimethyl ester. Other products present in the control samples (Lanes 1 to 3), (including Compound C) were not identified.

PBP

I - 2- 3-

12345678910 FIG. 5. Fluorograph of SDS-gel indicating hydroxylamino-

lysis of [‘4C]penicilloyl moiety from all four S. UUIWU PBPs. [‘4C]Penicillin G was bound to PBPs 1, 2, 3, and 4 (5 pg of protein) following which a lOO-fold excess of unlabeled penicillin G was added, as in Fig. 3. Neutral hydroxylamine was then added (final concentra- tion, 0.8 M), following which samples were incubated at 25°C and periodically precipitated with 4 volumes cold acetone. Electrophoresis and fluorography were as described under “Materials and Methods”. Lanes 1, 2, incubation with hydroxylamine in the presence of 3% SDS. Lanes 1 to 10, incubation with hydroxylamine for 0, 90,0, 1, 5, 10, 15, 30, 60, 90 min, respectively.

its bound penicilloyl moiety as penicilloic acid (14)) and in B. subtilis, where a mixture of cephalosporin-sensitive PBPs can be obtained free from the major PBP (PBP 5, the carboxypeptidase) by cephalosporin affinity chromatography (8). As with the carboxypeptidases, breakdown of these penicilloyl. PBP complexes proceeded with first order kinetics and re- quired native penicilloyl. PBPs. [“‘ClPenicilloyl. PBP complexes were isolated by gel filtration and the major product of release from these cephalosporin-sensitive PBPs was identified as [14C]phenylacetylglycine. The possibility that small amounts of other products were formed, e.g. from the relatively small amount of S. aureus PBP 1 or B. subtilis PBP 1


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12060 Penicillin-binding Proteins of S. aureus and B. subtilis

PBP

:-

5-

1234 5678 FIG. 6. Fluorograph of SDS-polyacrylamide gel indicating

release of 14C label from B. .subtiZk [‘%]penicilloyl l PBPs 1, 2, 4, and 5. ]‘4C]Penicillin G (15 &ml) was bound to a mixture of B. subtilis PBPs 1, 2, and 4 (10 pg) in 30 ~1 of 50 mM Tris-Cl, pH 7.5, 0.5 M NaCl. 1% Triton X-100 for 10 min at 25°C. followina which a 100- fold exckss of penicillin G was added. Penicillmase (0.5 unit) was then added with subsequent incubation for various times at 37°C. The release reaction was halted by preparation of samples for electrophoresis (see “Materials and Methods”). Lanes 1 to 8, incubation at 37°C for 0, 720, 0, 20, 120, 220, 300, and 720 min, respectively. Lanes I and 2, incubation in presence of 1% SDS (control). Half-lives for the [‘4C]penicilloyl.PBP complexes were estimated to be 10 min (PBP l), 300 min (PBP 2), 60 min (PBP 4), and 120 min (PBP 5) at 37°C.

present in the mixtures of PBPs, could not be excluded. In fact, the rapidity of the breakdown of B. subtilis [%]penicil- 10~1. PBP 1 (tllz = 10 min at 37°C) suggests that this PBP might catalyze release of penicilloic acid. Purification of PBP 1 from B. subtilis membranes should help to clarify this question. The fragmentation product(s) arising from the thiazolidine ring of penicillin were not identified in this study.

The apparent stability of the S. aureus penicihoyl. PBP 3 complex in membranes (not shown) as well as with purified PBP 3, indicates that the resistance to spontaneous degradation observed with the purified PBP does not result from denaturation of the fragmentation activity during purification. In contrast to the apparent stability of this penicilloyl.PBP complex to fragmentation, PBP 3 did catalyze hydroxylaminolysis of its bound penicilloyl moiety (Fig. 5). This suggests that the enzymatic reactions of hydroxylaminolysis and fragmentation (20,32) follow different catalytic pathways. Differ- ences between hydroxylaminolysis and the fragmentation reaction have also been observed with the B. stearothermophi- Zus penicilloyl a carboxypeptidase complex.’ In the presence of hydroxylamine at neutral pH, hydroxylaminolysis yields penicilloyl hydroxamate and reactivated enzyme, while hydrolysis of the bound penicilloyl moiety to yield penicilloic acid is prevented effectively. At lower concentrations of hydroxylamine the slower fragmentation reaction predominates with subsequent acyl transfer from phenylacetylglycyl enzyme (likely to be an intermediate (21, 27)) to water as the favored nucleophilic acceptor? This specific transfer of penicilloyl moiety to hydroxylamine and of phenylacetylglycyl moiety to water catalyzed by the B. stearothermophilus carboxypeptidase may, in part, reflect the specific exclusion of certain nucleophiles by particular conformations at an acceptor site. Different specificity profiles have also been observed for acyl (phenylacetylglycyl, penicilloyl, or substrate) transfer to several nucleophiles catalyzed by a Streptomyces carboxypeptidase (27).

The B. stearothermophilus and B. subtilis [‘4C]penicilloyl. carboxypeptidase complexes have been isolated by gel filtra-

tion and contain 0.8 to 1.0 mol of penicillin bound/m01 of purified enzyme (6, 33). That these covalent penicilloyl.PBP complexes can also be isolated in near stoichiometric yields after denaturation by any one of several procedures (6) sup- ports the claim that a covalent complex forms in the native enzyme. The penicillin-binding activity of these and other PBPs is sensitive to mild heat or SDS denaturation, indicating that the binding reaction requires a specific enzymatic confor- mation and is not simply an artifact resulting from nonspecific acylation of a nucleophilic protein group by the reactive p- lactam of penicillin. The demonstration that the hydroxylaminolysis of the penicilloyl moiety bound by PBPs is enzyme- catalyzed (32) and that carboxypeptidases catalytically release the bound penicillin (20) provides further evidence that penicillin binds to an activated site. The recent demonstration that both penicillin and diacetyl-L-Iys-D-Ala-D-lactate (substrate) bind to the same serine residue in each of two penicillin-sensitive carboxypeptidases (5, 6) provides direct evidence for the proposal that penicillin is an active site-directed ac- ylating agent (2).

The degradation of [‘4C]penicillin G to yield [14C]phenylacetylglycine catalyzed by the higher molecular weight PBPs of 5’. aureus and B. subtilis indicates that these PBPs, for which no carboxypeptidase or transpeptidase activity has been described, are enzymatically active in the penicillin fragmentation reaction. Although this fragmentation is most likely the consequence of inherent catalytic properties of these PBPs, it proceeds too slowly to be of major physiological significance. The fact that these higher molecular weight, cephalosporin-sensitive PBPs catalyze several reactions described for n-alanine carboxypeptidases, including this fragmentation reaction, hydroxylaminolysis of the bound penicilloyl moiety and acyl-enzyme formation (14), indicates that an in vitro transpeptidase (or carboxypeptidase?) activity will likely be found for these and other high molecular weight PBPs, believed to include penicillin-sensitive transpeptidases essential for normal cell growth and viability.

Purification of the individual cephalosporin-sensitive PBPs (30)3 and studies of their in vitro transpeptidase activities are currently being pursued in several laboratories.

Acknowledgments-We wish to thank Doctors John W. Kozarich, James R. Rasmussen, and Christine E. Buchanan for their helpful advice and discussions during the course of this study.

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11. 12.

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D J Waxman and J L Strominger[14C]phenylacetylglycine.

and Bacillus subtilis active in the conversion of [14C]penicillin G to Cephalosporin-sensitive penicillin-binding proteins of Staphylococcus aureus

1979, 254:12056-12061.J. Biol. Chem.

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