Isolation of the Penicillin-Binding Peptide from D-Alanine
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Proc. Natl. Acad. Sci. USA Vol. 74, No. 3, pp. 1009-1012, March 1977 Biochemistry Isolation of the penicillin-binding peptide from D-alanine carboxypeptidase of Bacillus subtilis (penicilloyl-carboxypeptidase/peptide fragments/ester bond) NAFSIKA GEORGOPAPADAKOU, SVEN HAMMARSTROM, AND J. L. STROMINGER The Biological Labhratories, Harvard University, Cambridge, Massachusetts 02138 Contributed by Jack L. Strominger, January 6, 1977 ABSTRACT The D-alanine carboxypeptidase of B. subtilis we report two such fragments resulting from a tryptic and is a membrane-bound enzyme which is inhibited by penicillins Pronase digestion, respectively. and binds them covalently. The enzyme has been labeled with [14C- or [a5SSpenicillin. After tryptic or Pronase digestion of the MATERIALS AND METHODS labeled, denatured, reduced, and carboxymethylated enzyme, a radioactive peptide was isolated in each case. The amino acid Materials. Deoxyribonuclease, lysozyme, and dithiothreitol compositions of these two peptides are reported. The Pronase were purchased from Sigma; L-1-tosylamido-2-phenylethyl peptide was a subset of the tryptic peptide. Neither contained chloromethyl ketone (TPCK)-trypsin from Worthington; a cysteine residue and the only amino acid in the Pronase pep- Pronase from Calbiochem; Sephadex G-50 from tide to which the penicillin could be bound was a serine resi- (fine) Phar- due. macia; guanidine hydrochloride from Heico; dansyl chloride, Chen-Ching polyamide sheets, standard amino acid mixture, The D-alanine carboxypeptidase (CPase) of Bacillus subtilis 6 M HCl, ninhydrin, fluorescamine, and triethylamine from is a membrane-bound enzyme which catalyzes the hydrolysis Pierce; [8-14C]penicillin G, potassium salt (specific activity: 54 of the terminal D-alanine residue of UDP-MurNAc-penta- mCi/mmol) from Amersham/Searle. [35S]Penicillin G, potas- peptide, the uridine nucleotide intermediate in cell wall pep- sium salt (specific activity: 1.6 mCi/mmol) was kindly syn- tidoglycan synthesis. The reaction is similar to the transpepti- thesized by Richard Thoma of E. R. Squibb and Sons. Pyridine dase reaction involved in cell wall biosynthesis, except that was purified by distillation after 2 hr of refluxing with ninhy- water rather than an amino group is the acceptor. Both reac- drin (5 g of ninhydrin per liter of pyridine). Iodoacetic acid was tions, it was suggested, proceed through an acyl-enzyme in- purchased from Eastman and purified by recrystallization from termediate (1), and both are inhibited by penicillins. CPase is petroleum ether. All other reagents were of analytical grade. a moderate-sized protein (50,000 molecular weight) (2), is the Preparation of Labeled CPase. CPase was isolated by af- major penicillin binding component in B. subtilis (3), and has finity chromatography (4) from B. subtilis (strain Porton) been purified to homogeneity by affinity chromatography (4). membranes obtained by lysozyme DNase treatment (11). In Its function is not known, but it might be involved in the reg- a typical experiment, 10 mg of enzyme was obtained from 300 ulation of crosslinking during cell wall biosynthesis. g of cells. The enzyme in 5 ml of 0.05 M Tris-HCI buffer (pH It was suggested earlier that the penicilloyl-enzyme complex 7.5) containing 1% (vol/vol) Triton X-100, 0.5 M NaCl and 1 might be a penicilloyl thioester involving a cysteine residue in mM 2-mercaptoethanol] was incubated with labeled penicillin the susceptible enzyme. This suggestion was based on the in- G ([8-14C]penicillin G, 25 ,uCi, or [35S]penicillin G, 0.8,Ci) for hibition of penicillin binding by thiol reagents and the release 10 min at 25°. The incubation was stopped by adding 4 volumes of a penicilloyl derivative by hydroxylamine and ethanethiol of cold acetone containing a 100-fold molar excess of unlabeled (5). However, the concentrations of thiol inhibitors which were penicillin G. The resulting suspension was centrifuged at 11,000 effective may have been too high to be specific (6). Further- X g for 20 min, and the pellet was dried under a stream of ni- more, an alternative interpretation of the release of penicilloyl trogen. by hydroxylamine has been obtained, namely, the release is an Denaturation, Reduction, Carboxymethylation, and enzymatically catalyzed transfer of penicilloyl to hydroxyl- Trypsin or Pronase Digestion. The precipitated carboxy- amine, and denatured penicilloyl enzyme is stable under con- peptidase, labeled with penicillin G (160 nmol, 1.1 X 107 dpm) ditions in which acyl thioesters do not migrate and are cleaved was dissolved in 2 ml of 6 M guanidine-HCI containing 2 mM by hydroxylamine (refs. 6-10; S. J. Curtis and J. L. Strominger, EDTA, and 0.2 M Tris-HCI (pH 8.2). After 6 mg of di- manuscript submitted for publication). The chemical reactivity thiothreitol had been added, the solution was incubated under of penicilloyl-enzyme, denatured under several conditions, was nitrogen at 370 for 1 hr. Then 20 mg of iodoacetate was added compatible with the presence of a serine ester (8). However, at and the solution was incubated under nitrogen and in the dark least in the case of Escherichia coli, D-alanine carboxypeptidase at room temperature for 1 hr. The solution was dialyzed against IA, a sulfhydryl group, presumably in the active site, is involved distilled water at 40 during which the carboxypeptidase pre- in the deacylation of the penicilloyl- and acyl-enzyme. Its cipitated. The resulting suspension was lyophilized. After lyo- substitution by thiol reagents results in stabilization of the philization, the carboxymethylated carboxypeptidase was penicilloyl-enzyme and in accumulation of an acyl-enzyme dissolved in 3 ml of 0.1 M NH4HCO3 and TPCK-trypsin was intermediate derived from a normal substrate (ref. 8; S. J. Curtis added to a final concentration of 10% (wt/wt) relative to the and J. L. Strominger, manuscript submitted for publication). carboxypeptidase. The hydrolysis was carried out at 370 for 1 These findings prompted a close examination of the peni- hr, and the reaction was stopped by freezing with dry ice-ace- cilloyl-enzyme linkage, by isolating and characterizing peptide tone followed by lyophilization. Pronase digestion was per- fragments containing bound penicillin. In this communication, formed under the same conditions as trypsin digestion. If longer incubation times were used, much of the bound penicillin was Abbreviation: CPase, D-alanine carboxypeptidase. released. 1009 Downloaded by guest on October 2, 2021 1010 Biochemistry: Georgopapadakou et al. Proc. Natl. Acad. Sci. USA (74) 1977 Carboxypeptidase [ 14C] penicillin G [14C] penicilloyl-CPase I 1. dithiothreitol 2. iodoacetate carboxymethylated [ 14C] penicilloyl-CPase (50%) * Pronase Trypsin mixture of peptides mixture of peptides 1. G-50 chromatography 1. G-50 chromatography (30%) 2. pH 6.5 electrophoresis 2. pH 6.5 electrophoresis (15%) 3. descending chromatography 3. pH 3.5 electrophoresis (5%) 4. pH 3.5 electrophoresis I 4. descending chromatography (2%) Pronase [14C] penicilloyl-peptide tryptic [14C]penicilloyl-peptide FIG. 1. Flow diagram for the isolation of tryptic and Pronase peptides from 14C-labeled CPase. Approximate yields for [14C]penicilloyl-CPase, based on radioactivity, are given in parentheses. Isolation of Labeled Peptides. Fractionation was first water/pyridine (15/3/12/10) (13). Analytical chromatography performed on a Sephadex G-50 (fine) column (1.2 X 100 cm) was carried out in isoamyl alcohol/pyridine/water (6/6/7) (14). with 0.1 M NH4HCO3 as eluent at a flow rate of 10 ml/hr at The purifications are summarized in Fig. 1. 4°. Fractions of 2 ml were collected and monitored for both 14C Transfers of peptide between fractionations were performed content (by counting 50 ,l aliquots in Aquasol) and peptide by the "sewing" method (15). Peptides were located by spraying content (by measuring A23o nm). The fractions comprising the guide strips with triethylamine/CH3Cl (1/20, vol/vol) followed major radioactive peak were pooled and lyophilized. Further by fluorescamine/acetone (0.1/100, wt/vol) and then visual- purification was achieved by high voltage electrophoresis at pH izing under long-wavelength ultraviolet light. Radioactivity 6.5 and 3.5 and descending chromatography on Whatman was located by autoradiography. The concentration of the ra- 3MM paper (46 X 57 cm) prewashed with 1% acetic acid. The dioactive peptides was calculated from the radioactivity electrophoresis buffers (all vol/vol) were pyridine/acetic measurements and the specific activity of the label. acid/water (50/2/950; pH 6.5) and pyridine/acetic acid/water Performic Acid Oxidation and Amino Acid Analysis. The (1/10/89; pH 3.5) (12), and the electrophoreses were carried purified radioactive peptides (usually 1 nmol), eluted from the out on a flat plate and in a tank, respectively. Chromatography paper with distilled water, were hydrolyzed in 6 M HC1 in of the peptides was carried out in 1-butanol/acetic acid/ evacuated tubes at 1100 for 20 hr. When analyses for cysteic acid were performed, separate samples were subjected to per- formic acid and oxidation by the procedure of Moore (16) prior to hydrolysis with acid. Amino acid analyses were performed on a Durrum d-500 or a Beckman 121 M amino acid ana- lyze. NH2-terminal Amino Acid Determination. Approximately 0.2-0.5 nmol of peptide was dansylated by the procedure of Gray and Hartley (17). The dansyl amino acids were identified 20 40 60 Fraction no. ' by comparison with a standard dansylated amino acid mixture SEPHADEX G-50 CHROMATOGRAPHY , / pH 6.5 ELECTROPHORESIS after two-dimensional polyamide thin-layer chromatogra- phy. RESULTS cm D. Properties of the penicilloyl-enzyme linkage o- -Origin _ _ _ _ _ Labeled penicillin was bound to the B. subtilis D-alanine car- 10-o4. boxypeptidase under conditions that led to complete inhibition -10- [35S] of the enzyme (18). After carboxymethylation, there was 0.5 -11~~~ -['4c+35SI mol of The 30- [k4C] 0 - Origin mol of labeled penicillin per enzyme.