Formation of 5,5-Dimethyl-~2-thiazoline-4-carboxylic Acid during Cleavage of Penicillin G by D-Alanine Carboxypeptidase from BaciZZus stearothermophiZus*

n-Alanine carboxypeptidase from Bacillus stearothermophilus is a membrane-bound enzyme which catalyzes a reaction resembling the cross-linking of peptidoglycan strands in the biosynthesis of bacterial cell walls. However, water rather than an amino agroup is the acyl acceptor. The enzyme is inhibited by penicillins which bind covalently to it (1). We have recently reported that the enzyme also catalyzes the degradation of penicillin G to phenylacetylglycine (2) during a self-catalyzed reactivation (3,4). The same conversion of penicillin G was observed with n-alanine carboxypeptidase from B. subtilis (4) and subsequently with a soluble carboxypeptidase from Streptomyces (5). The reactivation is relatively rapid (t,,2 = 10 min) for the thermophilic B. stearothermophilus enzyme but slower for the mesophilic B. subtilis enzyme (t 112 = 200 min) at the temperature optima for the enzymes (55” and 37”, respectively). The degradation reaction implies that the bonds between the a-carbonyl and N-4 and between C-5 and C-6 of the penicillin molecule are cleaved and that yet another product, derived from the thiazolidine ring, is formed (2). The nature of this product is the subject of the present report.

n-Alanine carboxypeptidase from Bacillus stearothermophilus is a membrane-bound enzyme which catalyzes a reaction resembling the cross-linking of peptidoglycan strands in the biosynthesis of bacterial cell walls. However, water rather than an amino agroup is the acyl acceptor. The enzyme is inhibited by penicillins which bind covalently to it (1). We have recently reported that the enzyme also catalyzes the degradation of penicillin G to phenylacetylglycine (2) during a self-catalyzed reactivation (3,4). The same conversion of penicillin G was observed with n-alanine carboxypeptidase from B. subtilis (4) and subsequently with a soluble carboxypeptidase from Streptomyces (5). The reactivation is relatively rapid (t,,2 = 10 min) for the thermophilic B. stearothermophilus enzyme but slower for the mesophilic B. subtilis enzyme (t 112 = 200 min) at the temperature optima for the enzymes (55" and 37", respectively). The degradation reaction implies that the bonds between the a-carbonyl and N-4 and between C-5 and C-6 of the penicillin molecule are cleaved and that yet another product, derived from the thiazolidine ring, is formed (2). The nature of this product is the subject of the present report.
was prepared by trifluoroacetic acid degradation of penicillin G methyl ester (7). The crystalline product gave an NMR spectrum in CDCl,, identical with that published before (7). Treatment of the ester with 1 eq of 5% KOH in 50% aqueous methanol (1 mgl ml) were incubated at 37" for 10 min. The preincubation with cephalothin inactivates the minor penicillin binding proteins but has no effect on o-alanine carboxypeptidase (1). ["SlPenicillin G, 0.15 ml (1 mg/ml), was then added and the incubation was continued for another 10 min at 37". The membrane-bound penicilloyl carboxypeptidase was separated from excess lR"Slpenicillin G by centrifugation at 100,000 x g for 1 h. The pellet was resuspended in cold Tris buffer, centrifuged again, and finally resuspended in 20 ml of 0.01 M sodium cacodylate buffer, pH 6.5, containing 2.4 mg of potassium 5,5-dimethyl-AZ-thiazoline-4-carboxylate and incubated at 55" for 50 min to release the bound penicillin (4). After centrifugation at 100,000 x'g for 1 h, proteins remaining in the supernatant were precipitated with acetone (80% v/v, final concentration) and removed by centrifugation at 11,000 x g for 20 min. out on Whatman No. 3MM papers (46 x 57 cm) at pH 3.5 or pH 8.2 in cooled tanks (8). Compounds were detected with 0.5% KMnO,, 1% Na2C03 or 0.2% ninhydrin, 0.5% acetic acid in butanol-l:water, 955, v:v. Radioactivity was detected by autoradiography or determined in a liquid scintillation spectrometer after cutting appropriate zones of the paper in pieces (1 x 1 cm) and suspending these in Omnifluor/ toluene.

AND DISCUSSION
Experiments with [8-14C]penicillin G and [?S]penicillin G showed that under the conditions described above equal percentages of radioactivity bound to n-alanine carboxypeptidase (21 and 18%, respectively). Moreover, incubation of [YJI-and the [YSlpenicilloyl carboxypeptidase at 55" caused release of similar percentages of bound radioactivity (81 and 88%, respectively). This indicates that the whole penicillin molecule binds to the enzyme and that stoichiometric amounts of 14C and 35S degradation products are released during the reactivation of the enzyme.
Analysis of the 14C-labeled product by thin layer chromatography after extraction and methylation showed that the radioactivity co-chromatographed with phenylacetylglycine methyl ester, as demonstrated previously (2). The YS-labeled material was nonextractable with ethyl acetate. It separated from solubilized proteins by acetone precipitation of the latter. High voltage paper electrophoresis at pH 3.5 (Fig. la) showed that about 60% of the radioactivity co-migrated with potassium 5,5dimethyl-AZ-thiazoline-4-carboxylate.
The radioactive components immediately in front of.and behind the reference com- pound each contained about 20% of the radioactivity. These components increased to about 30% each if the electrophoresis was carried out at 2.5 kV and 35 min instead of 5 kV and 17 min, indicating that degradation of the thiazoline occurred during the electrophoresis. Also, during high voltage electrophoresis at pH 8.2, the major radioactive component co-migrated with 5,5-dimethyl-AZ-thiazoline-4-carboxylate.
The radioactive components moved toward the anode, both at pH 3.5 and pH 8.2, in agreement with the pK values for the thiazoline (9). Acid hydrolysis of methyl 5,5-dimethyl-AZ-thiazoline-4carboxylate yields n-penicillamine hydrochloride (7). Two hundred micrograms of potassium 5,5-dimethyl-AZ-thiazoline-4-carboxylate plus 200,000 cpm of the Y&labeled degradation product were hydrolyzed under the same conditions. High voltage paper electrophoresis at pH 3.5 (Fig. lb) showed that the unlabeled thiazoline had been completely converted to a ninhydrin-positive component with the same electrophoretic mobility as authentic n-penicillamine and that the radioactivity was now homogenous and co-migrated with this component. Further evidence for identity of the [YSlpenicillin G degradation product and 5,5-dimethyl-AZ-thiazoline-4-carboxylic acid was obtained by crystallizing a mixture of the potassium salt of the thiazoline (12.5 mg) and 52,000 cpm of the degradation product from ethyl acetate:methanol, 9:1, v:v, at 4" ( Table I).
The results indicate that 13S1penicillin G is degraded to 5,5dimethyl-AZ-thiazoline-4-carboxylic acid, during its interaction with n-alanine carboxypeptidase from Bacillus stearothermophilus.
This gives additional support for our previously proposed mechanism of penicillin G cleavage (Fig. 2) by this enzyme (2) and indicates a further similarity between the enzymatic reaction and a chemical degradation of penicillin G methyl ester in trifluoroacetic acid (7). The results also exclude an alternative pathway for the enzymatic formation of phenylacetylglycine, in which cu-formyl phenylacetylglycine is an intermediate, since this pathway would lead to the formation of [3S]penicillamine from [35S]penicillin G. After the completion of this manuscript, it has been reported that soluble n-alanine carboxypeptidase from Streptomyces catalyzes a conversion of penicillin G to phenylacetylglycine plusN-formyl penicillamine (10). N-For-my1 penicillamine is a hydrolysis product of 5,5-dimethyl-AZ-thiazoline-4-carboxylate, which might have been formed during isolation or analysis (or both). This view is supported by the observed lability during electrophoresis of the thiazoline identified here and by the fact that unlabeled thiazoline was added in the present experiments to trap the radioactive product formed from [35S]penicillin G. Alternatively, the mechanism of penicillin G degradation may be different for the two enzymes or the Streptomyces enzyme might catalyze a further hydrolysis of the thiazoline.