Abstract
Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan. It was hypothesized (Tipper, D., and Strominger, J. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141) that 1) penicillin is a structural analog of the acyl-D-alanyl-D-alanine terminus of the pentapeptide side chains of nascent peptidoglycan, and that 2) penicillin, by virtue of its highly reactive beta-lactam structure, irreversibly acylates the active site of the cell wall transpeptidase. Although the cell wall transpeptidase has proven elusive, a closely related penicillin-sensitive cell wall enzyme, D-alanine carboxypeptidase, has been purified from membranes of Bacillus stearothermophilus by penicillin affinity chromatography. By amino acid sequence analysis of 14C-labeled cyanogen bromide peptides generated and purified from this carboxypeptidase covalently labeled with either [14C]penicillin G or the substrate, [14C]diacetyl-L-lysyl-D-alanyl-D-lactate, it was shown that the penicillin and substrate were both bound as esters to a serine at residue 36. Therefore, the second hypothesis stated above was proven to be correct for D-alanine carboxypeptidase. Several new methods were developed in the course of this work, including 1) a rapid penicillin-binding assay, 2) use of hydroxylamine to protect peptides against carbamylation during ion exchange chromatography in concentrated urea solutions, and 3) gel filtration chromatography in 70% formic acid, a universal solvent for peptides.
Highlights
Penicillin kills susceptible bacteria byspecifically thesizingcross-linkedpeptidoglycan (4-6), solubilization of inhibiting the transpeptidase that catalyzes the final these membranes with detergents causes immediate loss of step in cell wall biosynthesis, the cross-linking of pep- transpeptidase activity (6, 7)
Several new methods were developed in the course of this work, including 1) a rapid penicillin-binding assay, 2) use of hydroxylamine toprotect peptides against carbamylation during ion exchange chromatography in concentrated urea solutions, and 3) gel filtration chromatography in 70% formic acid,a universal solvent for peptides
Authentic [14C]penicilloic acid was generated by treating 200 p1 of 0.1 M ['4C]penicillin G with 1 unit of Bacillus cereus penicillinase (Sigma) for 2 h at 25°C in 50 mM NaP04,pH 7.0. ['4C]Penicilloicacid released from labeled peptides was identified by thin layer chromatography on Silica Gel F254 (Merck) along with the authentic compound using the solvent, water/acetic acid/n-butyl alcohol (1:2:4)
Summary
Active Site of catalysis is rapid compared to the acylation step, so the steady state concentration of acylenzyme intermediate is low. During the 1-h coupling reaction, CPase wassolubilizedfrom a thawed membrane suspension by addition of 4 M NaCl and 25% Triton X-100 to give final concentrations of 1.0M NaCl and 5%Triton X-100 This suspension was stirred 30 min at 0°C and centrifuged at 48,000 X g for I h to remove insoluble material. The washed 6-APA-Sepharose was added tothe solubilized crude CPase (1 ml of 6-APASepharose/lO ml of solubilized membranes) and the mixture was swirled gently at 37°C for 30 min. After drying over anhydrous sodium sulfate and filtration, the ethyl acetate solution was warmed and hexane added tothe point of cloudiness.The yield of crystalline product was 0.97 g 3. Purification of ['*C]penicillin- and ['4C]AczLALac-labeled CNBr peptides.B. stearothermophibs CPase (480nmol) was covalently labeled as described under "Experimental Procedures," cleaved with CNBr, and the resulting peptides were fractionated on a column (1.5 X 15 cm) of SP-Sephadex.
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