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Penicillinase-releasing protease of Bacillus licheniformis 749 Specificity for hydroxyamino acids
Abstract:
The membrane penicillinase of Bacillus licheniformis 749/C differs from the exopenicillinase in that it has an additional 24 amino acid residues and a phosphatidylserine at the NH2 terminus (Yamamoto, S., and Lampen, J.O. (1976) J. Biol. Chem. 251, 4095-4101). The conversion of the membrane penicillinase to the exo form is probably carried out by a specific penicillinase-releasing protease (PR-protease) whose properties are generally consistent with the properties of penicillinase secretion. The substrate specificity of the PR-protease was determined by identifying the NH2 and COOH termini of the peptides produced by hydrolysis of ribonuclease B and beef insulin. The enzyme hydrolyzed only peptide bonds involving the carboxyl groups of serine or thrombine. Similar bonds in synthetic di- or tripeptides of L-serine were not cleaved. The existence of seryl-lysine and threonyl-glucamic acid bonds in the protease-susceptible (phospholipopeptide) region of the membrane penicillinase and the presence of only lysine or glutamic acid at the NH2 terminus of the exoenzyme released in vivo are consistent with the specificity of PR-protease; hence, we propose that this enzyme has an essential role in the formation of exopenicillinase. The PR-protease is a potential tool for protein sequence determination because of its narrow and novel substrate specificity.
Insights
A novel protease (PR-protease) from Bacillus licheniformis releases exopenicillinase from its membrane-bound precursor. This protease specifically cleaves seryl-lysine and threonyl-glutamic acid bonds, aiding in enzyme secretion.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Bacillus licheniformis secretes exopenicillinase, a valuable enzyme.
- Membrane-bound penicillinase differs from exopenicillinase by additional amino acids and a lipid modification.
- A specific protease is hypothesized to convert membrane penicillinase to exopenicillinase.
Purpose of the Study:
- To determine the substrate specificity of the penicillinase-releasing protease (PR-protease).
- To elucidate the role of PR-protease in the secretion of exopenicillinase.
- To assess the potential of PR-protease for protein sequencing.
Main Methods:
- Hydrolysis of ribonuclease B and beef insulin using PR-protease.
- Identification of NH2 and COOH termini of resulting peptides.
- Analysis of synthetic di- and tripeptides of L-serine.
Main Results:
- PR-protease specifically cleaves peptide bonds at the carboxyl group of serine or threonine.
- Synthetic serine-containing peptides were not cleaved, indicating a requirement for specific adjacent residues.
- The identified cleavage sites align with the structure of membrane penicillinase and the released exopenicillinase.
Conclusions:
- PR-protease plays a crucial role in the formation of exopenicillinase by cleaving its membrane-bound precursor.
- The enzyme's specific cleavage activity is consistent with in vivo exopenicillinase release.
- PR-protease's narrow substrate specificity makes it a potential tool for protein sequence determination.