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A membrane enzyme from Staphylococcus aureus which catalyzes transpeptidase, carboxypeptidase, and penicillinase
Abstract:
Staphylococcus aureus H membranes were found to contain four major binding components: Mr = 115,000; Mr = 100,000 doublet; and Mr = 46,000. The low molecular weight protein bound penicillin reversibly and was purified by prebinding membranes with penicillin prior to affinity chromatography. The purified protein catalyzed transpeptidase and carboxypeptidase reactions using di[14C]acetyl-L-lysyl-D-alanyl-D-alanine as the substrate and glycine and hydroxylamine as the acceptors. In addition, the enzyme catalyzed a penicillinase reaction. Kinetic analysis of these reactions revealed similar Vmax values suggesting that, if there is a single active site, the rate-determining steps (i.e. deacetylation) are similar. Rapid denaturation of the enzyme.substrate complex resulted in the detection of covalent penicilloyl- and diacetyl-L-lysyl-D-alanyl.enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Insights
Researchers identified a low molecular weight protein in Staphylococcus aureus H membranes that binds penicillin. This protein, purified via affinity chromatography, exhibits transpeptidase, carboxypeptidase, and penicillinase activities, suggesting a shared active site for these reactions.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Staphylococcus aureus H membranes possess multiple penicillin-binding components.
- Understanding these interactions is crucial for developing targeted antimicrobial strategies.
Purpose of the Study:
- To identify and characterize the penicillin-binding proteins in Staphylococcus aureus H membranes.
- To investigate the enzymatic activities of the purified low molecular weight penicillin-binding protein.
Main Methods:
- Affinity chromatography using penicillin prebinding.
- Enzymatic assays for transpeptidase, carboxypeptidase, and penicillinase activities.
- Kinetic analysis and SDS-PAGE to study enzyme-substrate complexes.
Main Results:
- Four major binding components were identified in S. aureus H membranes.
- A low molecular weight protein (Mr = 46,000) was purified.
- The purified protein demonstrated transpeptidase, carboxypeptidase, and penicillinase activities.
- Kinetic data suggested a single active site for these reactions.
Conclusions:
- The low molecular weight penicillin-binding protein from S. aureus H exhibits multiple enzymatic functions.
- These findings provide insights into the mechanism of penicillin action and resistance.
- The study highlights a potential target for novel antibiotic development.