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Updated: Jul 30, 2026
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A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
Interaction of the pBR 322-coded RTEM beta-lactamase with substrates. Evidence for specific conformational
The Biochemical Journal
|February 1, 1982
Abstract:
The rate of inactivation of RTEM-1 beta-lactamase by Pronase is accelerated by class A ('resistant') penicillins. Other substrates (class S penicillin and cephalosporins) protect against the inactivation. Cefoxitin, a semi-synthetic cephamycin, induces a more extensive, hysteretic response. In its presence the enzyme is inactivated by trypsin as well as by Pronase.
Insights
Class A penicillins speed up RTEM-1 beta-lactamase inactivation by Pronase. Other penicillins and cephalosporins offer protection, while cefoxitin causes a prolonged, complex inactivation response with trypsin and Pronase.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- RTEM-1 beta-lactamase is a key enzyme conferring bacterial resistance to beta-lactam antibiotics.
- Understanding its inactivation mechanisms is crucial for developing strategies to overcome antibiotic resistance.
Purpose of the Study:
- To investigate the effects of different classes of penicillins and cephalosporins on the inactivation rate of RTEM-1 beta-lactamase by Pronase.
- To characterize the interaction of cefoxitin with RTEM-1 beta-lactamase and its impact on enzyme inactivation.
Main Methods:
- Enzyme kinetics assays were performed to measure the inactivation rates of RTEM-1 beta-lactamase.
- The enzyme was pre-incubated with various beta-lactam antibiotics (class A penicillins, class S penicillin, cephalosporins, cefoxitin) before exposure to Pronase or trypsin.
Main Results:
- Class A penicillins significantly accelerated the Pronase-mediated inactivation of RTEM-1 beta-lactamase.
- Other substrates, including class S penicillin and cephalosporins, protected the enzyme from inactivation.
- Cefoxitin induced a unique, hysteretic inactivation response, rendering the enzyme susceptible to both Pronase and trypsin.
Conclusions:
- The inactivation of RTEM-1 beta-lactamase is modulated by different beta-lactam substrates, with class A penicillins acting as accelerators and others as protectants.
- Cefoxitin exhibits a distinct mechanism of interaction, leading to a more profound and prolonged enzyme inactivation.
- These findings provide insights into the complex interplay between beta-lactam antibiotics and beta-lactamase enzymes, relevant to antibiotic resistance research.
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