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Updated: Sep 30, 2026
Homemade Site Directed Mutagenesis of Whole Plasmids
Published on: May 10, 2009
Site-directed mutagenesis of beta-lactamase leading to accumulation of a catalytic intermediate
W A Escobar1, A K Tan, A L Fink
1Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
Abstract:
Site-specific mutation of Glu-166 to Ala in beta-lactamase causes a millionfold reduction in catalytic activity toward both penicillin and cephalosporin substrates and results in the stoichiometric accumulation of a normally transient acyl-enzyme intermediate. Kinetic analysis indicated that substitution of Glu-166 by Ala leads to negligible effect on the acylation half of the reaction but effectively eliminates the deacylation reaction. Such differential effects on the rates of formation and breakdown of an enzyme-substrate intermediate have not been previously reported. Thus, unlike the situation for most transfer enzymes, e.g., the serine proteases, acylation and deacylation in beta-lactamase catalysis are not "mirror" images and must involve different mechanisms. The results suggest an explanation for the different catalytic activities between the beta-lactamases and the penicillin-binding proteins involved in bacterial cell-wall synthesis.
Insights
Mutating Glu-166 to Ala in beta-lactamase dramatically reduces catalytic activity and halts deacylation. This suggests distinct mechanisms for acylation and deacylation in beta-lactamase, differing from serine proteases.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Beta-lactamase enzymes are crucial for bacterial resistance to antibiotics like penicillin and cephalosporin.
- Understanding the catalytic mechanism of beta-lactamase is key to developing new antibacterial strategies.
- Enzyme-substrate intermediates are transient species formed during catalysis, and their properties offer insights into reaction mechanisms.
Purpose of the Study:
- To investigate the role of Glutamic acid 166 (Glu-166) in the catalytic mechanism of beta-lactamase.
- To determine the impact of specific mutations on enzyme activity and intermediate formation/breakdown.
- To compare the catalytic mechanism of beta-lactamase with other transfer enzymes like serine proteases.
Main Methods:
- Site-specific mutagenesis was used to replace Glu-166 with Alanine (Ala) in the beta-lactamase enzyme.
- Kinetic analysis was performed to measure the rates of acylation and deacylation reactions with penicillin and cephalosporin substrates.
- The accumulation of acyl-enzyme intermediates was monitored to assess their stability.
Main Results:
- The Glu-166 to Ala mutation resulted in a millionfold decrease in catalytic activity against both penicillin and cephalosporin.
- The mutation led to the stoichiometric accumulation of the acyl-enzyme intermediate, which is normally transient.
- Kinetic data showed negligible effects on the acylation step but a complete elimination of the deacylation step.
Conclusions:
- Acylation and deacylation in beta-lactamase catalysis are not symmetrical and likely involve different mechanistic pathways.
- The findings suggest that the catalytic mechanism of beta-lactamase differs significantly from that of serine proteases.
- This study provides insights into the distinct catalytic strategies of beta-lactamases compared to penicillin-binding proteins involved in bacterial cell-wall synthesis.
