Carboxy groups as essential residues in beta-lactamases

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Beta-lactamases are enzymes crucial for antibiotic resistance, classified into groups A, B, and C based on amino acid sequences.
• Understanding the active sites and inactivation mechanisms of beta-lactamases is vital for developing new therapeutic strategies against bacterial infections.

Purpose of the Study:

• To investigate the inactivation mechanism of beta-lactamases using a carboxy-group reagent.
• To determine the extent of enzyme inactivation and nucleophile incorporation in different beta-lactamase classes.
• To identify the specific amino acid residues involved in the inactivation process.

Main Methods:

• Incubation of beta-lactamases (two class A, two class C) at pH 4.0 with 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide and a colored nucleophile.
• Monitoring of enzyme inactivation and nucleophile incorporation.
• Amino acid sequencing to pinpoint the location of incorporated nucleophile.

Main Results:

• All four examined beta-lactamase enzymes (Bacillus cereus, Bacillus licheniformis, Enterobacter cloacae P99, Pseudomonas aeruginosa) were inactivated by the reagent.
• Complete inactivation correlated with the incorporation of approximately 2-3 moles of nucleophile per mole of enzyme.
• In beta-lactamase I from Bacillus cereus, 53% of the incorporated nucleophile was localized to glutamic acid-168.

Conclusions:

• The carboxy-group reagent effectively inactivates beta-lactamases from both class A and class C.
• Glutamic acid-168 in Bacillus cereus beta-lactamase I is a key site for nucleophile incorporation during inactivation.
• These findings contribute to understanding beta-lactamase structure-function relationships and potential inhibition strategies.