The pH-dependence of class B and class C beta-lactamases

Area of Science:

• Biochemistry
• Enzymology
• Microbiology

Background:

• Beta-lactamases are enzymes that hydrolyze beta-lactam antibiotics, conferring bacterial resistance.
• Beta-lactamases are classified into three main structural classes: A, B, and C.
• Understanding the kinetic properties of different beta-lactamase classes is crucial for developing effective antibiotic therapies.

Purpose of the Study:

• To investigate the pH-dependence of kinetic parameters for class B and class C beta-lactamases.
• To compare the pH-dependent behavior of class B and class C beta-lactamases with class A enzymes.
• To elucidate the reaction mechanism underlying the pH-dependent hydrolysis of beta-lactam antibiotics by these enzymes.

Main Methods:

• Kinetic analysis of beta-lactamase activity across a range of pH values.
• Characterization of beta-lactamase II (class B) from Bacillus cereus and a class C beta-lactamase from Pseudomonas aeruginosa.
• Determination of kinetic parameters (kcat and kcat/Km) for the hydrolysis of benzylpenicillin and cephalosporin C.

Main Results:

• The pH-dependence of kinetic parameters for class B and class C beta-lactamases differed significantly from each other and from class A enzymes.
• For beta-lactamase II (class B), kcat/Km plots against pH were symmetrical, while kcat plots were not.
• A similar observation of asymmetrical kcat plots and symmetrical kcat/Km plots was made for the class C beta-lactamase from Pseudomonas aeruginosa.

Conclusions:

• The observed pH-dependence suggests a reaction mechanism involving two ionic forms of an intermediate.
• These intermediate forms contribute to product formation at different rates, explaining the asymmetrical kcat profiles.
• The findings provide insights into the catalytic mechanisms of different beta-lactamase classes, aiding in the design of enzyme inhibitors.