Trapping the acyl-enzyme intermediate in beta-lactamase I catalysis

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Beta-lactamase I is crucial for antibiotic resistance.
• Understanding its catalytic mechanism, particularly the acyl-enzyme intermediate, is key.
• Cryoenzymology offers a method to stabilize transient intermediates.

Purpose of the Study:

• To trap and characterize the acyl-enzyme intermediate in beta-lactamase I catalysis.
• To investigate the role of water in the enzyme's rate-determining step.
• To assess the suitability of aqueous methanol as a cryosolvent for beta-lactamase I.

Main Methods:

• Cryoenzymology was employed using aqueous methanol cryosolvents.
• Enzyme stability and function were tested across a temperature range (-75 to 25°C).
• High-performance liquid chromatography (HPLC) and peptide digestion (pepsin, trypsin) were used for analysis.

Main Results:

• Beta-lactamase I remained stable and functional in 70% methanol at or below 0°C.
• The catalytic rate (kcat) decreased with increasing methanol concentration, indicating water's role in the rate-determining step.
• The acyl-enzyme intermediate was successfully trapped at -40°C, with covalent attachment confirmed at Ser-70.

Conclusions:

• 70% methanol is a suitable cryosolvent for studying beta-lactamase I.
• Water, shielded within the enzyme-substrate complex, is involved in the deacylation step.
• Ser-70 is identified as the active site residue for acyl-enzyme intermediate formation.