Substrate specificity of penicillin amidase from E. coli

Area of Science:

• Biochemistry
• Enzymology

Background:

• Penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11) from E. coli is crucial for beta-lactam antibiotic synthesis.
• Understanding its kinetic parameters is vital for optimizing enzymatic production.

Purpose of the Study:

• To determine kinetic parameters for 12 penicillin amidase substrates.
• To investigate the influence of free amino groups on reaction kinetics and enzyme binding.
• To identify hindrances in the enzymatic synthesis of specific beta-lactam antibiotics.

Main Methods:

• Determination of kinetic parameters (kcat, Km) for 12 E. coli penicillin amidase substrates.
• Study of pH-dependences of kcat/Km for enzymatic hydrolysis of specific p-nitroanilides.
• Analysis of substrate binding and reaction mechanisms under varying pH conditions.

Main Results:

• Most phenylacetyl-containing substrates exhibited similar catalytic constants (kcat ≈ 50 s-1).
• Substitution with 2-thienylacetyl groups slightly decreased kcat.
• Substrates with free amino groups showed strong kcat dependence on the leaving group, with the enzyme binding only the deprotonated substrate form.
• Low reaction rates, particularly at low pH, limit the enzymatic synthesis of penicillins and cephalosporins with free amino groups.

Conclusions:

• Enzymatic synthesis of beta-lactam antibiotics with free amino groups is hindered by low reaction rates under thermodynamically favorable conditions.
• The enzyme's preference for the deprotonated substrate form and the bimolecular reaction regime at low pH contribute to this limitation.