Penicillinamidohydrolase in Escherichia coli. I. Substrate specificity

Area of Science:

• Biochemistry
• Enzymology
• Microbiology

Background:

• Penicillin amidohydrolase (penicillinacylase) is an enzyme found in bacteria.
• Understanding its substrate specificity is crucial for enzyme applications.
• The enzyme from Escherichia coli is located in the periplasmic space.

Purpose of the Study:

• To determine the substrate specificity of bacterial penicillin amidohydrolase from Escherichia coli.
• To investigate enzyme hydrolysis rates for various phenylacetic and phenoxyacetic acid derivatives.
• To explore potential applications of enzyme substrates for activity assays.

Main Methods:

• Measuring initial hydrolysis rates of different substrates under zero-order kinetics.
• Utilizing toluenized bacterial cell suspensions and crude enzyme preparations.
• Testing N-phenylacetyl derivatives, amides of phenylacetic and phenoxyacetic acids, and structurally similar compounds.

Main Results:

• Significant differences in substrate hydrolysis rates were observed.
• N-phenylacetyl derivatives were identified as the most rapidly hydrolyzed substrates.
• Beta-phenylpropionamide and 4-phenylbutyramide were not utilized by the enzyme.

Conclusions:

• The bacterial penicillin amidohydrolase displays distinct substrate specificity.
• The enzyme preferentially hydrolyzes N-phenylacetyl derivatives.
• Colorless substrates yielding chromophoric products could enable rapid enzyme activity assays.