Affinity labeling of an Escherichia coli β-lactamase

Abstract

1. 1. A β-lactamase from Escherichia coli was inactivated by a substrate derivative, benzylpenicillin isocyanate. 2. 2. The inactivation process followed pseudo-first-order kinetics with K and k 3 values of 4 · 10 −5 M and 9 · 10 −2 min −1, respectively. 3. 3. The inactivation rate was reduced by the presence of benzylpenicillin or methicillin as would be expected for a specific reaction with a group in the active site of the enzyme. 4. 4. Benzylpenicillin isocyanate was hydrolyzed by the enzyme with a Michaelis constant identical with the K value described above. 5. 5. The inactivated enzyme was not reactiviated by dialysis. 6. 6. It is concluded that the inhibition by benzylpenicillin isocyanate was specific and irreversible through its binding to the catalytic site of the enzyme.