The relation between penicillin structure and penicillinase activity

Abstract Staphylococcal penicillinase was isolated and purified and its activity and properties were compared with Bacillus penicillinase. Relatively little change was observed in the Michaelis constants and maximum velocities for either enzyme when the phenyl, benzyl, and aliphatic penicillin series were employed as substrates. Compounds with a positively charged nitrogen in the side-chain (e.g., α-aminobenzylpenicillin, p -aminobenzylpenicillin, and 6-aminopenicillanic acid) produced evidence of a lowered affinity for the enzyme. The marked shift in the optimal pH for activity of the staphylococcal enzyme with 6-aminopenicillanic acid provided additional evidence that a decrease in binding results from the presence of a positive charge in the side-chain. With the Staphylococcus enzyme, the Michaelis constants increased with increasing acid strength of the parent side-chain acids when a series of phenyl penicillins was employed, providing evidence for the presence of an electrophilic or positively charged group at the binding site of penicillin to the enzyme. The participation of histidine in the active site of staphylococcal penicillinase was indicated by the pH-activity curve for benzylpenicillin. Methicillin showed a marked decrease in binding to the enzyme with a relatively small change in maximal velocity (about 4000-fold vs. 25-fold). An explanation for the behavior of the enzyme with methicillin is presented.