Inhibition of Bacillus subtilis aminopeptidase D by beta-lactam antibiotics

Penicillins and cephalosporins inhibit the hydrolysis of D-alanyl-beta-naphthylamide by aminopeptidase D (EC 3.4.11.-) from Bacillus subtilis. The inhibition is predominantly non-competitive, although the more effective inhibitors, i.e., those with the lower Ki values, seem to exhibit a tendency toward competitive kinetics, while penicillin V, the antibiotic with the largest Ki, exhibits a strong tendency toward uncompetitive kinetics. The removal of antibiotic from the enzyme by gel filtration on Sephadex G-25 restores 100% activity to the enzyme, and suggests that the inhibition does not derive from a covalent antibiotic-enzyme complex. The antibiotics which have been studied, with their respective Ki values (mM) and inhibition type are: methicillin (1.01 +/- 0.34, mixed non-competitive-competitive); cephaloridine (2.43 +/- 0.17, non-competitive); cloxacillin (3.19 +/- 1.29, mixed non-competitive-competitive); oxacillin (6.88 +/- 0.77, non-competitive); cephalothin (8.12 +/- 0.99, non-competitive); penicillin G (20.0 +/- 3.21, non-competitive) and penicillin V (32.1 +/- 4.90, mixed non-competitive-uncompetitive). An empirical correlation exists between the Ki value and the freedom of rotation about the bond between the phenyl ring and the atom alpha to the ring in the variable side chain portion of the penicillin molecule.