Purification of the membrane-bound DD-carboxypeptidase of the unstable spheroplast L-form of Proteus mirabilis by affinity chromatography. Non-competitive inhibition of the enzyme by penicillins and low stability of the enzyme-inhibitor complex.

Membrane-bound DD-carboxypeptidase of the unstable L-form of Proteus mirabilis was solubilized by the non-ionic detergent Genapol X-100 and purified to protein homogeneity by affinity chromatography on ampicillin bound to succinyl-aminododecyl-cellulose. The purified enzyme with a molecular weight of 43000 is inhibited non-competitively by penicillin G and carbenicillin, indicating a function of the penicillins as allosteric inhibitors. Sensitivity of the enzyme to penicillins is only moderate with a Ki of 1 muM for penicillin G. Breakdown of.the enzyme-inhibitor complex EI with different penicillins occurs rapidly with reappearance of active DD-carboxypeptidase. The half-life of EI with penicillin G is 5.5 min at 30 degrees C and 3.5 min at 37 degrees C, 10--1000-fold shorter than EI half-lives of DD-carboxypeptidases in several other bacteria. The low stability of the enzyme-inhibitor complex and the moderate penicillin sensitivity appear to be the basis for the continued activity of DD-carboxypeptidase during growth of the L-form and synthesis of peptidoglycan in the presence of high concentrations of penicillin.

D-alanyl-D-alanine carboxypeptidase in the bacterial form and L-form of Proteus mirabilis.

Membranes of the bacterial form and the stable and unstable L-forms of Proteus mirabilis contain LD and DD-carboxypeptidase. The DD-carboxypeptidase is inhibited non-competitively by penicillin G. The enzyme of the bacterial form is highly penicillin-sensitive (Ki - 4 X 10(-9) M penicillin G). Inhibition is only partly reversible by treatment with penicillinase or by dialysis against buffer. In contrast, the DD-carboxypeptidase of the unstable L-form, grown in the presence of penicillin, is 175-fold less penicillin-sensitive (Ki = 7 X 10(7) M penicillin G). Inhibition is completely reversed by penicillinase or dialysis. After inhibition by penicillin and subsequent reactivation the penicillin sensitivity of the bacterial DD-carboxtpeptidase is similar to the sensitivity of the enzyme of the unstable L-form. The hypothesis is proposed that P. mirabilis contains two DD-carboxypeptidases of different penicillin sensitivity and with different mechanisms of penicillin binding. Peptidoglycan synthesis in the cell walls of the unstable L-form is probably carried out with the help of only one DD-carboxypeptidase, viz. the completely reactivatable enzyme with the lower penicillin sensitivity.