The interaction between various penicillins and cephalosporins the carboxylate group of which at C-3 or C-4 had been esterified or amidated and different penicillin-recognizing enzymes was studied. In general, our findings reinforced the common assumption that an anionic group at that position is necessary for the effective acylation of these enzymes. However, the relative activities of the modified beta-lactams as inactivators of the Streptomyces R61 DD-peptidase or as substrates of the Bacillus licheniformis, Streptomyces albus G and Enterobacter cloacae beta-lactamases did not fit a general scheme in which the intrinsic electronic and geometric properties of the beta-lactam compounds would be sufficient to explain their substrate or inactivator properties towards the various types of enzymes investigated.

Titration of the active-site serine DD-peptidase of Streptomyces R61 shows that formation of acyl enzyme during hydrolysis of the substrate Ac,-~-Lys-~-Aia-~-Ala and enzyme inactivation by the p-lactam compounds benzylpenicillin, N-acetylampicillin and ampicillin relies on the acidic form of an enzyme's group of pK z 9.5. It is proposed that protonation of a lysine &-amino group facilitates initial binding by charge pairing with the free carboxylate of the substrate and the 8-lactam molecules. Lowering the pH from 7 to 5 has no effect on the secondorder rate constant of enzyme acylation by benzylpenicillin and N-acetylampicillin but results in a decreased rate constant of acylation by ampicillin and Ac,-~-Lys-~-Ala-~-Ala. Protonation of the side-chain amino group of ampicillin and a decreased efficacy of the initial binding of the peptide to the enzyme seem to be responsible for the observed effects. Whatever the molecule bound to the enzyme, there is no sign for the active involvement of an enzyme's histidine residue of pK 6.5 -7.0 in the hydrolysis pathway.Two families of bacterial enzymes specifically interact with p-lactam compounds. The p-lactamases, which hydrolyse the p-lactam ring, can play an important role in resistance. The DD-peptidases, which participate in the last stages of wall peptidoglycan metabolism, are the primary targets of these antibiotics [l, 21. Many of the /3-lactamases (classes A and C) and DD-peptidases characterized so far are serine enzymes and react with p-lactam compounds according to reaction (1).(1)where E = enzyme, D = p-lactam, k + and k , , = first-order rate constants, K = dissociation constant of E . D (Michaelis complex) and E -D* = serine ester-linked acyl enzyme [3]. Most often, the value of k , , is high or very high with the j-lactamases, and so low with the DD-peptidases that a slight stoichiometric excess of p-lactam compound causes longlasting immobilization of the protein at the level of the acyl enzyme and these latter enzymes behave as penicillin-binding and C, the active-site serine is followed by an Xaa-Xaa-Lys sequence (which suggests that this lysine probably plays an important role), little is known concerning the enzymes' functional groups involved in substrate binding and catalysis. where HY is H 2 0 or a suitable R-NH2 peptide or amino acid. The goal of the present work was to obtain further information on the possible functional groups involved in catalysis and penicillin binding by the Streptomyces R61 DD-peptidase. When this study was initiated, it was known [2] that at pH 7.0 and 37°C (unless otherwise stated) (a) the K , k + , and k+, values for the interaction with benzylpenicillin were 13 mM (25"C), 180s-I (25°C) and 1 . 4~1 0 -~s -' ; (b) the corresponding values for the interaction with ampicillin were 7.2 mM, 0.77 s-l and 1.4 x s-'; (c) the K,,, and k,,, values for the best substrate Ac,-~-Lys-~-Ala-~-Ala were about 10 mM and 50 S K I , respectively and (d) the activity of the enzyme decreased with increasing ionic strength [15]. MATERIALS AND...

The interaction between the Streptomyces R61 penicillin-sensitive DD-peptidase and deacetyl-cephalosporin C or its lactone derivative has been studied at different pH values. The results show the importance of an enzyme group of pK g 9 which might form an ion pair with the free carboxylate of the former compound. This electrostatic interaction is shown to contribute to the formation of the first, non-covalent enzyme-inactivator complex by a factor of at least 50.

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