Search

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.

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class.

The beta-lactamase of Enterobacter cloacae P99 consists of one polypeptide chain of Mr 39000 devoid of disulphide bridges and free thiol groups. It contains an unusually high proportion of tyrosine and tryptophan. The N-terminal sequence exhibits overlaps with the tryptic peptide obtained after labelling the active site with 6 beta-iodopenicillanate. The active-site serine residue is at position 64. The homology with the chromosomal beta-lactamase of Escherichia coli K 12 (ampC gene) is lower within the 25 residues of the N-terminal portion than around the active-site serine residue. The P99 beta-lactamase is inactivated by 6 beta-bromo- and 6 beta-iodo-penicillanate, with a second-order rate constant of 110-140M-1 X s-1 at 30 degrees C and pH 7.0, a value that is much lower than that observed with class-A beta-lactamases.

An inactivator of serine beta-lactamases, 6 beta-iodopenicillanate, can be utilized as a probe in the classification of beta-lactamases. It is a substrate for class-B Zn2+-containing beta-lactamase II. Although it inactivates enzymes from both classes A and C, it is much more efficient for the former group, with which it sometimes interacts following a branched pathway. On the basis of these observations, predictions are made concerning the class to which several enzymes belong.

A beta-lactamase produced by a penicillin-resistant strain of Serratia marcescens was isolated and purified. The kcat. value for benzylpenicillin was about 5% of that observed for the best cephalosporin substrates. However, the low Km of the penam resulted in a high catalytic efficiency (kcat./Km) and the classification of the enzyme as a cephalosporinase might not be completely justified. It also exhibited a low but measurable activity against cefotaxime, cefuroxime, cefoxitin and moxalactam. Substrate-induced inactivation was observed both with a very good (cephalothin) or a very bad (moxalactam) substrate. The active site was labelled by beta-iodopenicillanate. Trypsin digestion produced a 19-residue active-site peptide whose sequence clearly allowed the classification of the enzyme as a class C beta-lactamase.

Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between beta-halogenopenicillanates and some class A beta-lactamases. This suggested the stabilization of a highly charged intermediate by solvation. Those data could be interpreted on the basis of a reaction pathway where an episulphonium ion was transiently formed. The various mechanisms proposed for explaining the formation of the dihydrothiazine chromophore are discussed.

beta-Lactamase K1 was purified from Klebsiella pneumoniae SC10436. It is very similar to the enzyme produced by Klebsiella aerogenes 1082E and described by Emanuel, Gagnon & Waley [Biochem. J. (1986) 234, 343-347]. An active-site peptide was isolated after labelling of the enzyme with tritiated beta-iodopenicillanate. A cysteine residue was found just before the active-site serine residue. This result could explain the properties of the enzyme after modification by thiol-blocking reagents. The sequence of the active-site peptide clearly established the enzyme as a class A beta-lactamase.