Hydrolysis of 7-substituted cephalosporins catalysed by β-lactamases I and II from Bacillus cereus and by hydroxide ion

Abstract

Kinetic parameters are reported for the Bacillus cereus P-lactamase Iand P-lactamase II -catalysed hydrolysis of a series of thirty-seven cephalosporins substituted in the 7-position. These are compared with the second-order rate constants for the hydroxide ion-catalysed hydrolysis of these derivatives. There is no significant dependence of the rate of the base-catalysed hydrolysis upon the nature of the side-chain substituent. For P-lactamase I, kc,,/K, varies over 2 x lo5 but for plactamase II the variation with substituents is only 10. For alkyl substituents, k,,JK, increases with chain length and passes through a maximum, for p-lactamase I this is with the undecyl derivative and for P-lactamase II the octylcephalosporin. For p-lactamase I, but not for P-lactamase II, the tbutylcephalosporin is a very poor substrate. There is no evidence for a significant cavity in either enzyme to host aromatic residues. An ionised carboxylate residue on the side-chain significantly reduces reactivity with P-lactamase I but not p-lactamase II. It is suggested that a carboxy group on P-lactamase I acts as a general catalyst facilitating p-lactam C-N bond fission.The two major classes of p-lactam antibiotics are the penicillins (1) and the cephalosporins (2).t Bacteria which are normally susceptible to these antibiotics may be rendered insusceptible if they can produce P-lactamases, enzymes that catalyse the hydrolysis of the p-lactam.There are many different p-lactamases which have been classified on the basis of substrate profile (the ability to catalyse the hydrolysis of various p-lactam antibiotics e.g. penicillinase or cephalosporinase), on the basis of their sequence or on a mechanistic bask2 For example, the TEM2 P-lactamase from E. coli is very efficient and one molecule of the enzyme can catalyse the hydrolysis of 2 OOO molecules of benzylpenicillin in 1 ~e c o n d . ~ However, this enzyme is more than loo00 less reactive towards some cephalo~porins.~ Conversely, one molecule of the P99 P-lactamase can hydrolyse 2 OOO molecules of cephaloridine in 1 second. One mechanistic class comprises plactamases that are serine enzymes; this is subdivided into classes A and C based on the amino acid sequences.2 The other mechanistic class of p-lactamases is distinguished by the requirement for zinc(r1) ions and a lack of sequence homology to enzymes of the other classes. There are two well characterised but unrelated enzymes of this class B-p-lactamase I1 from B. cereus and p-lactamase L-1 from Pseudonomas maltophilia. RCONH RCONH An example of the systematic nomenclature system for the trivially named cephalosporins, includes 3-[(acetyloxy)methyl]-8-oxo-7-(2-pheny1acetamido)-5-thia-1 -azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid for benzylcephalosporin.

Inactivation of the RTEM-1 cysteine β-lactamase by iodoacetate. The nature of active-site functional groups and comparisons with the native enzyme

“…It seems likely in the native enzyme that the basic functional group present, the carboxylate group of Glu-166, would act as a general base catalyst of nucleophilic attack by the serine hydroxy group on the f-lactam carbonyl group of the substrate. This has also been proposed by Waley, Page and coworkers [35] on the basis of other evidence [8,[35][36][37] for the presence of a carboxylate group at the class A f-lactamase active site. Nucleophilic attack is apparently also aided, as in serine proteinases, by the presence of an oxy-anion hole [4,38].…”

The β-lactamase cycle: a tale of selective pressure and bacterial ingenuity

“…However, esterification of the carboxylic group at position C-3 in penicillins and C-4 in cephalosporins decreases the catalytic efficiency of the B. cereus II b-lactamase by two orders of magnitude. 170 The same phenomenon is observed when the carboxylic group of cephalosporins is modified in the corresponding lactone. Most class B enzymes exhibit high activity towards the 'b-lactamasestable' compounds carbapenems, cephamycins and thirdgeneration cephalosporins (Table 4).…”

Exploitation of Antibiotic Resistance as a Novel Drug Target: Development of a β-Lactamase-Activated Antibacterial Prodrug

“…Analysis of the literature identified the amide functionality at C-7 of the cephem ring as central to PBP and β-lactamase activity, − and therefore structural changes at this position provided the initial focus of investigation. By use of cephalothin 7 (Table ) as the starting point, analogues were prepared to explore bioisosteric replacement, , functionalities present in early generation β-lactam antibiotics, and to probe steric and electronic tolerance. , All compounds were synthesized according to the previously reported methods (Figure S1 in Supporting Information). , Antibacterial activity was assessed by determining the minimal concentration required to inhibit bacterial growth, known as the minimal inhibitory concentration (MIC), against the E. coli strain DH5α ± expression of the ESBL TEM-116 .…”

“…The aqueous layers were combined, washed with EtOAc, and acidified to pH 2 with 1 M HCl. 55 The desired product was isolated as described.…”