Transpeptidases 3-lactams

This insertion is accomplished by an enzyme called transpeptidase. -Lactam antibiotics function as substrates for the transpeptidase, thereby establishing selective inhibition of bacterial cell wall synthesis. The structural similarity between -lactam antibiotics and the alanylalanine unit is remarkable as can be seen in Figure 6.8. 

Resistance can also arise when target enzymes, ie, the PBPs, and in particular the transpeptidases, are modified. Target-mediated cephalosporin resistance can involve either a reduced affinity for an existing PBP, or the acquisition of a supplementary, P-lactam insensitive PBP (139). 

The antibacterial effectiveness of penicillins cephalospotins and other P-lactam antibiotics depends upon selective acylation and consequentiy, iaactivation, of transpeptidases involved ia bacterial ceU wall synthesis. This acylating ability is a result of the reactivity of the P-lactam ring (1). Bacteria that are resistant to P-lactam antibiotics often produce enzymes called P-lactamases that inactivate the antibiotics by cataly2ing the hydrolytic opening of the P-lactam ring to give products (2) devoid of antibacterial activity. 

P-Lactam antibiotics exert their antibacterial effects via acylation of a serine residue at the active site of the bacterial transpeptidases. Critical to this mechanism of action is a reactive P-lactam ring having a proximate anionic charge that is necessary for positioning the ring within the substrate binding cleft (24). 

Preliminary investigations involving a P-lactam-sensitive, bifimctional D-alanyl-carboxypeptidase—transpeptidase (C Pase—T Pase) from Streptomjces R61 have identified the three-dimensional stmcture and catalytic site of interaction with penicillins (63). 

Although most /3- lactam antibiotics bind covalently to some or all of the same six proteins, there are decided differences among them in terms of their relative affinities. For example, cefoxitin (see Table 1 for structures) fails to bind to protein 2 while cephacetrile binds very slowly to proteins 5 and 6. Cephaloridine binds most avidly to protein 1, the transpeptidase, and inhibits cell elongation and causes lysis at its minimum inhibitory concentration. On the other hand, cephalexin binds preferentially to protein 3 and causes inhibition of cell division and filament formation (75PNA2999, 77MI51002). 

By virtue of their fused /3-lactam-thiazolidine ring structure, the penicillins behave as acylating agents of a reactivity comparable to carboxylic acid anhydrides (see Section 5.11.2.1). This reactivity is responsible for many of the properties of the penicillins, e.g. difficult isolation due to hydrolytic instability (B-49MI51102), antibacterial activity due to irreversible transpeptidase inhibition (Section 5.11.5.1), and antigen formation via reaction with protein molecules. 

The biological activity of penicillins and cephalosporins is due to the presence of the strained /3-lactam ring, which reacts with and deactivates the transpeptidase enzyme needed to synthesize and repair bacterial cell walls. With the wall either incomplete or weakened, the bacterial cell ruptures and dies. 

Figure 6.8 Pendllins are similar to the bacterial peptidogiycan terminal alanylalanine moiety. Because of this similarity, the enzyme transpeptidase recognizes 8-lactam antibiotics as substrate. As a result of this the 8-lactam is incorporated in the peptide chain thereby making peptide-peptide cross-linking impossible. The occurrence of this phenomenon stops the construction of the bacterial cell wall.

In contrast to macrolides, the targets of (3-lactams, the penicillin binding proteins (PBPs) require several mutations in order to become resistant while simultaneously maintaining their viable function as cell wall transpeptidases/transglycosidases. Thus, in order to achieve clinically relevant resistance Streptococcus pneumoniae uses a unique strategy to rapidly accumulate several point mutations. Due to its natural competence for transformation during respiratory tract... 

Frere JM et al (1975) Kinetics of interactions between the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61 and (3-lactam antibiotics. Eur J Biochem 57 343-351... 

A comparison of the structures of penicillin and Dalanyl-Dalanine (cf. structures 41 and 42) shows that there is a great deal of similarity between the two molecules. Penicillin is essentially an acylated cyclic dipeptide of Dcysteine and Dvaline (84). As such, it contains a peptide bond, that of the /3-lactam ring, that can acylate the enzyme. Labeling studies of the peptidoglycan transpeptidase of Bacillus subtilis indicate that radioactive penicillin reacts with a sulfhydryl group of a cysteine residue of the enzyme (86). 

The /3-lactam bond is broken (instead of the equivalent peptide bond joining the alanine residues) but the remaining ring system in the /3-lactam (a thiazolidine in penicillins) is not released (Fig. 8.3). Instead, the transpeptidase remains linked to the hydrolysed antibiotic with a half hfe of 10-15 minutes. Whilst bound to the / -lactam, the transpeptidase cannot participate in further rounds of peptidoglycan... 

The antibiotic activity of certain (3-lactams depends largely on their interaction with two different groups of bacterial enzymes. (3-Lactams, like the penicillins and cephalosporins, inhibit the DD-peptidases/transpeptidases that are responsible for the final step of bacterial cell wall biosynthesis.63 Unfortunately, they are themselves destroyed by the [3-lactamases,64 which thereby provide much of the resistance to these antibiotics. Class A, C, and D [3-lactamases and DD-peptidases all have a conserved serine residue in the active site whose hydroxyl group is the primary nucleophile that attacks the substrate carbonyl. Catalysis in both cases involves a double-displacement reaction with the transient formation of an acyl-enzyme intermediate. The major distinction between [3-lactamases and their evolutionary parents the DD-peptidase residues is the lifetime of the acyl-enzyme it is short in (3-lactamases and long in the DD-peptidases.65-67... 

Fig. 5.2. Mechanism of transpeptidases in the bacterial cell wall, the target of inhibitory ji-lactam antibiotics (Fig. 5.1, Pathway a)...

Lactamases (EC 3.5.2.6) inactivate /3-lactam antibiotics by hydrolyzing the amide bond (Fig. 5.1, Pathway b). These enzymes are the most important ones in the bacterial defense against /3-lactam antibiotics [15]. On the basis of catalytic mechanism, /3-lactamases can be subdivided into two major groups, namely Zn2+-containing metalloproteins (class B), and active-serine enzymes, which are subdivided into classes A, C, and D based on their amino acid sequences (see Chapt. 2). The metallo-enzymes are produced by only a relatively small number of pathogenic strains, but represent a potential threat for the future. Indeed, they are able to hydrolyze efficiently carbape-nems, which generally escape the activity of the more common serine-/3-lac-tamases [16] [17]. At present, however, most of the resistance of bacteria to /3-lactam antibiotics is due to the activity of serine-/3-lactamases. These enzymes hydrolyze the /3-lactam moiety via an acyl-enzyme intermediate similar to that formed by transpeptidases. The difference in the catalytic pathways of the two enzymes is merely quantitative (Fig. 5.1, Pathways a and b). 

Lactamases have very high rates of deacylation compared to those of transpeptidases. This quantitative difference translates into very different qualitative results, since /3-lactams inactivate transpeptidases but are destroyed by /3-lactamases [18]. The mechanism underlying the differences in rate constant for deacylation between transpeptidases vs. /3-lactamases remains poorly understood [19]. 

An important molecular target of the B-lactam antibiotics is an enzyme that acts as a transpeptidase in the stepwise polymerization leading to a thickened, strong bacterial cell wall. Several amino acids are present in addition to the terminal -alanyl- -alanyl unit which the Strominger hypothesis suggests has the same overall shape and reactivity as... 

The (3-lactam antibiotics structurally resemble the terminal D-alanyl-D-alanine (o-Ala-o-Ala) in the pen-tapeptides on peptidoglycan (murein) (Fig. 45.1). Bacterial transpeptidases covalently bind the (3-lactam antibiotics at the enzyme active site, and the resultant acyl enzyme molecule is stable and inactive. The intact (3-lactam ring is required for antibiotic action. The (3-lactam ring modifies the active serine site on transpeptidases and blocks further enzyme function. 

In addition to transpeptidases, other penicillin-binding proteins (PBPs) function as transglycosylases and carboxypeptidases. All of the PBPs are involved with assembly, maintenance, or regulation of peptidoglycan cell wall synthesis. When (3-lactam antibiotics inactivate PBPs, the consequence to the bacterium is a structurally weakened cell wall, aberrant morphological form, cell lysis, and death. 

During the biosynthesis of the cell wall, the muropeptide is formed from acetylmuramyl-pentapeptide, which terminates in a D-alanyl-D-alanine. The synthesis of this precursor is inhibited by the antibiotic cycloserine (9.36), a compound produced by many Streptomyces fungi but which is not used clinically. During the crosslinking of the pen-tapeptide precursor, the terminal fifth alanine must be split off by a transpeptidase enzyme. This last reaction in cell wall synthesis is inhibited by the p-lactam antibiotics. 

Figure 9.4 Mechanism of penicillin. By means of its highly reactive lactam ring, penicillin is able to deactivate the transpeptidase enzyme. This in turn leads to a halting of cell wall construction within the bacterium, ultimately leading to bacterial death.

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