Aa PpAp Pe

Figure 11.6. Schematic representation of the a, p, and e sites of the large ribosomal subunit and the product shifts in the ribosomal peptidyl transferase center during transpeptidation.

as slight interdomain and intersubunit shifts, cannot be excluded. 11.5. Inhibitors

Numerous specific inhibitors of the peptidyl transferase reaction catalyzed by prokaryotic or eukaryotic ribosomes have been described (for reviews, see Gale et al., 1981; Garrett & Rodriguez-Fonseca, 1995). All the inhibitors, as expected, affect the large (50S or 60S, respectively) ribosomal subunit and have an affinity to it. Many antibiotics commonly used for treating bacterial infections are inhibitors of the peptidyl transferase of the prokaryotic 70S ribosome. Kirillov, Garrett et al. (1997) have suggested to classify the peptidyl transferase antibiotics into at least two categories: (1) those inhibitors that are to some degree "co-structural" with the 3'-end of aminoacyl-tRNA, bind to the acceptor (a) site of PTC or nearby, and thus prevent, competitively or non-competitively, binding or proper settling of the acceptor substrate at PTC (i.e., at the stage A in Fig. 11.13); (2) those that are bound in the vicinity of PTC and perturb the movement or positioning of the nascent peptide during transpeptidation (i.e., at the stage B ® C in Fig. 11.13).

11.5.1. Chloramphenicol

Chloramphenicol (also known as chloromycetin or levomycetin) is the most known inhibitor of peptidyl transferase in 70S ribosomes (Fig. 11.7). This is a broad-spectrum bacteriostatic antibiotic commonly used in pharmacological practice. It does not affect eukaryotic 80S ribosomes, but may attack mitochondrial ribosomes of Eukaryotes. The antibiotic binds loosely to the 70S ribosome or its isolated 50S subunit and can be washed away easily from the particles. Correspondingly, the action of the antibiotic is reversible. The antibiotic binds in the region of PTC and blocks the site responsible for the interaction with the acceptor substrate (a site); puromycin and 3'-terminal fragments of the aminoacyl-tRNAs compete with chloramphenicol for binding to PTC.

Chemically, chloramphenicol is the analog of N-blocked amino alcohol with an aromatic radical. The dichloromethyl group is not strictly required for activity and can be changed for many moderately massive radicals. The aromatic nitro-group may also be changed for a number of other electronegative groups without resulting in any loss of antibiotic activity. The amide bond and stereochemistry of the -CO-NH-group with adjacent atoms are noteworthy with respect to the antibiotic action mechanism: this part of the molecule may mimic the peptide group with the adjacent Ca-atom and side radical.

Chloramphenicol inhibits "natural" transpeptidation between the peptidyl-tRNA and aminoacyl-tRNA in the course of elongation, as well as the reaction of the peptidyl-tRNA or its analogs with puromycin. The simplest explanation for these effects is that chloramphenicol is an inactive analog of the acceptor substrate and after binding to PTC competitively interferes with the interaction of true acceptors. However, there is also evidence for a noncompetitive mode of chloramphenicol inhibition. In accordance with the above, chloramphenicol affects chemical reactivity of residues within the so-called PTC ring of domain V of 23S RNA (see Section 9.3 and Fig. 9.7): it enhances the ractivity of A2058 and protects A2059, A2062, A2451, and G2505. Mutations conferring chloramphenicol resistance are also located in the same PTC ring; their positions have been identified at G2032, G2057, A2058, G2061, A2062, G2447, A2451, C2452, A2503, and U2504.

11.5.2.Lincosamides

This group of drugs includes lincomycin (Fig. 11.8) and its derivatives, such as clindamycin and celesticetin. These are also bacteriostatic antibiotics widely used in therapy. They attack specifically eubacterial 70S ribosomes. The antibiotics interact with the peptidyl transferase center of ribosomes and seemingly block the acceptor-binding site (a site). Correspondingly, they compete with chloramphenicol for binding to the ribosome. In any case, the antibiotics inhibit the peptidyl transferase reaction. At the same time the translating ribosomes with long nascent peptides seem to be less sensitive to the drugs, as

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