Peptidyltransferase inhibition

Mechanism of Action. THie earliest studies on the mechanism of action of lincomycin showed that lincomycin had the immediate effect on Staphjlococcus aureus of complete inhibition of protein synthesis (23). TThis inhibition results from the blocking of the peptidyltransferase site of the SOS subunit of the bacterial ribosome (24). Litde effect on DNA and RNA synthesis was observed. 

Puromycin. Puromycin (19), elaborated by S. alboniger (1—4), inhibits protein synthesis by replacing aminoacyl-tRNA at the A-site of peptidyltransferase (48,49). Photosensitive analogues of (19) have been used to label the A-site proteins of peptidyltransferase and tRNA (30). Compound (19), and its carbocycHc analogue have been used to study the accumulation of glycoprotein-derived free sialooligosaccharides, accumulation of mRNA, methylase activity, enzyme transport, rat embryo development, the acceptor site of human placental 80S ribosomes, and gene expression in mammalian cells (51—60). 

Other antibiotics inhibit protein synthesis on all ribosomes (puromycin) or only on those of eukaryotic cells (cycloheximide). Puromycin (Figure 38—11) is a structural analog of tyrosinyl-tRNA. Puromycin is incorporated via the A site on the ribosome into the carboxyl terminal position of a peptide but causes the premature release of the polypeptide. Puromycin, as a tyrosinyl-tRNA analog, effectively inhibits protein synthesis in both prokaryotes and eukaryotes. Cycloheximide inhibits peptidyltransferase in the 60S ribosomal subunit in eukaryotes, presumably by binding to an rRNA component. 

Macrolides bind to the SOS ribosomal subunit of bacteria but not to the SOS mammalian ribosome this accounts for its selective toxicity. Binding to the ribosome occurs at a site near peptidyltransferase, with a resultant inhibition of translocation, peptide bond formation, and release of oligopeptidyl tRNA. However, unlike chloramphenicol, the macrolides do not inhibit protein synthesis by intact mitochondria, and this suggests that the mitochondrial membrane is not permeable to erythromycin. 

Chloramphenicol Binds to 50 S ribosomyl subunit and inhibits peptidyltransferase... 

Yes. Chloramphenicol inhibits the peptidyltransferase of the 50S ribosomal subunit of bacteria, while cycloheximide inhibits the analogous enzyme in the 60S subunit of eukaryotic ribosomes. Their structures are shown below. 

Chloramphenicol blocks translation in bacteria by inhibiting peptidyltransferase of the large ribosomal subunit. It does not interfere with peptidyltransferase in the large subunit of eukaryotic ribosomes. However, the mitochondrion of animal cells contains ribosomes that are similar to bacterial ribosomes, and chloramphenicol can block protein synthesis in this organelle. This could contribute to the side effects of this drug when used in the treatment of animals. 

Peptidyltransferase assays have also provided insight into the mechanisms whereby spermine promotes, and NHJ ions inhibit, polypeptide synthesis on the ribosomes of S. solfataricus, T. tenax and D. mobilis (see section 3.3). First, the 30S uncoupled peptidyltransferase activity is absolutely dependent on spermine while being totally unaffected by monovalent cations. Secondly, monovalent cations strongly inhibit the 30S subunit coupled reaction [66]. Thus, polyamines appear to be obligatorily required to convert the catalytic center of the spermine-dependent ribosomes into an active conformation, whereas monovalent cations inhibit polypeptide synthesis by preventing 30S subunits from interacting with the cognate SOS particles (ref. [66], see below). 

Two considerations argue against the idea [124,126] that spermine activates polypeptide synthesis in the thermophile Sulfolobus) systems by protecting ribosomes against thermal inactivation. First, spermine is absolutely required for the fimctioning of the spermine-dependent SOS subunits at low temperature (37°C). Secondly, polyamines are not required for (and in fact inhibit) the peptidyltransferase activity of SOS subunits from the hyper-thermophilic archaeon T. celer [66]. 

Chloramphenicol inhibits protein synthesis by binding the 50S ribosomal subunit and preventing the peptidyltransferase step. Decreased outer-membrane permeability and active efflux have been identified in Gram-negative bacteria however, the major resistance mechanism is drug inactivation by chloramphenicol acetyltransferase. This occurs in... 

Some antibiotics are active against both bacterial and mammalian cells. One example is chloramphenicol, which inhibits peptidyltransferase in both bacterial and mitochondrial ribosomes, although eukaryotic cytoplasmic ribosomes are unaffected. Such a drug may be clinically useful if a concentration range can be maintained in the patient in which the antibacterial action is substantial but toxic effects on host cells are minimal. However, because of the potential for toxicity, such antibiotics are used only in serious infections when other drugs fail. 

Formation of peptide bond Chloramphenicol (50S) Inhibit the activity of 1 peptidyltransferase j (-static) i... 

Chloramphenicol inhibits the activity of peptidyltransferase and is currently used primarily as a backup I j drug. Its activity dinical use, and adverse effects are considered. j... 

D. Tetracycline inhibits the activity of peptidyltransferase in bacterial protein synthesis... 

Some trichothecenes, a group of mycotoxins, have macrodiolide or macrotri-olide skeletons. Trichothecenes inhibited protein synthesis by binding to the ribosomal peptidyltransferase site [141]. Roritoxins (roritoxin A, 113) are 16-membered ring macrodiolides isolated from Myrothecium roridum [142]. Verru-carin A (114) is an 18-membered ring macrotriolide produced by Myrothecium spp. [143]. 

It is usually accepted that the 16-membered-ring macrolides inhibit peptidyltransferase activity [79, 88, 89], because they inhibit puromycin reaction although poorly. The reaction is used as an assay system for peptidyltransferase activity, because puromycin characteristically interrupts peptide bond formation by virtue of its structural similarity to the 3 end of aminoacyl-tRNA. Puromycin enters the A site (the so-called aminoacyl site) on the ribosome and is incorporated into either a nascent polypeptide or into A-acylaminoacylate, consequently causing premature release of puromycinyl polypeptide or A-acylaminoacyl-puromycin from the ribosome. 

Evidence also indicates that 16-membered-ring macrolides, as peptidyltransferase inhibitors, hinder the polyuridylic acid-dependent polymerization of phenylalanine, despite the fact that 14-membered-ring macrolides are not able to inhibit polyphenylalanine synthesis. In particular, 16-membered-ring macrolides containing at least one disaccharide-monoglycoside in their structures, such as leucomycin, spiramycin, carbomycin, and tylosin, may cause degradation of polyribosome [93,94]. 

On the other hand, the inhibitory effect of erythromycin, a 14-membered-ring macrolide, on such a peptidyltransferase reaction is markedly diminished in terms of the character of a substrate. Erythromycin inhibits poly(A)-dependent polymerization of a transferred substrate such as lysine residue linked to tRNA but not other oligonucleotide-dependent polymerization of an amino acid linked either to tRNA or to oligonucleotides such as CACCA and UACCA. It has been shown that the transfer of A-acylaminoacyl residues to puromycin (puromycin reaction) is usually stimulated by erythromycin [88, 89, 95]. Igarashi et al. [96] have also confirmed these findings. That is to say, they found that erythromycin inhibits the release of a deacylated tRNA from the P site of ribosome. The release of such a deacylated tRNA from the P site and the translocation of peptidyl-tRNA from the A site to the P site of ribosome occurs concomitantly when EF-G catalyzes the GTP-dependent movement of the ribosome and the codon-anticodon-linked mRNA-peptidyl-tRNA complex. 

Menninger and Otto [101] proposed a major inhibitory mechanism common to probably all macrolide antibiotics. In E. coli mutants with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase EC 3.1.1.29), they observed that peptidyl-tRNA accumulates at a nonpermissive temperature (40°C) and that the cells die. The accumulation at a high temperature was enhanced when the cells were pretreated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wild-type cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30°C). Based on their observations, they suggested that stimulated dissociation of peptidyl-tRNA from ribosomes is the major mechanism of action of macrolide antibiotics. Their observations agree with recent results showing that a macrolide antibiotic binds to peptidyltransferase in ribosome. 

Lovett and Rogers [201], in a recent review, discussed cotranslational regulation of protein synthesis. That is to say, a translational process conducted by the ribosome of the leader peptides (of the catA86 specifying chloramphenicol acetyltransferase and cmlA encoding a membrane protein that probably alters chloramphenicol transport operons) appears to take place concomitantly with the synthesis of the nascent peptide that is capable of binding to rRNA and inhibiting the pep tidy Itransferase. In addition, a nascent 5- or 8-residue leader peptide is found to inhibit ribosomal peptidyltransferase. 

Peptidyltransferase as a ribozyme may be inhibited by nascent-leader peptides capable of binding (probably in a deposition-like manner) to the center of the enzyme, because the nascent peptides repress the enzyme activity in vitro [201]. 

Inhibitors of translation - A number of the common inhibitors of prokaryotic translation are also effective in eukaryotic cells. They include pactamycin, tetracycline, and puromycin. Inhibitors that are effective only in eukaryotes include cycloheximide and diphtheria toxin. Cycloheximide inhibits the peptidyltransferase activity of the eukaryotic ribosome. Diphtheria toxin is an enzyme, coded for by a bacteriophage that is lysogenic in the bacterium Corynebacterium diphtheriae. It catalyzes a reaction in which NAD+ adds an ADP ribose group to a specially modified histidine in the translocation factor eEF2, the eukaryotic equivalent of EF-G (Figure 28.36). Because the toxin is a catalyst, minute amounts can irreversibly block a cell s protein synthetic machinery. As a result, pure diphtheria toxin is one of the most deadly substances known. 

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