H O

Figure 11.1. Transpeptidation reaction catalyzed by the ribosome.

Transpeptidation Reaction

Figure 11.2. Schematic representation of transpeptidation reaction between aminoacyl-tRNA and peptidyl-tRNA on the ribosome.

Figure 11.2. Schematic representation of transpeptidation reaction between aminoacyl-tRNA and peptidyl-tRNA on the ribosome.

acceptor substrate. The latter requirement needs a special comments. Depending on the specificities of different aminoacyl-tRNA synthetases, aminoacyl residues become originally attached either to 2' or 3' position of the ribose residue in the tRNA terminal adenosine (see Section 3.4 and Table 3.1). In solution, however, the aminoacyl residue migrates between the 2'- and 3'-positions. In PTC the aminoacyl residue is fixed only in the 3' position.

The free 2'-hydroxyl of the acceptor substrate ribose is not essential for transpeptidation; if it is substituted (e.g., methylated) or is absent (2'-deoxyderivatives), the acceptor activity of the substrate is retained. The 2'-hydroxyl is, however, indispensable to the activity of the donor substrate.

The catalytic mechanism of ribosomal transpeptidation has been the subject of numerous studies and discussions (for reviews, see Krayevsky & Kukhanova, 1979; Garrett & Rodriguez-Fonseca, 1995). There is evidence that the histidine imidazole of some ribosomal protein may be involved in the catalysis. However, all attempts at isolating an acyl-enzyme (acyl-ribosome) intermediate of the kind found in proteinases catalyzing hydrolysis and transpeptidation have been unsuccessful. Perhaps such an intermediate should not exist in principle, because peptidyl-tRNA is already an activated macromolecular acyl-derivative which may be regarded as a functional analog of the acyl-enzyme group. Therefore, many investigators believe that transpeptidation in the ribosome is catalyzed simply by an appropriate spatial orientation and alignment of the aminoacyl-tRNA and peptidyl-tRNA reacting groups without the catalytic involvement of special nucleophile groups of PTC. This hypothesis is strongly supported by experiments showing a low specificity of the ribosomal PTC with respect to the types of bonds formed in it. Indeed, if a hydroxyacyl residue (HO-CHR-CO-), rather than an aminoacyl residue (H2N-CHR-CO-), is attached to the tRNA or its 3'-end analog, the hydroxyderivative serves as a good acceptor substrate, and the ester bond is produced by the ribosome (Fahnestock et al., 1970). Similarly, when the acceptor substrate is a thioacyl derivative the ribosomal PTC catalyzes the formation of a thioester bond (Gooch & Hawtrey, 1975). Moreover, the donor substrate can also be modified and the ribosome is capable of catalyzing the attack on the thioester group of the donor by the amino group of the acceptor, thus forming a thioamide bond (Victorova et al., 1976). Finally, the phosphinoester analog of the donor has been shown to react with aminoacyl-tRNA in the ribosomal PTC, with the formation of an unnatural phosphinoamide bond (Tasussova et al., 1981). The latter fact is especially important, since the geometry of the attacked group and the transition state of the reaction (trigonal bipyramid) are significantly different from those in the case of a normal donor (see below, Section 11.3); the nucleophilic catalysis is hardly compatible with the realization of the reactions of both types by the same

Figure 11.3. Ball-and-sticks skeletal model (without hydrogens) enzymatic center.

of puromycin based on X-ray analysis (M. Sundaralingam & S.K. At the same time, however, the

Arora, J. Mol. Biol. 71, 49-70, 1972). Filled circles are carbons, ribosomal PTC under certain c°nditi°ns is hatched circles - nitrogens, open circles - oxygen. capable of using water and low-

Figure 11.3. Ball-and-sticks skeletal model (without hydrogens) enzymatic center.

of puromycin based on X-ray analysis (M. Sundaralingam & S.K. At the same time, however, the

Arora, J. Mol. Biol. 71, 49-70, 1972). Filled circles are carbons, ribosomal PTC under certain c°nditi°ns is hatched circles - nitrogens, open circles - oxygen. capable of using water and low-

molecular-weight alcohols, e.g., methanol and ethanol, as acceptor substrates, thus performing hydrolysis or alcoholysis of the peptidyl-tRNA. These observations cannot be explained easily within the framework of the purely orientational mechanism of ribosomal PTC action. Most reasonable is the hypothesis that the orientation effect of PTC is indeed instrumental to transpeptidation on the ribosome, but that this effect can be aided by the contribution of specific microenvironments facilitating the reaction. It may well be that some groups located near the substrates properly oriented on the ribosome pull a proton away from the NH2-group of the acceptor, thereby increasing its nucleophilic nature, on the one hand, and contribute to protonation of the carbonyl oxygen, thereby increasing the electrophilic properties of the attacked carbon of the donor ester group, on the other:

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