R H

Whatever the case may be, it seems certain that the reaction proceeds according to the mechanism of the Sn2 nucleophilic substitution through the so-called tetrahedral intermediate (see below, Section 11.3):

11.2. Energy Balance of the Reaction

The standard free energy of hydrolysis of the ester bond between the tRNA and the carbonyl group of the aminoacyl or peptidyl residue AG0 is equal to about -7 to -8 kcal/mole. The standard free energy of hydrolysis of the peptide bond in a polypeptide of infinite length is approximately -0.5 kcal/mole. Thus, if the reaction substrates come directly from solution and the reaction products are released into solution, the net gain of free energy due to transpeptidation under standard conditions should be about - .5 to -7.5 kcal/ mole:

Pept(n)-tRNA' + Aa-tRNA''-*- tRNA' + Pept(n+1)-tRNA'' (-7 ± 0.5 kcal).

Such a formal calculation is usually put forward as an argument for ribosomal transpeptidation being fully supplied with free energy and therefore thermodynamically spontaneous.

It should be pointed out, however, that such an approach is valid only in the evaluation of ribosome-catalyzed model reactions between low-molecular-mass substrates, e.g.:

ribosome

F-Met-(3')ACC(5') + Phe-A-*-(5')CCA(3') + F-Met-Phe-A.

Here the substrates come to PTC directly from solution and the products are immediately and spontaneously released into solution.

In the elongation cycle, one substrate is always associated with the ribosome while the other comes to PTC from a prebound state; one of the reaction products is not released into solution until translation has been completed, while the other product is released not as a result of transpeptidation but only after the next step of the elongation cycle. All this makes it impossible to give even an approximate estimation of free-energy change in the course of transpeptidation proceeding in the normal ribosomal elongation cycle. Of course, the reaction is fast, suggesting that there is a significant decrease in the free energy of the ribosomal complex in the course of transpeptidation. This decrease, however, must be less than -7 kcal/ mole under standard conditions. For one thing, the free energy of hydrolysis of the bound substrate, provided hydrolysis products are released, should be lower than that of the free substrate since some portion of the energy had to be released upon binding if binding was spontaneous. In addition, the free energy of substrate hydrolysis, provided the product remains bound, should be lower than that when the product leaves the complex since the bound state of the product is associated with the accumulation of free energy, if the release of the product is thermodynamically spontaneous in principle. It can be assumed that the free energy of -7 kcal/mole, which would be released in transpeptidation if the substrate and the products were free, is in fact partly distributed at the preceding stage of aminoacyl-tRNA binding and the subsequent stage of translocation, thus driving the entire elongation cycle.

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