Rs

(3) Aa-tRNA + X-Aa'-tRNA' —»X-Aa'-Aa-tRNA + tRNA'.

In the first reaction the amino acid carboxyl group reacts with the polyphosphate group of ATP, resulting in the replacement of a pyrophosphate residue by the aminoacyl residue; a mixed anhydride, the aminoacyl adenylate, is formed. In the second reaction the adenylate residue is exchanged for tRNA, an ester bond being formed between the carboxyl group of the aminoacyl residue and the ribose hydroxyl of the tRNA terminal nucleoside. The third reaction catalyzed by the ribosome is the substitution of tRNA residue (tRNA') by the aminoacyl-tRNA; this results in the formation of an amide (peptide) bond between the amino group of the aminoacyl-tRNA and the carboxyl group of the other aminoacyl residue (Aa'). If a complete protein molecule consists of n aminoacyl residues, the overall balance of the reactions may be written as follows:

ARSases, tRNAs, RS

Furthermore, pyrophosphate is hydrolyzed in the cell by pyrophosphatase to orthophosphate:

The free energy of the hydrolysis of ATP pyrophosphate bonds under standard conditions (AG0) is about -7 to -8 kcal/mole. The anhydride bond of the aminoacyl adenylate and the ester bond of the aminoacyl-tRNA possess similar values of free energy of the hydrolysis under standard conditions. The free energy of the hydrolysis of the peptide bond in an infinitely long polypeptide (protein) under standard conditions is equal to only -0.5 kcal/mole. It can therefore be seen that the whole process of protein synthesis involves releasing a considerable amount of free energy; in other words, protein synthesis is a thermodynamically spontaneous and energetically ensured process:

n Aa + n ATP-»-protein + n AMP + n PPi - n x 7 kcal.

If pyrophosphate hydrolysis is added, the overall energy balance will be -nx15 kcal per mole of protein. Thus, the free-energy gain under standard conditions for a protein of about 200 aminoacyl residues will be roughly 3000 kcal/mole.

An analysis of the energy balance of each of the three reactions shows that the first two reactions do not by themselves achieve any gain in free energy (under standard conditions), and therefore the pre-ribosomal stages should not be shifted markedly toward the synthetic side; the shift, however, will be generated provided pyrophosphate is hydrolyzed in a parallel reaction. The main difference in the free-energy levels between substrates and products is found in the third reaction. This implies that the shift of the overall reaction toward synthesis is provided mainly by the ribosomal stage.

It is surprising that despite the full energy support of protein biosynthesis at the expense of ATP (or the ester bond energy of aminoacyl-tRNA), the ribosomal stage still requires two GTP molecules per amino acid residue:

This gives an additional free-energy gain of about 15 kcal per mole of amino acid (under standard conditions).

Thus, the sum total of all the chemical reactions in protein synthesis may be written as follows:

ARSases, PPase, tRNAs, RS

n Aa + n ATP + 2n GTP + 3n H,O-»- protein + n AMP + 2n GDP + 4n P;.

The total energy balance of the overall reaction AG0 is equal to about -30 kcal per mole of amino acid or -6000 kcal per mole of protein with a length of 200 amino acid residues.

Here, only the chemical aspect of the process has been taken into account. It is important to analyze to what extent this estimate may be changed if we take into account entropy loss due to the ordered arrangement of the amino acid residues along the chain of synthesized protein, and due to the fixed three-dimensional protein structure. It seems that the entropy loss due to amino acid ordering in the polypeptide chain may introduce only a small correction, around 2.5 kcal per mole of amino acid. As regards the three-dimensional ordering of the chain in the protein molecule, the entropy loss (decrease) is significant here, but it is compensated by the enthalpy gain resulting from non-covalent interactions of amino acid residues. Thus, in any case, the protein synthesis is accompanied by dissipation of a large amount of free energy.

The meaning of the expenditure of such a tremendous excess of energy is an enigma and an extremely interesting problem in molecular biology. Energy excess which is dissipated into heat and not used for any accumulated useful work (in the form of chemical bonds or nonrandom arrangement of residues) should play an important part in the functioning of the protein-synthesizing system. It is likely that this energy excess is necessary to support the high rates and high fidelity of protein synthesis.

0 0

Post a comment