H

(In the equation. R represents the specific side group of the amino acid.} The enzymes that catalyze this type of reaction are called aminoacyl synthetases. Upon activation, an amino acid (AA) is fchermudynamically capable of being efficiently used for protein synthesis. Nonetheless, the AA-AMP complexes am not Ihe direct precursors of proteins. Instead, for a reason we shall explain in Chapter 14, a second group transfer must occur to transfer the amino acid, still activated at its Carboxyl group, to the end 6f a tRNA molecule:

A peptide bond then forms by the condensation of the AA-tRNA niolnculn onto the end of a growing polypeptide chain:

AA-tRNA + growing polypeptide chain (of /? amino acids)-*

tRNA + growing polypeptide chain (ofn + 1 amino acids)

(Equation 4-151

Thus, the final step of this "coupled reaction," like that of all other couplcd reactions, necessarily involves the removal of the activating group and ihc conversion of a high-energy bond into one with a lower free energy of hydrolysis. This is the source of the negative AG that drives the reaction in the direction of protein synthesis.

Nucleic Acid Precursors Are Activated by the Presence of © -O

Both types of nucleic acid, DNA and RNA, are built up from mononucleotide monomers, also called nucleoside phosphate. Mononucleotides, however, are thermodynamirslly even less likely 1o combine than amino acids. This is because the phosphodiester bonds that link the former together release considerable free energy upon hydrolysis (-6 kcai/mol). This means that nucleic acids will spontaneously hydrolyze, at a slow rate, to mononucleotides. Thus, it is even more important that activated precursors be used in the synthesis of nucleic acids tha^i in the synthesis of proteins.

The immediate precursors for both DNA and RNA are the nucleoside-5'-triphosphates. For DNA, these precursors are dATP, dGTR dCTP, and dTTP (d stands for deoxy); for RNA, the precursors arc ATP, GTP, CTP, and UTP. ATP thus, not only serves as the main source of high-energy groups in group-transfer reactions, but it is itself a direct precursor for RNA. The other three RNA precursors all arise by group-transfer reactions like those described in Equations 4-10 and 4-11, The deoxytriphosphates are formed in basically the same way: After the deoxymononurleotides have been synthesized, they aie transformed to the triphosphate form by group transfer from ATP:

Deoxynucleoside—© + ATP-» Deoxynucleoside— ©~© + ADP,

{Equation 4-16)

These triphosphates can then unite to form polynucleotides held together by phosphodiester bonds. In this group-transfer reaction, a pyrophosphate bond is broken and a pyrophosphate group released:

+ growing polynucleotide chain (of n nucleotides) ©~Q + growing polynucleotide chain

This reaction, unlike that which forms peptide bonds, does not have a negative AG. In fact, the AG is slightly positive (aboul 0-5 kcal/mol). This situation immediately poses the question — as polynucleotides obviously form—What is the source of the necessary free energy?

The Value of O-G Release in Nucleic Acid Synthesis

The needed free energy comes from the splitting of the high-energy pyrophosphate group that is formed simultaneously with the high-energy phosphodiester bond. All cells contain a powerful enzyme, pyrophosphatase, which breaks down pyrophosphate molecules almost as soon as they are formed:

[Equation 4-191

The large negative AG means thai the reaction is effectively irreversible. This means that once O-Q is broken down, it never reforms.

The union of the nucleoside monophosphate group (Equation 4-16), coupled with the splitting of the pyrophosphate groups (Equation 4-19), has an equilibrium constant determined by the combined AG values of the two reactions: (0.5 kcal/mol) + (-7 kcal/mol). The resulting value (AG ■= —6.5 kcal/mol) tells us that nucleic acids almost never break down lo reform their nucleoside triphosphate precursors.

Hero wo see a powerful example of the fact that often it is the free-energy change accompanying a group of reactions that determines whether a reaction in the group will take place. Reactions with small,, positive AG values, which by themselves would never take place, are often part of important metabolic pathways in which they are followed by reactions with large negative AG values. At all times we must remember that a single reaction (or even a single pathway) never occurs in isolation; rather, the nature of the equilibrium is constantly being changed through the addition and removal of metabolites.

©~© Splits Characterize Most Biosynthetic Reactions

The synthesis of nucleic acids is not the only reaction where direction is determined by the release and splitting of Q~Q. In fact, essentially alt biosynthetic reactions are characterized by one or more steps that release pyrophosphate groups. Consider, for example, the activation of an amino acid by the attachment of AMP. By itself, the transfer of a high-energy bond from ATP to the AA-AMP complex has a slightly positive AG. Therefore, it is the release and splitting of ATP's terminal pyrophosphate group that provides the negative AG that is necessary lo drive the reaction.

The great utility oi the pyrophosphate split is neatly demonstrated when we consider the problems that would arise if a cell attempted to synthesize nutileic acid from nucleoside diphosphates rather than triphosphates (Figure 4-5). Phosphate, rather than pyrophosphate, would be liberated as the backbone phospho-diester linkages were made. The phosphodiester linkages, however, are not stable in the presence of significant quantities of phosphate, because they are formed without a significant release of free energy. Thus, the biosynthetic reaction would be easily reversible; if phosphate were to accumulate, the reaction would begin to move in the direction of nucleic acid breakdown according to the law of mass action. Moreover, it is not feasible for a cell to remove ihr phosphate groups as soon as they are generated (thereby preventing this reverse reaction), as all cells require a significant internal level of phosphate to grow. In contrast, a sequence of reactions that liberate pyrophosphate and then rapidly break it down into two phosphates disconnects the liberation of phosphate from the nucleic acid biosynthesis reaction, and thereby prevents the possibility of reversing the biosynthetic reaction (see Figure 4-5). In consequence, it would be very difficult to accumulate enough phosphate in the cell to drive both reactions in the reverse, or breakdown, direction. It is clear that the use of nucleoside triphosphates as precursors of nucleic acids is not a matter of chance.

This same type of argument tells us why ATP, and not ADP, is the key donnr of high-energy groups in all cells. At first this preference seemed arbitrary to biochemists. Now, however, we see that many reactions using ADP as an energy donor Would occur equally well in both directions.

nucleoside diphosphate

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