BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 6. AMINO ACID METABOLISM AND FUNCTIONS. PROTEIN BIOSYNTHESIS

6.10. Protein biosynthesis

6.10.3. Amino acid activation and aminoacyl-tRNA formation

To perform its adaptor function, a tRNA molecule must be bound to its corresponding amino acid. This recognition and binding function is carried out by the aminoacyl-tRNA synthetase enzyme (ligase, EC 6.1.1). The necessity for precise and specific interaction between Aminoacyl-tRNA synthetases, tRNAs, and their corresponding Amino Acids has been recognized as the "secondary Genetic Code". Thus, Polypeptide chain synthesis proceeds with the participation not of free amino acids, but of activated (high-energy) amino acids involving highly specific enzyme systems. Aminoacyl-tRNA ligases recognize only one specific proteinogenic Amino Acid and the tRNAs capable of binding exclusively to it.

The stage of preparing amino acids for Translation (recognition) is divided into two steps:

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As can be seen from Fig. 6.34 (1), the first step involves a nucleophilic attack of The amino acid carboxyl group on the phosphoanhydride bond between the α- and β-phosphate groups in the ATP molecule, resulting in The formation of an intermediate product—aminoacyl adenylate, enriched with a high-energy anhydride bond—and the release of pyrophosphate. The hydrolytic Cleavage of the latter, mediated by pyrophosphatase (EC 3.6.1), ensures the irreversibility of the aminoacyl adenylate formation reaction, while the released energy is utilized by the translation system.

Fig. 6.34. Amino Acid Activation reaction involving a class II aminoacyl-tRNA synthetase

In the second step, catalyzed by the same enzyme, the aminoacyl residue is transferred from the aminoacyl adenylate to the corresponding tRNA, specifically to the 2' (3')-hydroxyl group of the terminal adenosine of the tRNA acceptor stem (3'-CCA), leading to the release of AMP and the formation of an aminoacyl-tRNA molecule (Fig. 6.34 (2)). This reaction product—aminoacyl-tRNA—is an element of the secondary genetic code and contains both the informational component and energy in the form of a high-energy ester bond (Fig. 6.35). Energy balance of the process: the activation of each amino acid is accompanied by the utilization of two high-energy phosphate bonds.

Fig. 6.35. General Structure of aminoacyl-tRNA

Aminoacyl-tRNA synthetases are labile Enzymes, and the presence of Mg²+ ions is required to exhibit their maximal catalytic activity. Three substrate-binding centers are identified in the enzyme molecule: for ATP (K =10^-4 mol/L), amino acids (K =10^-5 mol/L), and tRNA (K =10^-8 mol/L). Synthetases are relatively "slow" enzymes, as the turnover number does not

exceed a few hundred catalytic acts per minute. The following course of events has been proposed for the synthetase reaction: ATP is the first to bind to the aminoacyl-tRNA synthetase, adenylating a Histidine (Arginine) residue in the active center of the enzyme with the release of pyrophosphate. In the next stage, the amino acid interacts with the adenylated enzyme to form aminoacyl adenylate, which, in turn, transfers the aminoacyl group to the histidine radical of the enzyme's active center. Thus, an aminoacylated enzyme is formed:

Following the binding of the corresponding tRNA by the enzyme, the aminoacyl group is transferred to the OH group of the ribose of the adenosine residue within the 3'-CCA of the tRNA, forming aminoacyl-tRNA. Depending on whether the aminoacyl residue attaches to the 2'- or (3')-OH group of the ribose of the terminal adenosine in the tRNA acceptor stem, all aminoacyl-tRNA synthetases are divided into two classes. The first class includes enzymes that mediate transfer to the (2')-OH group and activate the following amino acids: Arg, Val, Gln, Glu, Ile, Leu, Met, Trp, Tyr, Cys. Through a transesterification reaction, the aminoacyl group is relocated to the 3'-OH position, as this is predominant during peptide bond formation. This class of synthetases contains a characteristic Rossmann fold: a specific arrangement of a parallel β-sheet and α-Helical structures typical of enzymes that catalyze reactions utilizing high-energy compounds. The second class of enzymes comprises synthetases that activate Ala, Asn, Asp, His, Gly, Lys, Pro, Ser, Thr, Phe. This class is characterized by an antiparallel β-sheet that forms the enzyme's active center. Thus, aminoacyl-tRNA synthetases are enzymes with differences in structural Organization depending on the substrate Specificity of the reactions they catalyze. The Molecular Weight of aaRSs ranges from 40 to 400 ·10³. For E. coli aminoacyl-tRNA synthetases, it has been established that one-third of the enzymes are α-monomers; 10 are α2-homodimers, while a tetrameric structure is characteristic of glycyl-(α2β2), phenylalanyl-(α2β2), and alanyl-(α4)-tRNA synthetases.

The high specificity of tRNA recognition by its synthetase is associated with specific elements of the tertiary L-STRUCTURE OF THE tRNA, as well as the anticodon structure and the composition of nucleotide residues preceding the 3'-CCA. Due to this specificity in selecting tRNA and amino acids, the error rate in aminoacyl-tRNA synthetase function is approximately 1 per 10⁴ incorporated amino acids. At this pre-ribosomal stage, a translation error-correction mechanism operates, mediated by aminoacyl-tRNA synthetases.

In eukaryotes, aminoacyl-tRNA ligases function as part of high-molecular-weight complexes known as chodosomes: 7-9 different aminoacyl-tRNA synthetases, methylases, acetylases, protein Kinases, inorganic pyrophosphatase, Lipids, Prostaglandins and Cyclic NUCLEOTIDES, CARBOHYDRATES, zinc, magnesium, and manganese ions. The functioning of the phosphorylation system within the complex regulates interaction with the Components of the protein-synthesizing system, whereas inorganic pyrophosphatase stimulates the process by cleaving the activation reaction inhibitor—pyrophosphate. These complexes associate with polyribosomes and The Endoplasmic reticulum membrane, ensuring the compartmentalization of the eukaryotic translation apparatus and thereby enhancing the efficiency of its functioning.