Amino acid-activating enzymes specificity

As we have noted, the outcome of a virus infection is the synthesis of viral nucleic acid and viral protein coats. In effect, the virus takes over the biosynthetic machinery of the host and uses it for its own synthesis. A few enzymes needed for virus replication may be present in the virus particle and may be introduced into the cell during the infection process, but the host supplies everything else energy-generating system, ribosomes, amino-acid activating enzymes, transfer RNA (with a few exceptions), and all soluble factors. The virus genome codes for all new proteins. Such proteins would include the coat protein subunits (of which there are generally more than one kind) plus any new virus-specific enzymes. 

Fig. 5.1 Simplified model representation of the activation of an amino acid (ASY) at an amino acid-activating enzyme (i.e., an amino acid-specific aminoacyl-tRNA synthetase)...

Jhe synthesis of proteins, as characterized by the in vitro incorporation of amino acids into the protein component of cytoplasmic ribonu-cleoprotein, is known to require the nonparticulate portion of the cytoplasm, ATP (adenosine triphosphate) and GTP (guanosine triphosphate) (15, 23). The initial reactions involve the carboxyl activation of amino acids in the presence of amino acid-activating enzymes (aminoacyl sRNA synthetases) and ATP, to form enzyme-bound aminoacyl adenylates and the enzymatic transfer of the aminoacyl moiety from aminoacyl adenylates to soluble ribonucleic acid (sRNA) which results in the formation of specific RNA-amino acid complexes—see, for example, reviews by Hoagland (12) and Berg (1). The subsequent steps in pro-... 

Experimentally, C14-aminoacyl sRNA was incubated with rat liver microsomes or ribosomes, GTP, various fractions obtained from the nonparticulate portion of rat liver homogenates, and buffered salt-sucrose medium in a total volume of approximately 2 ml. (6-10). The C14-aminoacyl sRNA was prepared by the phenol-extraction procedure from the pH 5 amino acid-activating enzymes, fraction of rat liver after incubation with C14-L-amino acids (9, 13). C14-leucyl sRNA (approximately 1000 c.p.m.), having a specific radioactivity of approximately 55,000 c.p.m. per mg. of RNA, and containing a complement of endogenous, unlabeled, bound amino acids, was used in most of these studies. The microsomes were sedimented from the post-mitochondrial supernatant at 104,000 x g (10) and the ribosomes were prepared from them by extraction with deoxycholate (16). 

Some amino acid activating enzymes have been prepared in highly purified form. These are listed in Table IV they are all specific for a single amino acid, but may be contaminated with 10-50 % of other protein material. 

In translating the message contained in m-RNA into protein, specific amino acids are recognised, assembled and joined together in the sequence specified by the m-RNA. The amino acids do not, however, enter directly into the system but are first activated in reactions involving ATP and amino acid activating enzymes (AAA enzymes) a separate specific enzyme for each of the 20 individual protein amino acids ... 

The active site of an enzyme is generally a pocket or cleft that is specialized to recognize specific substrates and catalyze chemical transformations. It is formed in the three-dimensional structure by a collection of different amino acids (active-site residues) that may or may not be adjacent in the primary sequence. The interactions between the active site and the substrate occur via the same forces that stabilize protein structure hydrophobic interactions, electrostatic interactions (charge-charge), hydrogen bonding, and van der Waals interactions. Enzyme active sites do not simply bind substrates they also provide catalytic groups to facilitate the chemistry and provide specific interactions that stabilize the formation of the transition state for the chemical reaction. 

Activation of the amino-acids. This stage takes place in the cytoplasm. Each of the 20 amino-acids is covalently attached to a specific tRNA at the expense of ATP hydrolysis (i.e. it is an energy-driven process). Each amino-acid has a specific enzyme for this reaction to ensure that the correct amino-acid is linked to the tRNA molecule. 

Formation of aminoacyl-tRNA. This is a two-step process involving a single enzyme that links a specific amino acid to a specific tRNA molecule. In the first step (1) the amino acid is activated by the formation of an aminoacyl-AMP complex. This complex then reacts with a tRNA molecule to form an aminoacyl-tRNA complex (2). 

The attachment of an amino acid to an appropriate tRNA is accomplished via aminoacyl-tRNA synthetase and the hydrolysis of ATP. There is a separate enzyme specific for each amino acid, and it will recognize all tRNAs for that amino acid. The reaction proceeds in two steps and requires Mg2+ (Fig. 17-8). The first step, amino acid activation, results in the formation of an aminoacyl-AMP-enzyme intermediate. In the second step, the aminoacyl group is transferred to its appropriate (cognate) tRNA, the amino acid being linked to tRNA through an ester bond. It appears that recognition between the synthetase and tRNA is achieved through very precise contact between the... 

Why do enzymes bind to substrates Enzymes and substrates are attracted to each other via noncovalent interactions, such as electrostatic attractions. The active site of an enzyme has amino acids in a specific orientation where they can bind to the substrate. The energy diagram will show that the energy of the ES complex is less than the energy of the E + S alone. 

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