Amino acid active site

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. 

Aminoacyl-tRNA Synthetases Have Highly Discriminating Amino Acid Activation Sites... 

Amino acid attachment site Each tRNA molecule has an attachment site for a specific amino acid at its 3 -end (Figure 31.6). The carboxyl group of the amino acid is in an ester linkage with the 3-hydroxyl of the ribose moiety of the adenosine nucleotide at the 3 -end of the tRNA. [Note When a tRNA has a covalently attached amino acid, it is said to be charged when tRNA is not bound to an amino acid, it is described as being uncharged.] The amino acid that is attached to the tRNA molecule is said to be activated. 

The 37-kDa 334-residue subunits of the dimeric type I tryptophanyl-tRNA synthetase238 are the smallest known the largest bacterial synthetase is an alanine-specific type II tetramer with 95-kDa 875-residue subunits.239 Gene deletions show that a much smaller core, comparable in size to that of the tryptophanyl-tRNA synthetase, is needed for amino acid activation. The synthetases share little sequence homology except for a short 11-residue part of the adenylate binding site near the N terminus.240 241 Some of the synthetases contain bound zinc ions.225,242... 

One ATP is used for charging of the tRNA, and then one GTP at each of the steps of binding aminoacyl-tRNA to the A site of the ribosome, and translocation. Thus, ignoring initiation, the equivalent of three ATPs are used for each amino acid incorporated. But remember that in amino acid activation, the products are AMP and PP(, the latter being hydrolyzed to Pj to drive the reaction to completion. Thus, the equivalent of four high-energy phosphate bonds are used for each amino acid incorporated. 

Enzymes in the aaRS family are a promising target for the development of novel antibiotics (17). Selective inhibition of just one essential aaRS would be lethal to the pathogen. The best example is mupirocin, a commercially marketed IleRS inhibitor. Mupirocin, also known as pseudomonic acid, originally was isolated from Pseudomonas fluorescens and is used as a topical antibiotic against gram-positive bacteria, particularly Staphylococcus aureus. It binds directly to the first lysine of the conserved KMSKS sequence in the amino acylation active site (18). 

The CCA terminus containing the amino acid attachment site extends from one end of the L. This single-stranded region can change conformation during amino acid activation and protein synthesis. 

Figure 30.7 Active site of threonyl-tRNA synthetase. Notice that the amino acid-binding site includes a zinc ion (green ball) that coordinates threonine through its amino and hydroxyl groups.

Little information is available on the active site of this enzyme. In the recombinant enzyme from E. coli two cysteine residues were recognized at the positions 129 and 329, one of which seems to be at or close to the active center, as was shown by inhibition with thiol-specific reagents partly in the presence of CTP. However, exchange of these cysteine residues for other amino acids by site-directed mutagenesis did not change the enzyme activity significantly [590]. 

Erythromycins bind reversibly with a single high-affinity site on the 50S subunit of susceptible bacterial ribosomes. The site appears to be proteins L-15 and L-16, two of the 34 proteins constituting the ribosomal protein mass of the 50S unit. Removal of several L-16 proteins (by LiCl extraction) from a 50S subunit eliminates its affinity for EM peptidyl transfer ability is also eliminated. Restoring the L-16 protein alone reestablishes both functions. By itself L-16 has no EM binding capacity L-15 possesses both the capacity to bind EM and to effect peptidyl transfer, participated in some way by L-16. Both events occur on the P-site. Whether the bacteriostatic antimicrobial action of EM is due to the drugs inhibition of peptide bond formation or by the prevention of its translocation following peptide formation has not been established. To clarify the picture somewhat, perhaps it should be pointed out which aspects of protein synthesis are not affected by EM. They are amino acid activation, synthesis of the amino acid /RNA derivative, ribosomal association with raRNA, and reassociation of the 30S and 50S subunits to the complete ribosome. 

---