Peptides bond synthesis

Nissen P, Hansen J, Ban N et al (2000) The structural basis of ribosome activity in peptide bond synthesis. Science 289 920-930... 

Peptide bond synthesis Protein synthesis (on ribosomes) ATP, GTP... 

It is interesting to note that serine peptidases can, under special conditions in vitro, catalyze the reverse reaction, namely the formation of a peptide bond (Fig. 3.4). The overall mechanism of peptide-bond synthesis by peptidases is represented by the reverse sequence f-a in Fig. 3.3. The nucleophilic amino group of an amino acid residue competes with H20 and reacts with the acyl-enzyme intermediate to form a new peptide bond (Steps d-c in Fig. 3.3). This mechanism is not relevant to the in vivo biosynthesis of proteins but has proved useful for preparative peptide synthesis in vitro [17]. An interesting application of the peptidase-catalyzed peptide synthesis is the enzymatic conversion of porcine insulin to human insulin [18][19]. 

Fig. 3.3. Major steps in the hydrolase-catalyzed hydrolysis of peptide bonds, taking chymo-trypsin, a serine hydrolase, as the example. Asp102, His57, and Ser195 represent the catalytic triad the NH groups of Ser195 and Gly193 form the oxyanion hole . Steps a-c acylation Steps d-f deacylation. A possible mechanism for peptide bond synthesis by peptidases is represented by the reverse sequence Steps f-a.

Nelson RJ, Ziegelhoffer T, Nicolet C, Werner-Washburne M, Craig EA (1992) The translation machinery and 70 kd heat shock protein cooperate in protein synthesis. Cell 71 97-105 Nissen P, Hansen J, Ban N, Moore PB, Steitz TA (2000) The structural basis of ribosome activity in peptide bond synthesis. Science 289 920-930... 

The steps of initiation occur on the isolated small subunit (30S) of the prokaryotic ribosome. Ribosomes contain two subunits, a 30S and 50S subunit, which associate to form a 70S particle. (The S values refer to the rate at which each component sediments in the ultracentrifuge they don t always add up.) In general, the 30S subunit is mostly involved in the decoding and tRNA-mRNA interaction process, while the 50S subunit is involved in actual peptide bond synthesis. Ribosomal subunits are dissociated prior to the initiation reaction. 

Most of the experiments on incorporating amino acid esters into proteins during the plastein reaction have been carried out with papain, indicating that it is one of the best enzymes for this purpose. Other enzymes such as chymotrypsin (40) or carboxypeptidase Y from Sac-charomyces cerevisiae (41) are potent catalysts for peptide synthesis in homogeneous systems using N-acylamino acid esters of peptides as substrates and amino acid derivatives or peptides as nucleophile components. Adding organic co-solvents favored peptide bond synthesis (42,43). 

The equilibrium-controlled synthesis (thermodynamic approach) is the direct reversal of proteolysis. For this reason, independent of the individual mechanism, aU proteases can be used. The thermodynamic barrier to peptide bond synthesis is predonoinantly due to the energy required for the transfer of a proton from the positively charged a-annino group to the negatively charged a-carboxy group. The equilibrium constant for tho conversion of the un-ionized reactants into the product is determined by the concentration of the unionized forms in (Scheme 1). 

Scheme 1 Equilibrium-Controlled Peptide Bond Synthesis...

In a continuation of some studies on acyl migrations in model compounds related to aminoacyl-S-RNA, a technique has been developed using NMR to determine the rate of acyl migration in some 3 -0-acyl ribonucleo-sides. It was estimated that the half-time of equilibration into 2 and 3 -isomers of an averag e aminoacyl-S-RNA derivative in H 7 buffer at 37° is 2 X 10 sec. It was concluded that equilibration of an average aminoacyl-S-RNA would most likely be much faster than peptide bond synthesis. 

Following peptide bond synthesis, the ribosome Is translocated along the mRNA a distance equal to one codon. This translocation step is promoted by hydrolysis of the GTP in eukaryotic EF2-GTP. As a result of translocation, tRNAj , now without its activated methionine, is moved to the E (exit) site on the ribosome concurrently, the second tRNA, now covalently bound to a dIpeptIde (a peptIdyl-tRNA), Is moved to the P site (Figure 4-26, step U). Translocation thus returns the ribosome conformation to a state in which the A site Is open and able to accept another amlnoacylated tRNA complexed with EFlct-GTP, beginning another cycle of chain elongation. 

Not only transpeptidation, but also a certain amount of peptide-peptide condensation, is possibly involved in the plastein reaction. With some of the proteases used for plastein formation, especially a-chymotrypsin (26, 52, 53, 54), the acyl-enzyme intermediate can be formed at pHs 5 from the reversal of the degradative reaction (Equation 1 E-OH + HOOC-CHR-NH E-O-OCCHR-NH- + H20). Once the acyl-enzyme intermediate is formed, the acyl group can be transferred to a nucleophile resulting in peptide bond synthesis. 

The mechanism of peptide bond synthesis is thought to resemble the reverse of the acylation step in the serine protease, with the base of A2486 (A2451 in E. coli) playing the same general base role as histidine-57 in chymotrypsin. This A is universally conserved within the central loop of domain V [28]. 

For a polypeptide of N residues, a minimum of 4N high energy phosphates (such as ATP or GTP) must be hydrolyzed. This corresponds to about 50,000 kJ/mol for a typical protein of 300 residues. Per peptide bond, therefore, it costs the cell about 160 kJ of free energy. Note, however, that the free energy change required for peptide bond synthesis in dilute aqueous solution is about 20 kJ/mol. The difference between cellular peptide bond synthesis (160 kJ/mol) and peptide bond synthesis in dilute solutions (20 kJ/mol) is that cellular peptide bond synthesis is not random. That is, making a defined sequence of amino acids comes with an energy cost. 

The direction of an enzymatic equilibrium reaction (hydrolysis or peptide bond synthesis) depends on the chemical conditions [40]. Besides the above-mentioned hydrolysis, the proteinase-catalyzed synthesis of peptide bonds [41] has also been widely investigated and will be discussed below. 

Aso et al. [95] studied a model system in order to obtain basic information on the mechanism of amino acid incorporation during an enzymatic modification reaction in the presence of papain. They found that the amino acid ester reacted as a nucleophile in the aminolysis of the acyl-enzyme intermediate to result in the formation of new peptides. Several proteases used in enzymatic peptide bond synthesis are known to form transitory acyl-enzyme intermediates during the hydrolysis of proteins. However, the acyl groups can be transferred to other nucleophiles (amino terminals of peptides or amino acids), synthesizing new peptide bonds [71]. With full knowledge of the above-mentioned facts, covalent amino acid enrichment of proteins can result in... 

Section 23.9 Strategy of Peptide Bond Synthesis N-Protection and C-Activation... 

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