1. There is little twisting possible around the amide linkage of the peptide bond because the peptide bond has substantial double bond character. As a result, the group of atoms about the peptide bond can exist in the cis or trans configurations (see here). If we trace the carbons on the amino acids in a peptide to determine the cis/trans nature of the peptide bond, we find the trans configuration is the one usually favored. One exception is bonds in the sequence X-Pro, where X is any amino acid and Pro is proline. In this case, the cis configuration may be favored at times.

2. A molecule of water must be eliminated for each peptide bond formed. In an aqueous environment, the formation of a peptide bond is not favored thermodynamically (G +10 kJ/mol at room temperature). Instead, the reverse reaction, hydrolysis of the peptide bond, is favored.

3. In the absence of a catalyst, peptide bonds are fairly stable. That is, the uncatalyzed reaction is exceedingly slow at physiological pH and temperature. Thus, polypeptides are metastable. They hydrolyze rapidly only under extreme conditions or when suitable catalysts are present.

4. Because peptide bond hydrolysis is favored thermodynamically, not peptide bond formation, additional energy is required to make peptide bonds in cells. Thus, peptide bond formation is coupled to the hydrolysis of high-energy phosphate bonds during the process of translation. Figure 5.19 shows that activating the amino acid via attachment to a transfer RNA (tRNA) is the first step. ATP is hydrolyzed to AMP and PPi concomitantly with the formation of a covalent bond between the -carboxyl group of the amino acid and the 3' hydroxyl group of the adenosine of the appropriate tRNA molecule. The aminoacyl-tRNA molecule thus obtained can then form the peptide linkage between the -amino group of its amino acid residue and the -carboxyl group of the growing peptide chain.

5. Peptide bonds can also be synthesized chemically in a laboratory.