Amino Acids- Peptide Bonds

Yup, yet another post about amino acids :( Looking at the lecture notes, this isn't going to be easy either...)

You should know the net reaction for peptide bond synthesis and the net hydrolysis reaction.

Okay, this bit I'm pretty sure I know. The net synthesis reaction is just a condensation reaction, where two amino acids bond together and water is released. The net hydrolysis reaction is the opposite: water is added in order to break the bond. (It's not that simple when broken down into individual steps, but that's the net reaction.)

You should know the principles of synthetic peptide synthesis but not detailed structures of the protecting groups and solid phase coupling group. You should understand that synthetic peptide synthesis proceeds from C-terminus to N-terminus.

One of the challenges of synthesising peptides is that you need to ensure that the correct amino acids bind to each other in the correct order. One way to achieve this is to use "protecting groups"- that is, to keep the ends covered until you want them to bind to something. Manipulating both ends can be a bit clunky, so the terminal amino acid is often bound to a polystyrene bead (the "solid phase) on the carboxyl terminus. The amino termini of all of the amino acids is then bound to an Fmoc group which protects amino acids from reaction until required.

When it's time for another amino acid to be added on, the N-terminus of the peptide already on the chain has its Fmoc group removed, and the amino acid to be added on is activated at its carboxyl group by another molecule called DCC. The alpha-amino group of the peptide then "attacks" the carboxyl group of the amino acid to be added. These steps are repeated as often as required, and then HF is used to wash the peptide off the bead.

You should know the amino acid activation reaction with ATP and the nucleophilic substitution reaction coupling the amino acid to tRNA. You should know how the peptide bond is formed between the alpha-amino group of the aminoacyl-tRNA and the carbonyl carbon of the ester link in the peptidyl-tRNA. You should understand that protein synthesis proceeds from N-terminus to C-terminus.

Aminoacyl-tRNA, simply put, is a tRNA with an amino acid attached to the 3' end.

To attach an amino acid to tRNA, the amino acid must first be activated. The alpha-carboxyl of the amino acid attacks the alpha-phosphate of ATP (i.e. the phosphate closest to the ribose sugar), forming 5' aminoacyl adenylate, or aminoacyl-AMP. From there, there are two slightly different paths that the amino acid can take that both lead to the formation of aminoacyl-tRNA. Class I aminoacyl-tRNA synthases will attach the aminoacyl-AMP to the 2' end of the tRNA, releasing AMP. Transesterification then moves the amino acid on to the 3' end. Class II aminoacyl-tRNA synthases, on the other hand, attach the aminoacyl-AMP directly to the 3' end of the tRNA.

Peptidyl-tRNA is tRNA with a polypeptide chain attached to the 3' end. When an aminoacyl-tRNA comes in to the ribosome, the alpha-amino terminus of the new amino acid nucleophilically attacks the carboxyl terminus of the polypeptide chain on the peptidyl-tRNA. This creates a new peptide bond between the amino acid and the polypeptide chain, causing the polypeptide chain to move onto what was originally the aminoacyl-tRNA.The original peptidyl-tRNA, now deprived of its polypeptide, is soon released from the ribosome.

You should know what exopeptidases/exoproteases and endopeptidases/endoproteases are and the differences between them. You should also understand that proteins are broken down, via peptide hydrolysis reactions ultimately into their constituent amino acids, which are most commonly re-used to synthesise proteins.

Peptidases/proteases are enzymes that break down polypeptides. Exopeptidases/exoproteases break off amino acids one by one from the end of the chain, whereas endopeptidases/exoproteases break down bonds in the middle of the chain. Exopeptidases are not specific to any particular amino acids, but endopeptidases are. For example, trypsin will only break bonds at the C-terminal of arginine or lysine.

You should be able to draw the resonance and resonance hybrid forms of the peptide bond and understand that the partial double bond character of the peptide bond prevents rotation about it in proteins and peptides.

The two resonance forms of the peptide bond are one in which the double bond is between the C and the O, and one in which the double bond is between the C and the N with a negative charge on O and a positive charge on N. Resonance leads to stability, and stability in this case means that there is less rotation allowed around the peptide bond.

You should be able to draw H-bonding interactions between the peptide bond and other peptide bonds and with amino acid side chains.

I wrote a bit about peptide bonds in my first post about amino acids.

You should know how disulfide bonds form and that only some proteins have disulfide bonds that stabilise their structures, mainly extracellular proteins in an oxidising environment.

Disulfide bonds are S-S bonds formed between two Cys residues in an oxidising environment. As these are covalent bonds, they play important roles in stabilising the structure. They can be broken in a reducing environment.

You should know what isopeptide bonds are and that they occur between the side chain amino group of Lys and most commonly the side chain carbonyl carbon of Asp/Asn/Gln, but sometimes with a terminal carboxyl carbon. You should understand that the role of intracellular isopeptide bonds in providing extra stability to proteins. You should be aware that ubiquitination tags proteins targeted for degradation with the protein ubiquitin.

Isopeptide bonds are, simply put, peptide bonds that aren't between the alpha-amino and alpha-carboxyl groups but rather between the side chains of amino acids. As these bonds are covalent and have resonance, they are quite stable.

I'm not really sure how ubiquitination is relevant here, but yes, ubiquitin is sometimes added onto proteins as a way of marking them for degradation in proteases etc.

You should be aware that there are other amino acids other than the common 20. Two of these, selenomethionine and selenocysteine, where selenium replaces sulfur in the amino acids, are incorporated during protein synthesis via aminoacyl-tRNAs. Selenomethionine appears to be randomly incorporated instead of methionine whilst selenocysteine is incorporated via a special aminoacyl-tRNA at specific points in protein synthesis. Details of selenocysteyl-tRNA synthesis need not be learnt.

There's not much I can elaborate on here, other than that selenomethionine does not appear to have an effect on protein structure or function, whereas selenocysteine may be important for the activity of some enzymes.

You should be aware that other amino acids are derived from normal amino acids that are chemically modified (through the actions of enzymes) in the synthesised proteins in a general process called post-translational modification.

Amino acids can be modified by adding other groups, such as hydroxyl groups, onto them. For example, a hydroxyl group can be added to proline to form 4-hydroxyproline. These modified amino acids can be important- for example, 4-hydroxyproline is often found in collagen fibres (if I remember correctly).