You should know the reaction for proline hydroxylation and the reason why ascorbate is required to maintain proline hydroxylase in an active state.
Well fffffffffffffffff-
Okay. Breathe. Look at notes. Look at lecture slides. You can do this.
Apparently the reaction is proline + alpha-ketoglutarate + O2 --> 4-hydroxyproline (a.k.a. 4-hyp) + succinate + CO2.
The enzyme that catalyses this reaction is proline hydroxylase. It associates with a cofactor, Fe2+, which oxidises to Fe4+ in the reaction. Ascorbate is required to reduce Fe4+ back to Fe2+ and therefore maintain proline hydroxylase in an active state.
You should understand why proline hydroxylation is essential for the correct folding and stability of collagen and that collagen that does not contain 4-hydroxyproline is unstable and this leads to the degeneration of connective tissue seen in Scurvy. You should know why ascorbate prevents Scurvy.
The ring structure of 4-hydroxyproline (4-hyp) has a slightly different conformation to that of proline (4-hyp is exo while normal proline is endo). The difference in structure makes the correct placement of Pro and 4-hyp essential for the correct folding of collagen, which is a fibrous protein found in many of the connective tissues in our body. As ascorbate is required to maintain proline hydroxylase and thus the production of 4-hyp, a lack of ascorbate (vitamin C), as seen in scurvy, can lead to the degeneration of connective tissue. This can be prevented by sufficient uptake of ascorbate.
You should be able to draw a protein phosphorylation reaction involving Ser/Thr or Tyr (you do not need to know the structure of ATP, other than the three phosphoryl groups). You should be aware that MgATP and MgADP are actually used as substrates by enzymes and be aware that phosphoryl group transfer from ATP is energetically favoured because of the charge separation that ensues.
Serine, threonine and tyrosine all have OH groups. The oxygen is nucleophilic and can attack the gamma phosphate (i.e. the phosphate furthest away) in order to become phosphorylated. Some other amino acids can also be phosphorylated, such as histidine, which can be phosphorylated on the N-H groups of the imidazole ring.
Now for a little bit more about ATP. ATP has three negatively-charged phosphate groups, all joined end to end. You might think that all of those negative charges would repel each other, and you're right. Part of the reason why phosphorylation is so energetically favourable is because of this repulsion between negative charges.
The negative charges between phosphate groups are somewhat "shielded" from other molecules by association with a cation, usually magnesium (Mg2+). This is why MgATP and MgADP, rather than pure ATP/ADP are usually used as substrates by enzymes. There are other reasons why Mg is useful here: it may help position phosphoryl groups, can facilitate binding by forming complexes with other amino acids in enzymes, and can withdraw electrons from the furthest phosphoryl group in order to make it more susceptible to nucleophilic attack.
You should know that protein phosphorylation is catalysed by protein kinases and that dephosphorylation (hydrolysis) is catalysed by protein phosphatases. You should know that there are Ser/Thr kinases and Tyr kinases and that each protein kinase phosphorylates only amino acids in particular amino acid sequences in a protein.
As stated above, kinases are a group of enzymes that add phosphate groups to proteins, whereas phosphatases remove them. Serine and threonine are reasonably similar in structure, which is probably why there are Ser/Thr kinases. Tyrosine, on the other hand, is quite different to the other two, so there are special kinases for tyrosine residues. The reason why protein kinases can be specific not only to Ser/Thr/Tyr but also to particular sequences is that the active site may bind to several amino acids at once.
You should be aware that phosphorylation/dephosphorylation can change the structure of a protein and thus change its biological activity e.g. it can activate or deactivate an enzyme. In addition, it can affect the way the protein interacts with other proteins leading to degradation or translocation within the cell for example.
As I've probably mentioned several times by now, structure is critical to function. Adding or removing a phosphate group changes the shape as well as introduces a negative charge. Hence phosphorylation and dephosphorylation can result in activation or deactivation of an enzyme.
One common misconception is that kinases always activate enzymes while phosphatases always deactivate them. This is not necessarily true. Some enzymes are active with a phosphate group added, while others are active once the phosphate group is removed. It really depends on the enzyme in question.
You should know the "in-line" nature of phosphoryl transfer reactions and be able to draw this.
Okay, this is something that I didn't know until I went back through the slides. Essentially, when a nucleophile attacks a phosphorus group, it attacks in line and opposite to the leaving group on the other side of the phosphorus. This causes the transition state to be planar bipyramidal- the "planar" part coming from the phosphorus and the oxygens being in a plane. The final product also has an inversion of configuration around the phosphorus group (i.e. from S-configuration to R-configuration or vice versa) though this is only really detectable if the phosphorus group is attached to different isotopes of oxygen. (I'll explain chirality and S/R-configurations in a later post.)
You should know the purpose of lipidation is to anchor proteins to membranes, as the lipid group inserts into the membrane bilayer.
Lipidation is the addition of a lipid group to a protein. As lipids are obviously lipid-soluble, lipidation provides a great "anchor" to attach the protein to the membrane.
You should know one example of a lipidation reaction in detail and be able to draw it, using the side chain or terminal group to which the lipid is attached (sketches of the protein structures are not needed) and you can represent the palmitoyl, myristoyl or farnesyl groups by their names, no need to learn their structures. You should know what the substrate is in each of the three lipidation reactions and the type of bond formed with the substrate protein.
Fggfdfaljksdgl I hate learning reactions in detail.
Palmitoylation is the addition of a palmitoyl group onto a protein. It appears to occur at -SH groups on proteins (probably at cysteine residues). Palmitoyl CoA (i.e. palmitoyl attached to coenzyme A) is attached here via palmitoyl acyl transferase, releasing CoA. A thioester bond is formed.
Myristoylation is the addition of a myristoyl group on a protein. It occurs at N-terminal glycine residues. Myristoyl CoA is attached here via myristoyl transferase, again releasing CoA. An amide bond is formed.
Farnesylation is the addition of a farnesyl group on a protein (yup, getting super creative here). It occurs at cysteine residues near the C-terminus of the protein. There may be several different recognisable motifs that follow the cysteine residue, such as AAX (alanine-alanine-any amino acid). Farnesyl transferase attaches farnesyl pyrophosphate onto the cysteine residue, releasing pyrophosphate in the process. Afterwards, a converting enzyme releases the last few residues of the protein.
You should be able to draw the sulfation reaction. You should know the general purpose of protein sulfation.
This is another one where I had to frantically look back through the lecture slides. Thankfully, this one isn't too difficult.
Sulfation is basically the addition of a sulfate group onto a protein- a bit like phosphorylation but with sulfate instead of phosphate. Instead of ATP, there's another molecule called PAPS: 3'-phosphoadenosine 5'-phosphosulfate. This molecule is a bit like ADP, but with a sulfate in place of the second phosphate, and with a phosphate group also attached to the 3' carbon of the ribose sugar. Tyrosyl protein sulfotransferase (TPST) catalyses the movement of the sulfate group from PAPS onto the protein.
Protein sulfation is thought to be involved in protein-protein and cell-cell interactions.
You should be able to draw the acetylation reactions. You should understand the different roles of N-terminus acetylation and the reversible acetylation of Lys residues in histones. You do not need to be able to reproduce the complex diagrams of nucleosome and chromatin structures.
Acetylation basically involves the movement of an acetyl group from acetyl CoA onto either the N-terminus of a protein or onto lysine residues, releasing CoA in the process. This is catalysed by N-acetyl transferase (in the case of addition onto the N-terminus) or histone acetyl transferase (in the case of addition to lysine residues). N-terminus acetylation is thought to be able to protect proteins from degradation, whereas acetylation of lysine residues in histones is thought to serve as "markers" that can be read by other proteins that regulate gene expression.
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