Proteins

• The side chain of proline is known as an imino acid and is often found in the bends of folded protein chains.
• Molecules can be classified as dextrorotatory (clockwise) or levorotatory (anticlockwise) depending on how they rotate in polarised light.
• Cysteine is structured similarly to other amino acids but the sulfur atom alters the interpretation of the Cahn-Ingold-Prelog priority rule.

  
• A peptide bond is a rigid and planar unit with partial double bond characteristics that limit the conformational flexibility of the polypeptide chain. The amino terminus is the beginning of the polypeptide chain.
• Protein structures:

  Primary: linear sequence of amino acids

  Secondary: regular folding of regions of the polypeptide chain

  Tertiary: 3D arrangement of amino acids that are biologically active, maintained by non-covalent bonds
• Alpha helix:

  • Right handed coil
  • 3.6 residues per turn
  • Pitch of 5.4 A
  • Average length of 12 residues / 18 A over three helical turns
  • Core is tightly packed with favourable hydrogen bonding patterns and torsion angles with minimal steric interference
  • Side chains project outwards to avoid steric interactions with the backbone
• Beta-pleated sheet:

  • Uses the full hydrogen bonding capacity of the backbone
  • Can be parallel or anti-parallel
  • Parallel sheets can form beta barrels

  
• Keratin:

  • Fibrous protein that is unreactive and durable
  • Two alpha helices to form a left handed coil with a pitch of 5.1 A
  • Tilted helix causes an apparent reduction in pitch
  • Dimer is typically 450 A
• Beta-keratin:

  • Less disulphide bonds than alpha keratin allowing for stretching
  • Assumes beta-sheet conformation
• Alpha-keratin:

  • Present in mammals
  • Rich in cysteine residues to co-link adjacent chains
  • Hard or soft depending on sulphur content
  • Disulphide bonds are cleaved with mercaptans (thiols) and re-established with oxidising agents

  
• Collagen:

  • Triple helix that forms stress bearing components of connective tissues
  • Nearly a third of residues are glycine, and 15-30% proline
  • Three non-standard residues found
• Non-standard residues in collagen:

  • 4 - hydroxyproline
  • 3 - hydroxyproline
  • 5 - hydroxylysine

  
• Non-standard residues are formed after collagen peptides are synthesised. Pro residues are converted to Hyp in a reaction catalysed by prolyl hydroxylase (requires vitamin C).

  
• The amino acid sequence of collagen peptides consist of repeating triplets of G-P-Hyp/Hyl. Repeating proline residues prevent formation of an alpha helix, forming a left handed helical formation with 3 residues per turn
• Collagen is covalently cross-linked but not by disulphide bonds (no cysteine residues). Links are between lysine and histidine catalysed by lysyl oxidase and tend to occur near the N and C terminus of the molecule.
• Membrane proteins are globular that have motifs:

  • Four helix bundle
  • Helix - turn - helix
  • EF hand
  • Coiled coil
  • Greek key
  • Beta - alpha - beta
  • Beta - hairpin

  
• Chymotrypsin catalyses hydrolysis of proteins in the small intestine, part of the serine protease family. The serine is critical for catalytic activity. The catalytic triad:

  • Asp-102
  • His-52
  • Ser-195
• Ser-195 in chymotrypsin is activated by hydrogen bonding with His-57, with its activation as a result of hydrogen bonding with Asp-102.

  • Proton transfer from Ser-195 to His-57
  • Positively charged imidazole ring stabilised by electrostatic interaction with negatively charged Asp-102

  
• Substrate specificity is determined by the properties and spatial arrangement of the amino acids forming the active site. Small changes in amino acid residues forming the active site will have a large effect on the substrate specificity of an enzyme.

  
• The mechanism of the active site of chymotrypsin:

  1. Acetylation (Td state dissociates, leaving an acyl-enzyme intermediate)
  2. Deacylation (the acyl intermediate is hydrolysed by water)
  3. Reconstruction (the catalytic triad is reconstituted)

  
• Methyltransferases catalyse alkylation reactions, with substrates including DNA, RNA, proteins and small molecules.
• There are three methods of DNA methylation:

  • N6 adenine
  • N4 cytosine
  • C5 cytosine

  
• Prokaryote utilise all three methylated bases, whereas eukaryotes only form C5-methylcytosine.

  
• Specific methylases:

  • DAM (DNA adenine methylase) = GA*NT
  • CcrM (cell-cycle regulated DNA methylase) = GTA*NT
  • Hhal (DNA cytosine methylase) = CGC*G
• Hhal DNA MTase is part of the R/M system of Haemophilus haemolyticus. This is organised into two domains, the restriction domain and the modification domain.

  
• 5-fluorouracil (5-FU) is a pyrimidine analogue used as a methylation agent for cancer treatment that inhibits thymidylate synthase.

  • 5-FU is converted into metabolites in the cell that are incorporated into DNA and RNA
  • Causes cell cycle arrest and apoptosis
  • 5-FU containing DNA can inhibit C5 MTases

  
• The presence of fluorine on 5-FU prevents the elimination reaction and thus the DNA strand becomes covalently bonded to C5 MTase.

  
• There are four human C5 MTases known:

  • DNMT1
  • DNMT2
  • DNMT3a
  • DNMT3b
• Each DNMT enzyme cooperatively maintain all methylation in cancer lines. This can result in the inactivation of tumour suppressor genes through hypermethylation.

  • This is essential for neoplastic proliferation (cancer tumour formation)
  • Elimination of these enzymes by sequence deletion reduces genomic methylation by more than 95%

  
• When [S] is higher than [E], the reaction rate becomes independent of [S]. The RDS is [ES]. The overall rate of production of ES is the difference between the rates of the elementary reactions leading to its appearance and dissapearance.

  
• ES maintains a steady state and can be treated as having a constant value. This is related to the Michaelis constant KM.

  
• The initial velocity of the reaction can be expressed in terms of [E]T and [S].

  • Ts is the time when steady state is first achieved
  • This minimises such complicating factors as the effects of reversible reactions, product inhibition and progressive inactivation of the enzyme

  
• There are two forms of aldehyde dehydrogenase with a low KM mitochondrial and a high KM cytoplasmic form. Some individuals have a point mutation in their mitochondrial enzyme rendering it less active.

  
• Lineweaver-Burke plots:

  • Used to observe kinetic effects
  • Slope = Km / Vmax
  • y-intercept is 1 / VMax
  • x-intercept is -1 / KM
  • Competitive inhibitors will have steeper slopes

  
• Inhibitor binding:

  • Irreversible = tightly bound, covalent, residues include serine and cysteine e.g., di-isopropyl phosphofluoridate
  • Reversible = at high substrate concentration, the inhibitor is competed out of the active site
  • Noncompetitive = cannot be overcome by increasing the substrate concentration so enzyme affinity remains unchanged as well as KM

  
• All cancer cells are glycolytic, producing a lot of lactic acid and have a higher demand for glycolysis substrates e.g., glucose.
• Phosphoryl-transferase reactions involve the synthesis and hydrolysis of ATP.

  • AMPK senses the ratio between ATP and AMP
  • AMP kinase stops ATP usage and increases glucose transport to generate ATP
  • Phosphoanhydride bonds are high-energy whereas phosphodiester is not

  
• Redox reactions are carried out by enzymes, with coenzymes such as nicotinamide adenine dinucleotide (NAD+). C-H bonds can be visualised as hydride transfers. Each substrate must be aligned at the correct position in the enzyme active site so that orbitals overlap sufficiently.

  
• Step 1 of glycolysis:

  • Glucose -> gluose-6-phosohate (G6P)
  • Catalysed by hexokinase
  • With cofactor Mg2+ to induce kinase activity by making the gamma phosphate more susceptible to attack

  
• Step 2 of glycolysis:

  • G6P -> F6P
  • Catalysed by phosphoglucose isomerase (PGI)
  • Ring contraction by breaking hemiacetal bond, giving anomers, then tautomerisation