Please enable JavaScript.

Coggle requires JavaScript to display documents.

Amino acids - Coggle Diagram

- - - - amino acid analysis
        
        determines which aa are present (composition), quantity, but not the sequence
        
        steps
        
        acid hydrolysis of peptide into aa
        
        separate acid hydrolysis in mixture by HPLC
        
        quantify the aa by conversion to chromophores and measuring light absorbed with uv-vis
        
        high performance liquid chromatography (HPLC)
        
        take aa mixture and mixes with buffer
        
        force into column
        
        mix with ninhydrin to stain them
        
        goes through heater and intensity is recorded by photometer
        
        1 more item...
        
        column has stationary face that make hydrophobic aa and + charge aa stick and move slower
        
        polar aa and - aa move fast
      - determining sequence
        
        Edman
        
        partial hydrolysis makes fragments (some have their amid bonds)
        
        requires small portions because there are byproducts and can have limited cycles performed at a time
        
        chops each aa with n-terminal seq.
        
        first undergo nucleophilic addition of the peptide to phenylisothiocyanate (PITC)
        
        molecule undergoes intramolecular nuc. acyl substitution cleaving the first aa off
        
        generate a unique PTH derivative for each amino acid which through properties in TLC, can determine the aa identity
        
        N-terminus identification
        
        uses DNFB(tag) to create coloured products through a nucleophilic aromatic substitution reaction
        
        the N-terminus can be identified by the DNFB tag
        
        the tag only reacts to primary amines
        
        in aa with nucleophilic R groups like lysine, we look for 2 DNP tags instead of 1. The side chain reacts with the tag
      - C-terminal sequencing
        
        chopping of the peptide from the C-terminus using carboxypeptidase
        
        not efficient as the enzyme quits eventually
        
        on the graph, we measure the concentration of each aa as it is cleaved
        
        the ones that are cleaved first, will observe the first increase in concentration (after identifying with HPLC)
    - - The R groups and the carbonyl groups follow an up/down pattern to limit steric hinderence
      - also respects the planar nature of the molecule
        
        The C and N have double bond character
      - The planar groups can swivel about the alpha carbon giving different shapes
    - - if a big mutation such as charge change occurs, the properties of the protein changes
  - - - stabilized by H-bond
        
        depending where they are can form different structures
        
        alpha-helix
        
        3.6 aa/turn
        
        each NH bonds to a carbonyl 4 aa away holding the helix together
        
        R groups stick out for recognition purposes
        
        beta-sheath
        
        can be antiparallel
        
        can be parallel
        
        have longer linkages than antiparallel
        
        H bonds occur between NH and neighboring segment carbonyl
  - - - hydrophobic interaction
        
        non-covalent
      - H-bonds
      - electrostatic interaction
      - disulfide bonds
        
        type of covalent interaction
        
        disulfide can be reduced by another thiol
- - - - H-bonding
        
        only H-bond between O and N are biologically significant
        
        there is usually an H-bond in the enzyme and with the substrate
        
        to form enzyme substrate H-bond, must break the other H-bonds
        
        we can breaking 2 bonds, while forming 2, meaning the net energy is 0
        
        if the substrate is misoriented, we need energy to revert it as it is unfavourable
      - hydrophobic forces
        
        when enzyme and substrate are apart, the water is more ordered around the non polar molecules
        
        as they come together, H2O are surrounded by less non-polar molecules, favoring their disorder
        
        the water has become more disordered, favoring entropy, but the enzyme and substrate coming together has become more ordered
        
        causes a - G value
      - Ionic interactions
        
        water forms a shell around the charged atoms, preventing them from fusing when the enzyme and substrate are apart
        
        this is a polar environment
        
        when the enzyme and substrate comes together, is less polar allowing charged group to attach
  - - - cleaving occurs after the target aa from N to C terminus
      - an enzyme is required because little OH is present at physiological pH, so uses serine for catalysis
        
        the enzyme also stabilizes the tetrahedral intermediate(H-bond) and protonates the bad LG as it leaves
        
        in other words, the enzyme is using a diff mechanism
    - - step 1: the serine acts as a nuc., removing the C-terminus portion and the N-terminal fragment covalently binds to the enzyme as an ester
        
        these rxns are a series of nuc acyl substitution
      - Step 2: the enzyme uses H2O to hydrolyze the enzyme bond and N-term fragment as well as regen the enzyme
    - - serine is a a poor nuc so His deprotonates it and ser forms a cov bond to the substrate
        
        Tetrahedral intermediate collapse, causing LG to leave but LG gets protonated
        
        the substrate is bond ot the enzyme and is called an acyl enzyme
        
        water replaces the amine, but gets deprotonated by His. At same time H2O does a nuc attack on the ester
        
        oxyanion is stabilized by oxyanion hole, but tetrahedral structure collapse, regenerating the C=O which gets protonated shorty after
        
        enzyme is returned to original state and the R1 fragment is cleave from the enzyme
  - - - can be inorganic ions or small organic molecules
        
        the small organic molecules are called coenzymes
        
        most coenzymes come from vitamins
- - - - makes carbonyl more electrophilic by protonating the oxygen
        
        protonate LG to make it better so can break off the NHR group
    - - forces the bad LG to leave with a slow reaction
- - - - allows amide or carboxyl to react
    - - use a protecting group to protect the amide and carboxyl on the sides and not the two in the center
        
        take protecting group off afterwards
        
        solution phase synthesis
        
        protecting the COOH
        
        use a benzyl alcohol
        
        forms a benzyl ester through Fischer esterification
        
        deprotecting the COOH
        
        use a strong acid and acetic acid
        
        breaks the ester linkage, but not the amide bond due to esters having a higher acid-liability
        
        protecting the NH2
        
        protecting as a t-butyloxycarbamate using tert-butyloxycarbonyl anhydride by nuc acyl substitution
        
        creates a BOC group
        
        deprotecting the NH2
        
        use trifluoroacetic acid
        
        not strong enough to remove COOH's protecting group or cleave the amide bond
        
        forms an alkene through E1 and deprotects the amide
  - - - use coupling reagent to turn it into a more reactive acid derivative, but not acidic enough like acid chloride
        
        Use DCC
        
        DCC activates the COOH group, making it more reactive to couple
        
        more reactive group now uses with the amine, forming the amide linkage
        
        DCU is made as a byproduct due to it being a good LG
        
        Use TFA after to deprotect NH2 to add another peptide
        
        add DCC to form amide bond again
        
        finally add HBr/HOAc tp remove both protecting groups
      - cannot be acid chloride
        
        has two forms due to the very electronegative carbon, forming a racemic/ impure amino acid (enolization)
  - - - groups can now be added onto the N terminus, while filtering out any unwanted reagents
        
        the vessel will then only remain with the beads with the peptide chain attached
        
        can use same reagents as solution phase synthesis to extend the peptide chain
    - - requires a very high efficiency
        
        low efficiency makes lots of impurities, resulting in very little of desired final product after many round of synthesis
        
        lots of byproducts can also be made as well
- - - - D
        
        NH3 points to the right
      - L
        
        NH3 points to left
        
        natural amino acids are usually this form
      - D and L conformation are enantiomers
- - - - codons have 3 nucleobases and they are degenerate such that some aa have the same code
    - - recognizes the codons and binds them with anticodons
      - binds to the aa on the 3' ribose OH end
        
        the bond is a ester linkage made by a aminoacyl-tRNA synthetase that is specific to all 20 aa
        
        the bond is made by turning the COOH into a strong acid catalyst using ATP
        
        in this reaction a mixed anhydride is made between the amino acid and AMP (mechanism similar to nuc. acyl substitution)
        
        by adding AMP, it provides a handle for the aa to be bound by the tRNA
    - - to make amide bonds, ribosomes catalyze a peptidyl transferase reaction
        
        the entire complex + tRNA moves over to P site and the same rxn occurs to add the whole chain onto the new aa
        
        peptidyl transferase reaction mechanism
        
        first thought histidine in active site of ribosome functions as a base to deprotonate the amino group, causing a nucleophilic acyl sub.
        
        with x-ray crystallography and obtaining ribosome crystal structure, found that nucleobases acted as the base
        
        specifically it was an adenine and was found the ribosome is a ribozyme (catalytic RNA), not an enzyme