Amino Acids, Peptides & Proteins Flashcards
(25 cards)
Structure of L amino acids
- side chain
- Amine group —> Nucleophilic, pKa of 9-10 and gets protonated at pH 7
- Carboxylic acid group —> can replace OH, pKa of 2 and gets deprotonated at pH 7
What are the proteinogenic L-alpha-amino acids
Aromatic
- Phenylalanine (Phe)
- Tryptophan (Trp)
- Tyrosine (Tyr)
- Histidine (His)
Basic
- Histidine (His)
- Lysine (Lys)
- Arginine (Arg)
Acidic
- Aspartic acid (Asp)
- Glutamic acid (Glu)
Shape and features of amide bonds in primary structure
- Amide bond is planar
- Parial double bond character
- Restricted rotation about C-N bond
- Resonance is possible around the N
Features of the restricted rotation of amide group
- Geometric isomers exist
- Trans is preferred
- In proline, Cis and Trans isomers are closer in energy
- In amides, the bond restriction is good for hydrogen bonding
Secondary structure
Has alpha helixes and Beta-sheets
These can be antiparallel or parallel (the beta sheets)
Properties of acids and bases
At more acidic pH, the NH2 is protonated to become NH3+ with a pKa of 9.7
At a more basic pH, the carboxyl group gains a negative charge
Zwitterion is formed at the isoelectric point (pH when there no net charge) = pH 6.02
Effects of side chains
Can have basic or acidic properties/physical and chemical properties that dominate protein interaction and enzyme catalysis
Eg. Causes Pi stacking
Electrostatic effects of side chains
Forms charged/partially charged side chain groups with polar substrate groups
Hydrophobic effects of side chain
Causes exclusion of water driven by entropy
Hydrogen bonding as an effect of side chains
Hydrogen bonding between charged residues forms salt bridges
Can be between any backbone/side chain groups
Dilsulfide bridges as an effect of side chains
Forms dislufide bridges via covalent bonding and redox reactions
These can help stabilise proteins (normally extracellular)
Hydrolytic stability of amide groups
Under basic conditions
- first step is addition
- second step is elimination
- overall it is a Nucleophilic substitution at C=O
- the C-N bond is broken
- tetrahedral sp3 intermediate collapses to sp2 carboxylate
- stronger nucleophile is more reactive = PUSH
Under acidic conditions
- resonance structures exist of the initial molecule
- addition and elimination steps = substitution
- tetrahedral sp3 intermediate collapses to sp2 carboxylate
- protonation makes the carbonyl more reactive = PULL
Reactivity of carbonyl groups
In order from most to least reactive]
1. Acid chloride
2. Acid anhydride
3. Ester
4. Amide
Rate of initial attack and breakdown depends on XR
A more electrophilic carbonyl (C=O) or a better leaving group ability of XR leads to faster hydrolysis
What are proteases?
- Enzymes that catalyse amide hydrolysis
- Accelerate rate of hydrolysis at ambient temperatures by many orders of magnitude
- Use a combination of effects
- electrostatic and metal ion catalysis
- general acid and base catalysis
- Nucleophilic/covalent catalysis in some cases
- general hydrophobic and proximity effects
Common proteases
- Trypsin —> cleaves C-terminal side of Lys and Arg
- Chymotrypsin —> cleaves C-terminal side of Phe, Tyr and Trp
- Elastase —> cleaves C-terminal side of Ala and Gly
- Carboxypeptidase —> cleaves C-terminal amino acid
Role of proteases
Make the nucleophile (water) more reactive (= PUSH) and/or makes the carbonyl of the amide more reactive, stabilising the tetrahedral intermediate (= PULL)
What are the 4 main mechanistic classes of proteases?
- Metalloprotease
- Aspartyl protease
- Serine protease
- Cysteine proteases
Describe metalloproteases and aspartyl proteases
Both activate water nucleophile using carboxylate general and activate amide carbonyl carbon for Nucleophilic attack
Metalloproteases have a metal ion in the active site —> ‘pull’ from Zn2+ and ‘push’ from Glu. This activates the carbonyl and stabilises the intermediate oxyanion
In Aspartyl proteases, base and acidic catalysis occurs at the same time and the life cycle of HIV viruses depends on these proteases —> activates carbonyl by general acid protonation of oxyanion
Describe serine and cysteine proteases
Serine proteases are the most common class of proteases
Cysteine proteases hydrolyse proteins
In both:
- enzyme group reacts with amide carbonyl first = covalent catalysis occurs
- forms an enzyme-substrate covalent intermediate
- water is the second nucleophile
- Ser/Cys are the active sites
- activates nucleophile in both stages by making OH or SH more Nucleophilic
- activates carbonyl by stabilising intermediate oxyanion
Chymotrypsin structure and features
Is a serine protease which only cleaves at C-terminal side of aromatic amino acids (Phe, Tyr, Trp)
Majority is made up of beta sheets and it has a boronic acid inhibitor
Asp and Ser are the active sites
Serine protease mechanism - Chymotrypsin
- Enzyme-substrate complex forms —> serine nucleophile (deprotonated by Asp) attacks the carbonyl and the His (general base) deprotonates serine. Enzyme has R group (can be Phe, Tyr or Trp) and has a hydrophobic binding pocket in which aromatic side chains fit
- Tetrahedral intermediate —> stabilised by ‘oxyanion hole’ and His (general acid) assists in C-N cleavage
- Acyl-enzyme intermediate —> amine is lost and water replaces amine. Histidine acts as general base and water as the nucleophile
- New tetrahedral intermediate —> elimination step
- Enzyme product complex forms —> final product
Synthesis of peptides in nature
- DNA is transcribed to give mRNA which is then translated by the ribosome to give polypeptides (proteins)
- Peptide bond formation is catalysed by the ribosome and follows the same general nucleophilic substitution mechanism
- Where there is an addition of NH2 onto the C=O resulting in a tetrahedral intermediate followed by elimination of the tRNA
Chemical synthesis of peptides
- Uses chemical reagents to couple amine and acid groups together
- Dipeptide synthesis —> any amine and any acid group can react = 4 possible products for each pair of amino acids
- Temporary protection of the amine and carboxyl during synthesis by the synthesis solution
- SINGLE amine bond only can be formed
- Deprotects at the end of synthesis
What is automation in chemical synthesis?
If C-terminus is first linked to a solid support, this chemistry can be carried out iteratively using an automated peptide synthesiser
