BI1014 - enzymology and proteins

enzymes

- Catalysts for reactions under mild conditions

- High specificity for substrates and reactions

- Reversible binding to substrate → enzyme-substrate complex

- Reaction occurs → product released

low substrate concentration

few enzymes bound to substrate

high substrate concentration

saturation, most enzymes have substrate bound

initial velocity

V₀ (initial velocity) vs. substrate concentration = hyperbolic curve

Vmax

Vmax: max velocity at enzyme saturation

Km

Km: substrate concentration at ½ Vmax (approximates affinity)

- Michaelis constant

Kcat

Kcat: catalytic constant (turnover number)

Vmax equation

Vmax = Kcat × [Etot]

Lineweaver - burk plot

(1/v vs. 1/[S]):

x-intercept = -1/Km

y-intercept = 1/Vmax

Michaelis - Menten equation

kinetic parameters

V0

Vmax

Km

Kcat

enzyme inhibition

reversible and irreversible

reversible inhibition

Competitive: inhibitor binds active site

- Vmax unchanged, Km appears increased (KmApp)

- Overcome by increasing substrate

Ki: inhibitor constant

irreversible inhibition

- Covalent bond to enzyme

- Vmax decreases, Km unchanged

clinical relevance

Myasthenia gravis → treat by inhibiting acetylcholinesterase

enzymes

- Facilitate reactions

- Make/break bonds

- Facilitated electron distribution

KmApp

- With a competitive inhibitor, Km stays the same but seems to have changed when measured because more substrate is needed to reach Km/2 (known as KmApp)

determining Ki

ki is the inhibitor constant

functions of proteins

- Defence (keratin, antibodies)

- Structure (muscles, skin)

- Catalysis (enzymes)

- Transport (haemoglobin)

structures of proteins

- Polymers of L-amino acids

- Side chains (R groups) define identity

- Peptide bonds (partial double-bond character, no rotation)

- Written N- to C-terminal

amino acids

- d-amino acids exist but not in proteins encoded by DNA

- Amino acids in proteins all have L-configuration

side chains

- Side chains are made up of the R group

- The R groups contain different groups

- The name of the amino acid is determined by its side chain -> We shorten the name to the first 3 letters to make it easier

peptide bonds

- amino acids are joined by peptide bonds

- A delocalization of electrons across this region gives double bond like characteristics and stops the bond from rotating

- A polypeptide is multiple proteins bonded together

- Always write from the N-terminal end to the C-terminal end (carboxyl group)

secondary structures

- alpha helix

- beta pleated sheets

alpha helix

- Right-handed, 3.6 residues/turn

- H-bonds: N to N+4

- Side chains project outward

Proline breaks helix (no H-bonding, rigid)

beta sheets

- Antiparallel: strong H-bonds, alternating directions

- Parallel: weaker H-bonds, same direction

- R groups alternate up/down

Example: spider silk (Gly-Ala)n → flexibility

folding in tertiary structure

- Driven by hydrophobic effect (entropic gain)

- Side chain interactions (hydrophobic, hydrophilic)

stabilisation in tertiary structure

- Van der Waals, H-bonds, disulphide bonds (oxidized cysteines)

- Salt bridges (Asp-Lys, pH-sensitive)

domains in tertiary structure

- Independently folding regions

- Often linked to specific functions

quaternary structure

- Assembly of multiple polypeptide chains

- Examples: haemoglobin, RNA polymerase, HIV protease

- Critical for function, drug targeting, protein arrangement

sidechain interactions in tertiary structure

- Some are hydrophobic (valine, leucine, phenylalanine)

- some are hydrophilic (aspartate, lysine, serine)

hydrophobic effect

- Oil and water do not mix

- Hydrophobic effect: most important determinant of tertiary structure

- Hydrophobic collapse -> hydrophobic chains interact with other hydrophobic chains, hydrophilic with hydrophilic - entropic effect

entropic effect

- Hydrophobic molecules make water molecules form a "cage"

- Water molecules thus are more ordered than if the hydrophobic molecule was not there

- More order = less entropy

disulphide bonds

- Covalent bonds between two cysteine side chains

- Lose a proton and form a disulphide bond

- Oxidization reaction to form the bond, usually in secreted or Golgi apparatus proteins because of oxidising environments

salt bridge

- Important but pH sensitive

- Aspartate residues can form salt bridges with lysine residues

summary

Primary structure – sequence of amino acids

Secondary structure – helices and sheets

Tertiary structure – 3d structure – folded polypeptide

Quaternary structure – assembly of more than one folded polypeptide

protein purification

- recognition and assay

- criteria of purity

recognition and assay

identify and quantify target protein

criteria of purity

high-resolution separation methods

techniques of protein purification

SDS page, ammonium sulphate precipitation, isoelectric focusing

SDS page

- Denatures proteins with SDS

- Separates by mass

- Visualized with Coomassie/silver stain

ammonium sulphate precipitation

"Salting out" → reduced solubility separates proteins

isoelectric focussing

pH gradient separates proteins at their isoelectric point (pI)

chromatography

size exclusion, ion exchange, affinity chromatography

size exclusion

(SEC):

Separates by size; larger elute first

ion exchange

Separates by charge (cation or anion exchange)

affinity chromatography

High specificity (e.g., enzyme-substrate, antibody-antigen)

determining structure

Combine SEC + SDS-PAGE to deduce quaternary structure (e.g., dimers)