Biochemistry S1 Y1 Flashcards
What do the reactions in living systems obey?
Thermodynamic laws
- What is thermodynamics?
- What can make a reaction more thermodynamically favourable?
- The direction of a process
- Coupling of reactions
What is kinetics?
The rate of a process
What is a system?
A part of the universe
What are the surroundings?
The rest of the universe outside of a system that interacts with a system
What is a boundary?
The barrier between the system and the surroundings
What is a process?
Any change in a system
What are the two main types of energy transfer?
- Heat (random motion = energy transfer)
- Work (organised motion = energy transfer)
Difference between open and closed system?
Open has matter and energy in and out, closed only has energy in and out
- What is the first law of thermodynamics?
- What does it imply?
- “Energy is never created or destroyed but it can be changed to another form or be transported elsewhere”
- Reaction can occur both forward and backwards and deals with energy balance
What is a state function?
Something that only depends on the current state of a system e.g. energy
What is meant by enthalpy change?
The heat change of a reaction when pressure is constant (it also reflects the number and types of chemical bonds present)
Equation for enthalpy change (involving internal energy)?
ΔH= ΔU+pΔV
Enthalpy change = change in internal energy + (pressure x change in volume)
What is the second law of thermodynamics?
Spontaneous change in a system increases the combined entropy of the system and surroundings
What does Gibbs free energy (G) measure?
Driving force of process
- Equation for free energy change?
- Units?
- What is ΔG?
- ΔG = ΔH – TΔS
- Units:
ΔG and ΔH in J mol^-1 or kJ mol^-1
T in K
ΔS in J mol^-1 K^-1 - Change from free energy of reactants to free energy of products
What will a system far from equilibrium experience to return to stable equilibrium?
An irreversible process
ΔG > 0 is …? (what does this mean)
ENDERGONIC (forward unfavourable, reverse spontaneous)
ΔG = 0 is …? (what does this mean)
EQUILIBRIUM (no change)
ΔG < 0 is …? (what does this mean)
EXERGONIC (forward favourable and spontaneous)
Entropy equation?
S = K x ln x w
Entropy = Boltzmann constant x natural log x number of ways of arranging system
What is the equation for ΔG relating to equations like aA + bB –> cC + dD?
ΔG = ΔG° x R x T x loge x [products]/[reactants]
- What does ΔG° show?
- ΔG°’?
- All reactants at 1M concentration
- pH=7
Equation to find ΔG°?
ΔG° = -RT ln Keq (eq is subscript)
(ΔG = 0 and Keq is found by [products]/[reactants])
If ΔG° is…, Keq is…
1. ΔG° < 0
2. ΔG° = 0
3. ΔG° > 0
- Keq > 1
- Keq = 1
- Keq < 1
6 reasons why ATP is the major energy currency?
- The highly charged phosphate group make as a negative ΔG of ATP hydrolysis
- Electrostatic repulsion is relieved when last phosphate bond is hydrolysed
- Inorganic phosphate stabilises as a resonance hybrid
- Thermodynamically unstable but kinetically stable
- High activation energy for ATP hydrolysis (not hydrolysed before use)
- Cell can regulate energy distribution carried out by ATP by regulating the enzymes that work on it
5 aliphatic amino acids?
Alanine (Ala, A)
Valine (Val, V)
Leucine (Leu, L)
Isoleucine (Ile, I)
Methionine (Met, M)
all are hydrophobic and drive folding
3 aromatic amino acids?
Phenylalanine (Phe, F)
Tyrosine (Tyr, Y)
Tryptophan (Trp, W) (has 2 phenyl rings, others have 1)
2 acidic amino acids?
Aspartic acid (Asp, D)
Glutamic acid (Glu, E)
3 basic amino acids?
Histidine (His, H)
Lysine (Lys, K)
Arginine (Arg, R)
5 polar amino acids?
Serine (Ser, S)
Threonine (Thr, T)
Cysteine (Cys, C)
Asparagine (Asn, N)
Glutamine (Gln, Q)
2 unique amino acids?
Glycine (Gly, G) - flexible, creates many conformations
Proline (Pro, P) - creates kink in chain
PRACTICE USING CHEMDRAW TO DRAW PEPTIDES
Light absorption in amino acids:
- Amino acids that can?
- What can it be used for?
- Equation used?
- Aromatics
- Measuring protein concentration
- Beer-lambert law
Ionisation in amino acids:
- What occurs at low pH?
- As pH increases?
- What is an amino acid like at high pH?
- What is the isoelectric point?
- Amine is protonated (H3N+)
- Carboxylic acid is deprotonated (COO-)
- Amine normal (NH2), carboxylic acid is deprotonated
- The point of no overall electric charge (zwitterion)
Chirality in amino acids:
- What are chiral stereoisomers?
- Why do enzymes bind to one of the D or L stereoisomers but not the other?
- Molecules with the same bonds but a different spatial arrangement of them to form non-superimposable mirror images
- They have a different shape, so the enzyme is only specified to one
General equation for weak acid?
HA + H2O <–> H3O+ + A-
How can you tell the difference between whether a molecule is a L or D stereoisomer?
If you go clockwise around the molecule:
CORN (COOH - R group - NH2) = L
CONR = D
Which end of a polypeptide is classed as the beginning?
The amino end
How does the R - S system of classification of amino acids work?
If priority 1-4 is:
Clockwise = R
Anticlockwise = S
- What does the double bond between N and C in peptides prevent?
- Does a cis or trans configuration minimise steric clashes the most?
- Rotation
- Trans (R groups on opposite faces so are as far away as possible - but X-Pro linkages have steric clashes cis or trans)
What are the two types of rotation about bonds in a polypeptide?
- Phi = bond between N and alpha-carbon
- Psi = bond between alpha-carbon and carbonyl carbon
BUT, only 1/4 of Phi and Psi configurations are possible as steric clashes are very high
- What stabilises alpha helix?
- 2 types of alpha helix?
- What is their dipole moment and what does it allow?
- H-bonds
- Clockwise (most common) and anticlockwise
- Positive amino end and negative carbonyl end. Allows binding of negatively charged molecules
Beta-pleated sheets:
- How are polypeptide chains joined?
- 2 types and how they differ?
- H bonds between extended Beta sheets
- Antiparallel (H bonds connect each amino acid to a single amino acid on other chain) OR parallel (one amino acid bound to 2 amino acids on other chain)
Beta loops:
- What do they allow?
- 2 types and how they differ?
- Reversals in polypeptide chain and 1st and 4th amino acids form H bond
- Type I = AA, proline, AA, AA (proline allows kink)
Type II = AA, AA, glycine, AA (AA = amino acid)
What does amphipathic mean?
Protein folding to create inner hydrophobic, non-polar core to exclude hydrophobic residues from water
What is tertiary structure controlled by?
Interactions of side-chains (disulfide bridges, ionic bridges, H bonds, hydrophobic interactions)
What are disulfide bridges reduced to?
SH groups
How is the thermodynamically favoured structure of active ribonuclease found?
Native disulfide pairings stabilise it by breaking and reforming until the protein folds into the preferred structure due to a decrease in free energy
What are the 5 steps of the life cycle of influenza neuraminidase?
- Flu particle binds to sialic acid on proteins on cell membrane
- Endocytosis
- Viral Replication
- Budding
- Release
Structure of neuraminidase?
Tetrameric (alpha helices and beta ribbons)
How is flu treated?
Competitive inhibitor of neuraminidase prevents it from binding to sialic acid
What is the proteome?
Entire complement of proteins expressed in a cell OR set of proteins expressed under specific conditions
4 post-translational modifications?
- Addition of small chemical groups
- Addition of small proteins
- Addition of complex molecules
- Amino acid processing
8 steps of protein purification?
- Transformation (plasmid into E. coli)
- Selection (of colonies)
- Cell growth and protein production
- Cell lysis
- Column chromotography
- Dialysis
- SDS-PAGE (electrophoresis to check protein purity - only single protein should show)
- Protein assay (checks activity - should not have changed)
3 types of column chromatography?
Gel filtration, ion exchange, affinity
Gel filtration chromatography:
- How are proteins separated?
Separated based on molecular size whereby gel beads trap small molecules so big ones move through and are separated
Ion exchange chromatography:
- How are proteins separated?
Separated based on charge whereby negatively charged gel beads bind to positive proteins so negative proteins move through
Affinity chromatography:
- How are proteins separated?
Separated based on specific binding interactions whereby antibodies recognise a protein and bind so all the others run out and then the column is washed and all the proteins unbind and are obtained
What do enzymes increase?
Rate of chemical transformation
How do proteases degrade proteins?
Cleave peptide bonds by hydrolysis
6 classes of enzymes?
Oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases
- Role of oxidoreductases?
- E.g.?
- Catalyse redox reactions and transfer H and O atoms between substrates
- Alcohol dehydrogenase
- Role of transferases?
- E.g.?
- Transfer functional groups between donors and acceptors (compounds)
- Hexokinase
- Role of hydrolases?
- E.g.?
- Catalyse hydrolysis of substrates (hydrolytic cleavage of C-O, C-N, C-C)
- Carboxypeptidase A
- Role of lyases?
- Catalyse removal of a group by means other than hydrolysis or oxidation (e.g. elimanation) and catalyse addition to double bonds
- Pyruvate decarboxylase
- Role of isomerases?
- E.g.?
- Catalyse inter-molecular rearrangement (geometric and structural changes) and addition to double bonds
- Maleate isomerase
- Role of ligases?
- E.g.?
- Catalyse union of two molecules (e.g. C-S, C-O, C-N bonds) using chemical energy
- Glutamine synthetase
Why does enzyme specificity arise?
Precise interaction between enzyme and substrate and the intricate structure of enzyme
What does ΔG determine?
If a reaction is spontaneous
What will the concentration of reactants and products always reach?
Equilibrium position
How do enzymes use the transition state (S‡) of a substrate to work?
They lower the activation energy of the transition state (which is between the substrate forming the product) by using binding energy to stabilise the intermediate
What are enzymes complimentary to?
The transition state (NOT substrate)
2 factors of why enzymes can generate large rate?
- Non-covalent interactions between enzyme and substrate in ES complex (weak interactions+small energy release = stabilised interaction)
- Rearrangement of covalent bonds (as water is not a strong enough nucleophile to hydrolyse peptide bonds, proteases allow rearrangement of bonds and catalytic triads can transform molecules and split it into its groups)
4 ways binding energy contributes to catalysis?
- Enzyme holds reactants close in right orientation (proximity effect - closer = greater rate, orientation effect - reaction always occurs at right orientation)
- Weak bonds between enzyme and substrate = desolvation of substrate (water molecules blocking active site removed)
- Binding energy in transition states compensates for thermodynamically unfavourable ΔG associated with substrate distortion
- Enzyme undergoes conformational change for induced fit
What do enzymes have different levels of?
Specificity (involving geometric specificity and stereospecificity)
What does increasing [S] increase?
Amount of product until equilibrium
What is V0?
Initial rate of catalysis
What is V0 like at low [S]?
Increases linearly as [S] increases
What is V0 like at high [S]?
It plateaus
What does the michaelis-menten equation link?
Enzyme rate of reaction and [S]
What are the two parts of the steady state assumption?
First = pre-steady state (too short for observation)
Second = steady state ([ES] steady)
- V0 equation?
- Variations?
V0 = Vmax x [S]/[S]+Km
- If [S] «_space;Km then V0=(Vmax x [S])/Km AND enzyme rate is less than Kcat AND [E] = [Et]
- If [S]»_space; Km then V0=Vmax AND rate of catalysis = Vmax
- If [S] = Km then V0=Vmax/2
How does Km vary?
Enzymes have different Km values for substrates - low Km = better enzyme processing of substrate
What does Km depend on?
Substrate, pH, temperature
What does Km = [S] mean?
Half of active sites full
What is K1, K2 and K-1?
K1 = E + S –> ES
K2 = ES –> E + P
K-1 = ES –> E + S
Km equation?
Km = K-1 + K2 / K1
What does K-1»_space; K2 mean?
ES dissociates to E + S quicker than product is formed
What does high or low Km mean?
High = weak binding
Low = strong binding
What is the turnover number of an enzyme?
Number of substrate molecules converted into product by an enzyme molecule in an unit time when enzyme is fully saturated
Equation for Vmax?
Vmax = K2 [Et]
WHICH IS Vmax = Kcat [Et]
([Et] is total enzyme conc.)
- What is Kcat?
- Equation?
- Turnover
- Kcat = Vmax / [Et]
What is Kcat / Km a measure of?
Catalytic efficiency