Enzymes / Enzyme Kinetics Flashcards
1
Q
What is an enzyme
A
- Biological catalyst
- Increase reaction rates without being used up
- Most are globular proteins
2
Q
Describe the basic principles of enzyme function / biocatalysis
A
- Reaction specificity / selectivity (avoid side products)
- Milder reaction conditions conducive to cell conditions
- High reaction rate
- Regulation of biological pathways
- Allows entropically unfavourable reactions to complete
- Do not affect equilibrium, increase rate of reaction
- Lower Ea, increases spontaneity, organise reactive groups into close proximity and correct orientation
- Speed up reactions that are reversible by the same degree in both directions
3
Q
Describe enzyme structure
A
- Active Site: Where substrate binds, induced fit model, high specificity, where inhibitors can bind
- ES Complex: Active site of the enzyme is non-covalently bound to the substrate molecule
- Transition State: Highest energy that must be overcome in reaction pathway, enzymes lower this
4
Q
Why is a negative G required for a reaction to proceed
A
- Positive: Unfavourable, requires energy to begin
- Negative: Favourable, doesn’t require energy, releases energy
5
Q
What is the Michaelis-Menten equation and relationship to Line-Weaver Burke plot
A
Michaelis Menten (Km): - Enzyme reversibly forms an intermediate enzyme-substrate complex - [S] (substrate concentration) - V0 (initial velocity) - Vmax (maximum velocity) - Km = ½ Vmax, efficiency of enzyme - Km = [S] when V0 = 1/2 Vmax - Cannot determine Vmax and Km values Lineweaver Burk Plot: - Linearised double reciprocal plot - More accurate, real values, determine Vmax and Km - 1/V0 (y-axis) and 1/[S] (x-axis)
6
Q
What are key factors that affect enzyme kinetics
A
- Conc: Substrate / enzyme, maximum rate at which enzyme can catalyse, increase till it plateaus
- Protein Structure: Denaturation and renaturation
- Temp: Increasing temp increases rate, too high = denature protein / unfolding
- pH: Optimal pH, different for different enzymes
- Co-Factors: Additional components, enzymes can be limited by nature of AA side chains or can increase function (coenzymes)
7
Q
What is the difference between reversible and irreversible enzyme inhibition
A
- Irreversible: React with the enzyme, one inhibitor molecule can permanently shut off one enzyme molecule, often powerful toxins but also may be used as drugs
- Reversible: Bind to and can dissociate from the enzyme, they are often structural analogs of substrates or products, they are often used as drugs to slow down a specific enzyme
- Can bind to the free enzyme and prevent the binding of the substrate or the enzyme-substrate complex and prevent the reaction
8
Q
List the types of enzyme inhibition
A
- Competitive
- Un-Competitive
- Non-Competitive
- Mixed
9
Q
What is the difference between exergonic and endergonic reactions and what is energy coupling
A
- Endergonic: Synthesis of complex molecules, require energy, thermodynamically unfavourable / high Ea
- Exergonic: Breakdown of metabolites, releases energy, favourable, cellular concentration is higher than equilibrium concentration
- Energy Coupling: Chemical coupling of exergonic and endergonic reactions allows unfavourable reactions, high-energy molecule (ATP) reacts directly with the metabolite that needs activation
10
Q
What factors affect rate of reaction
A
- Temperature: Higher temperatures, decreases stability of macromolecules
- Concentration: Increasing conc of reactants, costly
- Altering Reaction: Coupling with a fast reaction, universally used by living organisms
- Catalysis: Lowering activation barrier, universally used by living organisms, offers acceleration under mild conditions, high specificity and possibility for regulation
11
Q
What is the lock and key vs induced fit models
A
- Lock and Key: Assumes that complementary surfaces are preformed
- Induced Fit Model: Conformational changes may occur upon ligand binding, allows for tighter binding of ligand and high affinity for different ligands, both protein and ligand undergo changes
12
Q
What is competitive inhibition
A
- Impair reaction progress by binding to enzyme at AS
- Prevent substrate from binding, doesn’t affect catalytic function
- Increase Km, Vmax unchanged
- LBP: Same intercept gradient changes
- Sildenafil (viagra)
13
Q
What is un-competitive inhibition
A
- Binds to ESC, doesn’t affect substrate binding
- Inhibits catalytic function
- Decrease in Vmax and Km
- MM: Doesn’t reach Vmax. plateaus early
- LBP: No change in relationship (parallel)
- Fomepizole (treat methanol poisoning)
14
Q
What is mixed inhibition
A
- Binds enzyme with or without substrate at regulatory / allosteric site
- Inhibits substrate binding and catalysis
- Increase Vmax, Km decreases as a result of Vmax, lines intersect left of y axis, different y and x intercept
- Penicillin (binds to bacterial enzyme when bound to peptidoglycan)
15
Q
Provide a summary of inhibition types and changes of line-weaver burke plot
A
- Competitive: Binds to active site, inhibit binding, increase Km, Vmax unchanged
- Un-Competitive: Bind to ESC, inhibit catalytic, decrease Vmax and Km
- Non-Competitive: Prevent conformational change, no catalysis, Vmax reduced, same Km
