Enzymes / Enzyme Kinetics

Question 1

What is an enzyme

Answer 1

• Biological catalyst
• Increase reaction rates without being used up
• Most are globular proteins

Question 2

Describe the basic principles of enzyme function / biocatalysis

Answer 2

• 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

Question 3

Describe enzyme structure

Answer 3

• 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

Question 4

Why is a negative G required for a reaction to proceed

Answer 4

• Positive: Unfavourable, requires energy to begin

- Negative: Favourable, doesn’t require energy, releases energy

Question 5

What is the Michaelis-Menten equation and relationship to Line-Weaver Burke plot

Answer 5

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)

Question 6

What are key factors that affect enzyme kinetics

Answer 6

• 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)

Question 7

What is the difference between reversible and irreversible enzyme inhibition

Answer 7

• 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

Question 8

List the types of enzyme inhibition

Answer 8

• Competitive
• Un-Competitive
• Non-Competitive
• Mixed

Question 9

What is the difference between exergonic and endergonic reactions and what is energy coupling

Answer 9

• 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

Question 10

What factors affect rate of reaction

Answer 10

• 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

Question 11

What is the lock and key vs induced fit models

Answer 11

• 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

Question 12

What is competitive inhibition

Answer 12

• 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)

Question 13

What is un-competitive inhibition

Answer 13

• 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)

Question 14

What is mixed inhibition

Answer 14

• 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)

Question 15

Provide a summary of inhibition types and changes of line-weaver burke plot

Answer 15

• 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

Question 16

What are the types of catalytic mechanisms

Answer 16

• Acid-Base: Receiving and donation of protons
• Covalent Catalysis: A transient covalent bond between enzyme and substrate, change pathway, requires a nucleophile on the enzyme, one path
• Metal Ion Catalysis: Metal ion bound to enzyme, interacts with substrate to facilitate binding, stabilises -ve charges, participates in oxidation reactions, use redox cofactors, pKa shifters
• Chymotrypsin: Digestive protease, cuts peptides at specific locations on peptide backbone, uses almost all enzymatic mechanisms

Question 17

What is enzyme kinetics

Answer 17

• Kinetics: The study of the rate at which compounds react
• Enzyme Kinetics: Allows a quantitative description of biocatalysis, determines the order of binding of substrates, elucidate acid-base catalysis, understand catalytic mechanism, find effective inhibitors and understand regulation of activity
• Rate of Reaction: Rate of enzymatic reaction is affected by enzyme, substrate, effectors and temperature

Question 18

What are non-covalent and covalent modifications to enzymes

Answer 18

Non-Covalent:
- Allosteric
- Positive, improve catalysis, increase velocity (smaller Km)
- Negative, inhibit, decrease velocity (larger Km)
- Conformational change, change in affinity
Covalent:
- Irreversible (zymogen) or reversible
- Positive or negative
- Phosphorylation, adenylation, acetylation

Question 19

What is non-competitive inhibition

Answer 19

• Type of mixed
• Inhibitor binds to enzyme prohibiting conformational change
• Substrate can still bind but will not be catalysed
• Lower Vmax, same Km, different gradient / y intercept