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Flashcards in Enzymes / Enzyme Kinetics Deck (19)
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1

What is an enzyme

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

2

Describe the basic principles of enzyme function / biocatalysis

- 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

Describe enzyme structure

- 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

Why is a negative G required for a reaction to proceed

- Positive: Unfavourable, requires energy to begin
- Negative: Favourable, doesn't require energy, releases energy

5

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

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

What are key factors that affect enzyme kinetics

- 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

What is the difference between reversible and irreversible enzyme inhibition

- 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

List the types of enzyme inhibition

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

9

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

- 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

What factors affect rate of reaction

- 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

What is the lock and key vs induced fit models

- 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

What is competitive inhibition

- 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

What is un-competitive inhibition

- 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

What is mixed inhibition

- 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

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

- 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

16

What are the types of catalytic mechanisms

- 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

17

What is enzyme kinetics

- 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

18

What are non-covalent and covalent modifications to enzymes

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

19

What is non-competitive inhibition

- 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