S4) Enzymes Flashcards
What are enzymes and what do they do?
Enzymes are protein catalysts that mediate metabolic pathways by increasing the velocity of chemical reactions while not being consumed during the reaction
Identicy 5 characteristics of enzymes
- Highly specific
- Unchanged after reaction
- Increase rate of reaction
- Proteins
- Does not affect reaction equilibrium
What is an active site?
An active site is the special cleft on an enzyme containing amino acid side chains that bind substrates and trigger catalysis
Identify 4 characteristics of active sites
- Cccupies a small part of the enzyme
- Formed by amino acids
- Clefts/crevices
- Bind to substrates through weak bonds
Identify 2 different hypotheses which govern substrates binding to an enzyme
- Lock and key hypothesis – active sites have a complementary shape to the substrate
- Induced fit hypothesis – binding of substrates can induce chages in the comformation of the active site
Describe the 5 steps involved in enzyme catalysis
- Substrate binds to active site on enzyme
- Conformational change occurs and ES complex forms
- Catalysis occurs
- ES complex is converted to EP complex
- EP compex dissociates to enzyme and product
What is an allosteric site?
An allosteric site is a cleft on an enzyme where an activating/inhibiting molecule may bind, changing the shape of the enzyme and influencing its ability to be active
Outline the specificity of enzymes
- Enzymes are highly specific, interacting with 1/few substrates and catalysing only one type of chemical reaction
- The set of enzymes made in a cell determines which metabolic pathways occur in that cell
Can enzymes be regulated?
Why is this needed?
Enzyme activity can be regulated (increased or decreased) so that the rate of product formation responds to cellular need
Why are many enzymes localised in specific organelles within the cell?
Compartmentalisation serves to:
- Isolate the reaction substrate/product from other competing reactions
- Provides a favourable environment for the reaction
- Organises thousands of cell enzymes into purposeful pathways
What is activation energy?
Activation energy is the minimum energy required by a chemical system to start a chemical reaction
How do enzymes impact activation energy in a chemical reaction?
- Activation energy is usually very high, leading to slow rates in uncatalyzed chemical reactions
- Enzymes work to lower activation energy, so more particles are able to overcome the transition state and react
- Rate of reaction increases
What is the rate of reaction?
The rate of a reaction is the number of substrate molecules converted to product per unit time ( [product]/time )
How does the rate of reaction vary in the presence of an enzyme?
The rate of an enzyme-catalysed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached
Why does the rate of reaction level off in enzyme-catalysed reactions?
The levelling off of the reaction rate at high substrate concentrations reflects the saturation of all available enzyme active sites with substrate
Identify and describe 2 factors which increases the rate of reaction
- Temperature: increases no. of molecules with activation energy
- Concentration: increases chance of molecular collisions
In Michaelis-Menten kinetics, what type of curve is shown when measuring the rate of reaction for an enzyme-catalysed reaction?
Rate of reaction is hyperbolic
For allosteric enzymes, what type of curve is shown when measuring the rate of reaction for an enzyme-catalysed reaction?
Rate of reaction is sigmoidal e.g. oxygen dissociation curve of haemoglobin
Discuss the effect of temperature on reaction velocity
- Initially, an reaction velocity increases with temperature due more molecules surpassing the energy barrier of the reaction
- Reaction velocity then decreases due to the temperature-induced denaturation of the enzyme
What is the optimum temperature for most human enzymes?
Between 35 and 40°C
Discuss the effect of pH on enzyme function
Extremes of pH lead to enzyme denaturation due to a conformational change the structure of enzyme (active site)
What is the optimum pH for human enzymes?
The optimum pH varies for different enzymes and often reflects the [H+] at which the enzyme functions in the body
State the Michaelis-Menten equation
- V = initial reaction velocity
- Vmax = maximal velocity
- Km = Michaelis constant
- [S] = substrate concentration
What is Km in Michaelis-Menten kinetics?
- Km is numerically equal to the substrate concentration at 1⁄2 Vmax
- Km does not vary with the concentration of enzyme
What is the significance of Km?
- Km is characteristic of an enzyme and its particular substrate
- It reflects the affinity of the enzyme for that substrate
Compare and contrast the enzyme affinity in terms of a small Km and large Km
- Small Km: high affinity of the enzyme for a substrate as a low [substrate] is needed to half-saturate the enzyme
- Large Km: low affinity of enzyme for substrate as a high [substrate] is needed to half-saturate the enzyme
Describe the relationship of velocity to enzyme concentration in Michaelis-Menten kinetics
The rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations
E.g. if the [enzyme] is halved, the rate of reaction is halved
What is a Lineweaver-Burk plot?
A Lineweaver-Burk plot plots 1/V0 versus 1/[S] to obtain a straight line graph for Michaelis-Menten kinetics
- It is used to calculate Km and Vmax, as well as to determine the mechanism of action of enzyme inhibitors
What are the values of the x and y intercepts in a Lineweaver Burk plot?
- X-intercept is equal to −1/Km
- Y-intercept is equal to 1/Vmax
What is an inhibitor?
An inhibitor is any substance that can diminish the velocity of an enzyme-catalysed reaction
Distinguish between the bonding of irreversible and reversible inhibitors
- Irreversible inhibitors bind to enzymes through covalent bonds
- Reversible inhibitors bind to enzymes through non-covalent bonds
Identify 2 forms of reversible inhibition
- Competitive inhibition
- Non-competitive inhibition
Outline competitive inhibition
- Inhibitor binds reversibly to the same site the substrate would occupy
- Inhibitor competes with the substrate for the active site
- Increasing [substrate] reduces the effect of inhibitor
Describe the effect of competitive inhibition on the Vmax and Km
- Effect on Vmax: effect is reversed by increasing [S] & rate of reaction reaches Vmax
- Effect on Km: increases the Km for a given substrate as more substrate is needed to achieve 1⁄2 Vmax
Describe the Lineweaver-Burk plot for competitive inhibition
- Inhibited and uninhibited reactions intersect on the y-axis at 1/Vmax (Vmax is unchanged)
- The inhibited and uninhibited reactions show different x-axis intercepts (Km is increased)
Outline non-competitive inhibition
- Inhibitor binds reversibly to allosteric site on the enzyme
- Inhibitor does not compete with the substrate for the active site
- Increasing [substrate] has no effect on inhibition as active site has changed shape
Describe the effect of competitive inhibition on the Vmax and Km
- Effect on Vmax: decrease the Vmax of the reaction as inhibition is not overcome with increasing [substrate]
- Effect on Km: same value of Km as inhibitors do not interfere with the binding of substrate to enzyme
Describe the Lineweaver-Burk plot for non-competitive inhibition
- Inhibited and uninhibited reactions intersect on the x-axis at 1/Km (Km is unchanged)
- The inhibited and uninhibited reactions show different y-axis intercepts (Vmax is decreased)
What are amyloid fibres?
An amyloid fibre is the misfolded, insoluble form of a normally soluble protein
Describe the structure of amyloid fibres
- Highly ordered with a high degree of ß-sheet
- Core ß-sheet forms before the rest of the protein
- Stabilised by hydrophobic interactions between aromatic amino acids
