enzymes + enzyme kinetics Flashcards
What is S⟶P?
from substrate to product
What is G‡?
free energy to overcome transition state
What is ∆G‡ₛ⟶ₚ?
energy to get from substrate to product
What is ∆G‡ₚ⟶ₛ?
energy to get from product to substrate
What is ∆G’ᵒ?
standard free energy
enzyme
a catalyst that lowers activation energy but is not used up in reaction
cofactor
an inorganic ion/coenzyme required for enzyme activity
What are the classes of enzyme classification?
1) oxidoreductases
2) transferases
3) hydrolases
4) lyases
5) isomerases
6) ligases
oxidoreductases
oxidation/reduction (transfer of electrons)
transferases
transfer of functional groups
hydrolases
breaking of single bonds through addition of water (hydrolytic cleavage)
Give an example of hydrolytic cleavage.
transfer of functional groups to water
lyases
breaking of bond(s) by means other than hydrolysis + oxidation, often forming a new double bond/ring structure
isomerases
rearrangement of groups (producing isoforms)
ligases
formation of new bond
zymogen
inactive form of enzyme
holoenzyme
apoenzyme + cofactor
lock-and-key
active site of unbound enzyme is complimentary to shape of substrate
induced fit
active site complimentary to shape of substrate only after substrate binds (enzyme changes shape when substrate binds)
collagen
major structural components of connective tissues
specificity
always cut in the same place
SDS-PAGE
separates proteins based on their mass (lighter proteins travel further than heavier proteins)
How can collagenolysis be assessed?
SDS-PAGE
What are the factors affecting enzyme activity?
1) pH
2) temperature
3) [substrate]
4) presence of inhibitors
How does pH affect enzyme activity?
pH outside optimum range will change conformation + may denature enzymes
How does temperature affect enzyme activity?
1) affects kinetic energy of substrate + enzyme
2) determines frequency of substrate + enzyme interaction
3) increases rate of reaction until enzyme is saturated
How does [substrate] affect enzyme activity?
1) determines frequency of substrate + enzyme interaction
2) increases rate of reaction until enzyme is saturated
coenzyme
organic (non-metal) cofactor required for action of certain enzymes
V₀
initial velocity
Vₘₐₓ
maximum velocity of enzymatic reaction when binding site is saturated with substrate
Kₘ
1) substrate concentration at which enzyme-catalysed reaction proceeds at 1/2 maximum velocity
2) inverse measure of substrate’s affinity for enzyme
rate, v =
1) ∆[P]/∆t (product formation
2) -∆[S]/∆t (substrate depletion)
ES
enzyme-substrate complex
EP
enzyme-product complex
What is the following equation simplified?
E + S ⇌ ES ⇌ EP ⇌ E + P
E + S ⇌ ES ➝ E + P
Why is the initial velocity the maximum rate?
enzyme + substrate concentrations are at highest
What is the relationship between {S] and rate?
as [S] increases, enzyme becomes saturated with substrate + rate reaches Vₘₐₓ
Michaelis-Menten equation, V₀ =
Vₘₐₓ[S]/Kₘ + {S]
reversible/irreversible inhibitors
depends on strength of bond between inhibitor + substrate
What are the types of inhibitors?
1) reversible
2) irreversible
3) competitive
4) uncompetitive
5) non-competitive
What happens in the presence of a competitive inhibitor?
1) Vₘₐₓ unchanged (sufficient [S] out-competes inhibitor)
2) Kₘ increased (reduced binding affinity as ES can dissociate + EI form, due to competition)
What happens in the presence of an un-competitive inhibitor?
1) Vₘₐₓ reduced (some unproductive ESI complex will always be present)
2) Kₘ reduced (formation of ESI to maintain equilibrium, more S binds to E + lower [S] required)
What happens in the presence of a non-competitive inhibitor?
1) Vₘₐₓ reduced (like un-competitive inhibitor)
2) Kₘ unchanged (no change in position of equilibrium)
What does less signal mean on a spectrophotometer?
more product
What does more signal mean on a spectrophotometer?
less product
Why are spectrophotometers used?
to measure product formation
What does a small Kₘ indicate?
high affinity (rate will approach Vₘₐₓ more quickly)
