Enzyme Kinetics And Inhibition Flashcards
What is enzyme rate?
No. Of moles of product produced per unit time
What is first-order?
Dependent on the concentration
What is zero order?
No relationship
What is second-order?
Relationship between the two concentrations
Why to study enzymes must first order kinetics be present?
This is because as velocity increases the concentration increases to the point where the enzyme is saturated with the substrate (Vmax)
Why at Vmax is the rate of reaction unaffected by the increase of concentration?
All active sites are used up
Why does the rate of reaction increases the enzyme concentration increases?
it is directly proportional as there’s a great availability to catalyse the reaction
What is the max enzyme velocity?
Vmax
What is the Michaelis-Menten equ?
Vo= Vmax [s]/ Km + [s]
What are the assumptions for Michaelis-Menten kinetics?
Eqm-association + dissociation of substrate + enzyme is rapid
ES immediately comes stead state + constant
At early time point velocity = 0
What does Km represent?
Dissociation constant of ES
What does it mean if KM low value?
ES complex held tightly together
What is the physiological relevance of KM?
Utilisation of glucose in liver
At Vmax with high [S], what is rate determined by?
[E]
What is Kcat?
Catalytic constant
= max no. of S converted to P per second by each active site
What does Kcat measure?
How fast a given enzyme can catalyse a specific reaction
What are the units for Kcat?
S-1
What does it mean if there is a larger Kcat?
More rapid catalytic event at enzyme’s active site
What is the Lineweaver Burke plot?
1 Km 1 1
—- = —– —- + ——-
V0 Vmax [S] Vmax
What is the Woolf-Hanes plot?
[S] 1 Km
—- = + ——- [S] + ——–
V0 Vmax Vmax
What does Michaelis-Menten rely on?
Law of mass action
= assumes free diffusion + thermodynamically-driven random collision
What is are the real-world limitations of heterogenous enzyme reactions?
Molecular mobility of E or S can be restricted = immobilisation or phase-separation of reactants
What is are the real-world limitations of homogenous enzyme reactions?
Mobility of E or S may be limited
What are multisubstrate reactions?
2 or 3 substrates
In multisubstrate reactions what is the order of biochemical steps determined by?
Mechanism of reaction
Describe random substrate binding
Multisubstrate reactions
Independent binding of substrates + products
2 independent binding sites; substrate binding independent of other substrate
Describe ordered substrate binding
Multisubstrate reactions
1 substrate must bind before 2nd substrate can bind effectively
Describe ping-pong mechanism
Multisubstrate reactions
Enzyme binds to substrate A then releases product
Intermediate form carriers A fragment then binds B
Product Q released + E returns to original state
How can type of reaction be evaluated?
[S1] = varied [S2] = constant
What does intersecting lines at increasing [S2] indicate?
Ternary complex
= both donor + acceptor in active site
What does parallel lines at increasing [S2] indicate?
Ping-pong mechanism
= both donor + acceptor in active site
Describe to competitive inhibition
Bind to enzyme’s active site
Reversible + similar structure to S
Compete with S for active site
How can competitive inhibition be reversed?
Increase [S]
Describe non-competitive inhibition
Bind to allosteric set
Structure different to S
Distorts shape of E + active site, prevents S binding
Can non-competitive inhibition be reversed by increasing [S]?
NO
Describe uncompetitive inhibition
Bind only to ES complex
Reduces effectiveness of ES
Takes long for S or P to leave
What happens to Km and Vmax in uncompetitive inhibition?
BOTH decrease
When does uncompetitive inhibition work best?
[S] high
Describe irreversible inhibition
Bind covalently
Inhibit permanently
Contain reactive electrophilic groups
Bind to nucleophile residues
Describe penicillin in irreversible reaction
Transition-state analogues bind more tightly to enzyme than S or P
Inhibits glucopeptidyl transferase
Kills growing cells by inactivating enzymes
What are the uncompetitive exceptions?
Should only occur if active site occupied by S
BUT can have affinity for unoccupied enzymes
What are the non-competitive exceptions?
Inhibition affinity should be unchanged regardless whether S bound or not
BUT affinity for inhibitor usually changes when substrate bound
