Enzymes 2 Flashcards
What is kinetics ?
The study of rate of change from reactants to products
What is velocity?
Velocity(v) is the chance in conc. Of substrate or product per unit time
What is Rate(k)?
Rate(k) is the change in total quantity (of reactant or product) per unit time
What is the initial velocity(Vo)?
Initial velocity (Vo) is the change in reactant or product conc. during the linear phase of a reaction
What are the 3 basic assumptions of Michaelis Menten Kinetics?
- At anytime, there are more substrates than enzymes- the percentage of the total substrate bound by enzyme at any one time is small
- Steady-state assumption: the rate of formation of ES is equal to that of breakdown of ES(to E+S and to ECP).
concentration of ES doesn’t change with time - If all enzymes in the ES /active sites of enzymes are bound, then rate of the product formation is maximal:
Vmax = k2[ES]
Give the equation of the steady state equation
K1: E+S -> ES
K-1: ES -> E+S
K2: E+P -> ES
What is the Michaelis Menten equation?
A quantitative description of the relationship between the rate of an enzyme catalyzed reaction v1, substrate concentration [S], the M-M rate constant (Km) and maximal velocity (Vmax)
Give the Michealis Menten equation
v1= Vmax[S]/ (Km+[S])
What is the Michealis constant/ Km?
Km is equal to the concentration of substrate required to attain half maximal velocity for any given reaction
What is Vmax/ max velocity?
The max velocity- all the active sites on all enzymes are being used
How to find Km on a velocity substrate concentration graph?
Km = [S] where velocity is 1/2 Vmax
What is a first order reaction?
When substrate concentration is much less than Km, the velocity of the reaction is approximately proportional to the substrate concentration
What is a zero order reaction?
When the substrate concentration is much greater than Km, the velocity is constant and equal to Vmax. The rate of reaction becomes independent of substrate concentration. This demonstrates enzyme saturation
What is the reaction order at low concentrations and why?
At low concentration [S]«_space;Km the velocity of the reaction is first order-proportional to substrate concentration
At high concentrations[S] what order is the reaction?
At high concentrations of substrate [S] »_space;Km, the velocity is constant and independent of substrate concentration
How does reaction order change as reaction proceeds?
Reactions starts at first order, dependent on amount of substrate bound to enzymes, but as all enzymes become bound, the reaction become zero order and constant velocity is independent to [S] as all enzymes active sites are occupied
Define the correlation of Km and enzyme affinity
Large Km- reflects low affinity of enzyme for the substrate
Small Km- reflects a high affinity of enzyme for the substrate
Km and Vmax are__________
Independent
Enzyme A starts the reaction with less substrate, what does this indicate?
Enzyme A has a higher affinity for the substrate than enzyme B/ A has a smaller Km
How can Km be changed?
It is characteristic of an enzyme and cannot be changed
How can Vmax be changed?
Vmax increases with higher enzyme concentration but Km stays the same
Describe the lineweaver -Burk analysis
- Lineweaver and Burk manipulated the MM equation by taking its reciprocal values generating a double reciprocal plot
- leads to a linear graph of the reciprocals of velocity and substrate concentration
What is the lineweaver Burk equation?
1/Vo= Km/vmax x 1[S]+ 1/Vmax
What are inhibitors?
Substances that reduce the rate of enzymatic reactions
Decreases velocity by binding to the enzyme
How do enzyme inhibitors work?
They are usually specific, work at low concentrations
They block the enzyme, but usually do not destroy it
What are real life examples of enzyme inhibitors?
Many drugs and poisons inhibitors of enzymes in the nervous system
Elg. Sedatives, metal complexes, substrate analogues
What is irreversible inhibition?
Irreversible inhibitor drugs or poisons bind covalently to the enzyme
This leads to an abnormal protein and degraded enzymes and permanently inactivating the enzymes
Give examples of irreversible inhibitors
Heavy metals (Hg2+, Pb2+, Cd2+)
What is reversible inhibitors?
Drugs bind noncovalently to the enzyme and so, can dissociate
What are the classes of reversible inhibitors?
Competitive inhibitors(common)
Non competitive inhibitors
What are competitive inhibitors?
Inhibitors that compete for the same active site as substrate and resemble normal substrate, e.g. protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV
What are non competitive inhibitors?
A substance that interacts with the enzyme, but usually not at the active site
How can competitive inhibitors be dislodged?
By increasing the amount of substrate
Give examples of competitive inhibitors
- methanol and ethylene glycol compete with ethanol for the binding sites on alcohol dehydrogenase
- methotrexate competes with folic acid for dihydrofolate reductase
(Hence why cancer patients need more folic acid for red blood cell production)
How does competitive inhibition affect Vmax?
Vmax remains the same in competitive inhibition
NB: in LB graph, thus means the pass through y axis at same point, MM graph, same Vmax
How does competitive inhibition affect Km?
Km is increased in the presence of a competitive inhibitor (on both graphs Km and 1/Km is pushed to the right on the X axis)
What is a medical application of competitive inhibitors?
Statins- e.x. Lovastatin(or other statins) in treating hypertension or heart disease
What is the mechanism of action of statin
Medication increases hyperlipedaemia
Same configuration as HMG-CoA(substrate) and lovastatin binds to HMG CoA reductase, preventing HMG CoA from binding
Where do non competitive inhibitors bind?
Binds to somewhere else from active site and changes how performs
How do non competitive enzymes affect Km?
Michealis constant does not change in the presence of inhibitor
On LB graph, both lines start at the same point (1/Km) and crosses y axis and different points
How is Vmax affected by non competitive inhibition?
Maximal velocity is decreased at non competitive inhibition
Non competitive inhibitor reaction will have a higher 1/Max as a result(cross the y axis at different points)
What is uncompetitive inhibition?
Inhibitor binds to ES complex, usually reversible
How does uncompetitive inhibition graph look?
1/v and 1/Km are all different, set of parallel lines
Give examples of irreversible inhibitors?
Permanently inactive enzymes
Heavy metals
Aspirin acetylates- used to reduce risk of heart attack
Fluorouracil- used for cancer patients
Orangophosphates- farmers use this for wheat control(can poison us)
What is product inhibition?
Product of a reaction, will decrease the rate of reaction(is reversible)
This is used to regulate pathways, such as metabolism
What is the kinetic effect of uncompetitive inhibitors?
Appearing Vmax decreased and Km is decreased
Differentiate non competitive inhibitor and uncompetitive inhibitor via their binding sites
Noncompetitive-binds to E or ES complex other than catalytic site
Enzyme cannot form products
Uncompetitive - binds only to ES complexes at locations other than the catalytic site
Modified enzyme structure, preventing binding site from being available
Which inhibitors can be reversed by substrate?
Only competitive inhibition
