Post on 04-Jan-2016
Where Effectors BindWhere Effectors Bind
Effector
where does it bind?
At theActive Site
At another site
substrateproduct
competitiveinhibitor
irreversible inhibitor
"designed site"
"incidental site"
allostericinhibitor
allostericactivator
irreversibleinhibitor
noncompetitiveinhibitor
irreversibleinhibitor
Uncompetitive inhibitor
Inhibitors - Simple EnzymesInhibitors - Simple Enzymes
Competitive Inhibitor - Has a shape similar to that of the substrate and binds to the active site of the enzyme and prevents the substrate from binding.
Noncompetitive Inhibitor - Binds at a another site on the enzyme other than the active site not dependent on substrate binding not the same as allosteric!
Uncompetitive Inhibitor - Binds at a another site on the enzyme other than the active site dependent on substrate binding not the same as allosteric
Irreversible Inhibition - Occurs when an inhibitor forms a covalent bond to the enzyme and inhibits its activity.
Competitive Inhibition ICompetitive Inhibition I
•Inhibitor and substrate have portion of shape in common•Compete for active site•Large amounts of inhibitor swamp out substrate binding and product formation is suppressed•Likewise large amounts of substrate swamp out inhibitor binding and product formation is minimized
Competitive Inhibition IICompetitive Inhibition II
•As the amount of inhibitor increases, there is more competition at the active site
• most pronounced at low levels of S
•At higher [S], inhibition is “swamped out”•Vmax does not change
• processing rate not effected•“Km” increases!!!
• the enzyme does not bind the substrate as well in the presence of inhibitor
Competitive InhibitionVelocity vs [Substrate]
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
0.00 0.50 1.00 1.50 2.00
substrate concentration [S]
Init
ial v
elo
city
[I] = 0 [I] = x [I] = 2x
Competitive InhibitionLineweaver-Burk Inverse Plot
y = 0.2457x + 1.0103
y = 0.3983x + 0.9819
y = 0.6194x + 0.9995
0
1
2
3
4
5
6
7
8
-5 0 5 10 15
1/substrate concentration [S]-1
1/in
itial
vel
ocity
Competitive Inhibition IIICompetitive Inhibition III
[I] = x
[I] =2x
A Lineweaver-Burk plotallows the importantparameters, Km and Vmax, to be determined directlyfrom an equation ratherthan from an extrapolation
1/Vmax = y intercept
Km/Vmax = slope of line
Get Vmax first, don’t forget inverserelationship!!!Highest line most inhibited!!!!
[I] = 0
Noncompetitive Inhibition INoncompetitive Inhibition I
•Inhibitor and substrate DO NOT have common shape•Binding is NOT at active site but causes a change in the substrate binding site•Large amounts of inhibitor prevent access of substrate to binding site•Large amounts of substrate CANNOT overcome inhibitory effect
Noncompetitive Inhibition IINoncompetitive Inhibition II
•As the amount of inhibitor increases, the efficiency of processing the product decreases
• same relative effect at all levels of S
•Higher [S] cannot “swamp out” effect; the “problem” is not at active site•Km does not change
• binding/dissociation not effected
•“Vmax ” decreases •the enzyme is less efficient in processing product
Noncompetitive InhibitionVelocity vs [Substrate]
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.900
0.000 0.200 0.400 0.600 0.800 1.000 1.200
substrate concentration [S]
Initi
al v
eloc
ity
[I] = 0
[I] = 2x
[I] = x
Noncompetitive InhibitionLineweaver-Burk Inverse Plot
y = 0.2461x + 1.0077
y = 0.307x + 1.2354
y = 0.4504x + 1.7139
0
1
2
3
4
5
6
7
-5 0 5 10 15
1/substrate concentration [S]-1
1/in
itia
l ve
loci
ty
Noncompetitive Inhibition IIINoncompetitive Inhibition III
A Lineweaver-Burk plotallows the importantparameters, Km and Vmax, to be determined directlyfrom an equation ratherthan from an extrapolation
1/Vmax = y intercept
Km/Vmax = slope of line
Get Vmax first, don’t forget inverserelationship!!!Highest line most inhibited!!!
[I] = 0
[I] = 2x
[I] = x
Uncompetitive Inhibition IUncompetitive Inhibition I
•Inhibitor and substrate DO NOT have common shape•Binding is NOT at active site •Binding of substrate causes a change in the shape of the enzyme that allows inhibitor to bind•Inhibitor binding prevents processing of substrate to product•Larger amounts of substrate cannot overcome inhibitory effect
Uncompetitive Inhibition IUncompetitive Inhibition I
As the amount of inhibitorincreases:
• the binding is effected• the efficiency of processing product decreases
Km increases•binding/dissociation effected
“Vmax ” decreases •the enzyme is less efficient in processing product
BC Online: 6B - Models of Enzyme Inhibition
Controls of Enzymatic ActivityControls of Enzymatic Activity
Inductive effects changing the amount of active enzyme present genetic control; irreversible covalent modification
Regulatory effects changing the activity of enzymes already present LeChatelier control; allosteric control; reversible
covalent modification; hormonal control
Inductive EffectsInductive Effects
Genetic control Turn genes on make mRNA make enzymes
Irreversible covalent modification Proteolytic activation zymogen ----> active enzyme very common for digestive enzymes
Regulatory effectsRegulatory effects
LeChatelier every system responds to the levels of substrates and products
Allosteric feedback: product of pathway providing information about the status of
metabolism forward activation: the product of an earlier reaction telling a later enzyme to
get ready Reversible covalent modification
reversible phosphorylation usually on serine extra ramping up of enzymatic activity, sometimes related to hormonal
response; control points in metabolic pathways Hormonal control
control from a distance (on organism level) binding to outside of cell OR entry into cell causes effect
Feedback InhibitionFeedback Inhibition
“Information” from a later step in pathway Usually product of
downstream reaction Amount of
product has to build up before effect is “felt” Utilization of
downstream product decreases effect
Forward ActivationForward Activation
“Information” from an earlier step in pathway Usually substrate of
upstream reaction Signal that lots of stuff
is on the way Amount of substrate
has to build up before effect is “felt” Utilization of upstream
substrate decreases effect
Factors Influencing the Rates of Enzyme-Catalyzed ReactionsFactors Influencing the Rates of Enzyme-Catalyzed Reactions
Concentration of the substrateConcentration of the substrate
0.0
0.2
0.4
0.6
0.8
1.0
Rat
e
0.0 5.0 10.0 15.0 20.0 25.0 30.0
Substrate Concentration
Rate versus Concentration of Substrate
Most Enzymes
Rate
Substrate Concentration
Rate versus Concentration of Substrate
Allosteric Enzyme
Allosteric Regulation of Enzyme ActivityAllosteric Regulation of Enzyme Activity
Allosteric Enzymes - Enzymes composed of two or more protein chains which contain separate regulatory sites and active sites. Often the regulatory site is on one chain and the active
site on another. Binding of a molecule at the regulatory site changes the
shape of the enzyme and affects its activity. Activator - a positive regulator Allosteric inhibitor - a negative regulator
not the same as noncompetitive inhibition
Concerted Model of Allosteric EffectConcerted Model of Allosteric Effect
T
T R
R
L
• The substrate, , can only bind to the Relaxed (R) form of the enzyme• The Taut (T) and Relaxed (R) forms are in equilibrium with each other with an equilibrium constant value of L
L = [R]/[T]
• The value of L is much less than 1• As the concentration of increases, equilibrium is shifted to the right• Since L remains constant, more of the T form is converted to the R form and there is more enzyme in the R form and the reaction speeds up
R
R
Sequential Model of Allosteric EffectSequential Model of Allosteric Effect
T
T R
R
L• The substrate, , can bind to both the Relaxed (R)and Taut (T) forms of the enzyme• The R form is bound preferentially• Only the site that has bound the substrate changes conformation by induced fit•The binding of the initial substrate passes on the conformational change to other subunits• These units will now bind the substrate with greater ease•As the concentration of increases, the number of relaxed active sites increases and the reaction speeds up sigmoidally form and the reaction speeds up
R
R
R
T
“Sense” of Cooperativity“Sense” of Cooperativity
every protein is in its lowest energy configuration given the prevailing conditions
the "resting" form of an enzyme is in the taut form either in equilibrium with relaxed form or can be changed to the relaxed form when substrate is added (or increased) this is the “turtle in its shell mode” -it is safe
the relaxed form becomes more favored upon the addition of substrate (or activator) the prevailing conditions have changed! “turtle in its moving and eating mode”- also more vulnerable!