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Bio 98 - Lecture 10
Enzymes III:
Competitive Inhibition &
Enzyme Regulation
Note: [E], concentration of free enzyme, is not the same as [E]t
k-1
k1 k2E + S ES E + P
VI. A better way to plot vo vs [S] data.
[S]
vo
Vmax
vo vs [S] plot
?
Km 1/[S]
1/Vmax
-1/Km
1/vo
Lineweaver-Burk plot
Vmax [S]vo = ––––––––– Km + [S]
1 Km 1 1–– = –––– ––– + ––––– vo Vmax [S] Vmax
The Lineweaver-Burk plot reduces uncertainty in estimating Vmax and Km.
Vmax [S]vo = ––––––––– Km + [S]
Take reciprocal of both sides of equation
Expand
= ––––––––vo Vmax [S]
Km + [S]1
1 Km 1 1–– = –––– ––– + ––––– vo Vmax [S] Vmax
Thus y = ax + b
Lineweaver-Burk
= ––––––––vo Vmax [S]
Km1 +[S]
Vmax[S]
Vmax [S]vo = ––––––––– Km + [S]
1 Km 1 1–– = –––– ––– + ––––– vo Vmax [S] Vmax
y = a x + b
Solve for y at x=1/[S]=0: y =
Solve for x at y=1/v0=0: x =
1/[S]
1/Vmax
-1/Km
1/vo
Lineweaver-Burk plot
1 1–– = –––– = b vo Vmax
1 1–– = - –––– [S] Vmax
Vmax b –– = - –– Km a
How do you measure competitive inhibition?
Vmax [S]vo = ––––––––– Km + [S]
[E][I]KI = –––––– [EI]
where [I]
= 1 + ––– KI
Vmax remains unchanged, but apparent Km increases with increasing [I]
-1 Km
-1 Km
K-I
K1
K-1 [I] = KI
[I] = 0
[I] = 2KI
Modes of Enzyme Regulation
1. Allosteric* control/regulation
• homotropic allostery (O2 for hemoglobin)
• heterotropic allostery (H+, CO2, BPG for Hb)
2. Covalent modification
• group addition - often reversible, ie phosphorylation
allosteric* = allo (other); steric (shape, object)
Regulation of Enzyme Activity
It would be wasteful to continue to turn substrate into product if enough is available for proper cellular function. Therefore, enzymes often are highly regulated by binding small molecule regulators that can either decrease or increase activity.
A classic example is in amino acid metabolism. Several enzymes are required to convert simple substrates into more complex amino acids:
In this example, five enzyme-catalyzed steps are required to convert Thr to Ile. When there is sufficient Ile available, Ile will “feedback” inhibit enzyme 1 (E1) that converts Thr to intermediate B. Such inhibition effectively shuts down the entire pathway. There must be a careful balance between the concentration of Ile required for normal function and the concentration of Ile required to inhibit enzyme E1. FEEDBACK INHIBITION!
E1Thr E2
threoninedehydratase
E3 E4 E5 IleB
A Simple Model of Heterotropic Allosteric Regulation(activation)
inhibitor activator
Heteroallosteric Regulation 2 subunit enzyme model
T-state R-state substrate
Heteroallostery: Effect of inhibitor or activator on vo vs [S] plot
[S]
vo
Vmax
Vmax
2
Km KmappKm
app
R-state
T-state
Phosphofructokinase-1 is regulated by both activators and inhibitors
+ PPP
F 6-P (mM)
+ ADP
F 6
-bis
F
pyruvate + ATP
When the concentration of phosphenolpyruvate (PPP) reaches a certain level, then P6-F kinase activity is lowered by feedback inhibition from phosphenolpyruvate. On the other hand, ADP is the allosteric activator; this may seem strange but the net objective of glycolysis is generation of ATP. If the ATP levels drop (ie [ADP] goes up), glycolysis needs to be stimulated. Thus ADP binds to and activates phosphofructokinase-1 to increase ATP production.
PEP
deactivates (phosphoenolpyruvate)
activates (ADP)
phosphoenol-pyruvate
Most allosteric enzymes are multi-subunit enzymesPhosphofructokinase has 4 subunits
Inactivated state (T-state)Activated state (R-state)
ADP
F6bisP + ADP
Covalent modifications
Phosphorylation - a reversible modification
protein phosphatase
H20HOPO3
=
activity state 1
ATP ADP
-OPO3
= activity state 2
protein kinase
Enz Enz
Phosphorylation is reversible and is used in many pathways to control activity.
Enzymes that add a phosphate to a hydroxyl side chain are commonly called kinases.
Enzymes that remove a phosphate from a phosphorylated side chain are called phosphatases.
Examples: pancreatic enzymes
Proteolysis – an irreversible modification
inactive enzyme
protease +
active enzyme*