Академический Документы
Профессиональный Документы
Культура Документы
Competitive Inhibition
Fig 8-15
Competitive Inhibition
COMPETITIVE Equilibria Scheme -Ic structrually resembles S, but is not an S E + S ES P + E Km -Ic binds to free E at active site where S binds + -Ic competes with S for free E Ic -High S overcomes inhibition because all E is bound in ES complex; since rate [ES] and Kc [ES] is max, rate is max; no EI c is present EI c slope = K m . (1+ [I c ] / K c ) / Vma x +I c
-I c
1 / Vo
+Ic
Vo
y-int = 1/Vmax
-I c
So x-int = -1/Km
1 / So slope = K m /Vmax
c
] / K c ))
Noncompetitive Inhibition
Fig 8-15
Noncompetitive Inhibition
NONCOMPETITIVE -Inc -Inc Equilibria Scheme P + E E + S ES is not structurally similar to S; is not an S Km + binds to free E or ES at a site where S + I nc I nc does not bind -Inc does NOT compete with S for free E K nc K'nc -High S cannot overcome inhibition because Inc binds to ES complex, inactivating it EI nc + S ESI nc
(inactive)
- I nc + I nc
Vo
y-int = 1/Vmax
O + CH3 C CO2-
O C NH2
ORDERED BI BI MECHANISM
NADH PYR LAC NAD
+
S1
S2
P1
P2
ES1
ES1S2
EP1P2
EP2
O + CH3 C CO2-
O C NH2 + CH3
N R **NAD
(oxidized form)
OH C CO2H
L-lactate
O H2N C CO2oxamate
INHIBITORS:
O CH3CH2HN C CO2ethyloxamate
For multisubstrate rxns, the type of inhibition depends upon the substrate that is varied in the inhibition experiment!
LACTATE DEHYDROGENASE O H H H2N C N pyruvate R **NADH (reduced form) HO H O C NH 2 N R NAD-OH H+ H CO2N R **NAD
(oxidized form)
O + CH3 C
O C NH2
INHIBITORS:
NADH
Which plot describes the inhibition of lactate dehydrogenase by NAD-OH when NADH is the varied substrate?
O H H HNC
2
H+
O C NH2
ORDERED BI BI MECHANISM
N R **NADH
(reduced form)
N R **NAD
(oxidized form)
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
ES1
ES1S2
EP1P2
EP2
NH2
Noncompetitive +I 1 / Vo
1/NADH
1/NADH
Which plot describes the inhibition of lactate dehydrogenase by NAD-OH when pyruvate is the varied substrate?
O H H HNC
2
H+
O C NH2
ORDERED BI BI MECHANISM
N R **NADH
(reduced form)
N R **NAD
(oxidized form)
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
ES1
ES1S2
EP1P2
EP2
NH2
Noncompetitive
1 / Vo
-I
1/PYRUVATE
1/PYRUVATE
Which plot describes the inhibition of lactate dehydrogenase by oxamate when NADH is the varied substrate?
O H H HNC
2
H+
O CH3 C CO2pyruvate
O C NH2
ORDERED BI BI MECHANISM
N R **NADH
(reduced form)
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
O H2N C CO2oxamate
R **NAD
(oxidized form)
ES1
ES1S2
EP1P2
EP2
Noncompetitive +I 1 / Vo
1/NADH
1/NADH
Which plot describes the inhibition of lactate dehydrogenase by oxamate when pyruvate is the varied substrate?
O H H HNC
2
H+
O CH3 C CO2pyruvate
O C NH2
ORDERED BI BI MECHANISM
N R **NADH
(reduced form)
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
O H2N C CO2oxamate
R **NAD
(oxidized form)
ES1
ES1S2
EP1P2
EP2
Noncompetitive
+I 1 / Vo -I
-I
1/PYRUVATE
1/PYRUVATE
Uncompetitive Inhibition
This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind
Uncompetitive Inhibition
This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind
UNCOMPETITIVE -Iu -Iu -Iu Equilibria Scheme P+ E is not structurally similar to S; is not an S E + S ES Km binds to ES only; S opens up a site for u I + binding site may be in active site but binding Iu of Iu requires prior binding of S Ku -High S cannot overcome inhibition because presence uI of S is required to provide a site for binding of EIu
1/Vo Vo
- Iu
+I -I slope = Km / Vmax
1 / So y-int = 1 / Vmax
O H H H2N C N R **NADH
(red uce d fo rm)
O +
C O P-O O H C OH CH2OPi
g lyceraldeh yd e-3-Pi
O INHIBITOR: HO As OOH
S1 S2 S3
P1
P2
ES 1
ES' 1 P 2
ES' 1 P 2
EP 1 P 2
EP 2
Predict the type of inhibition by H2 AsO4 - when each of the substrates is varied in inhibition experiments This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE H O C N R **NAD
(oxid ized fo rm )
O H H H2N C N R **NADH
(red uce d fo rm)
C O P-O O H C OH CH2OPi
g lyceraldeh yd e-3-Pi
O INHIBITOR: HO As OOH
S1 S2 S3
P1
P2
ES 1
ES' 1 P 2
ES' 1 P 2
EP 1 P 2
EP 2
Predict the type of inhibition by H2 AsO4 - when each of the substrates is varied in inhibition experiments
ORDERED TRI BI MECHANISM
NAD + GAP Pi
O
NADH
O HO P OOH
1,3-BPG
S1 S 2 S 3
P1
P2
HO As OOH
ES1
ES'1P2
ES' 1P2
EP1P2
EP2
.
Noncompetitive +I
1 / Vo -I 1 / Vo -I
Competitive +I
1 / Vo
Uncompetitive +I
-I
1/So
1/S o
1/S o
If So = Pi
If So = NADH or GAP
Product Inhibition
Equilibria Scheme PRODUCT INHIBITION E + S ES Km -Ip is structurally similar to S + -Ip binds to free E at active site where S binds P -Ip competes with S for free E Kp -At low S, resembles competitive inhibition -However, at high S, the inhibition is not overcome EIp because higher levels of P are generated which inhibit the enzyme 1 / Vo Vo slope = Km / Vmax So x-int = -1 / Km 1 / So P+ E
Suicide Inhibition
This type of enzyme inhibition results in the stoichiometric covalent modification of a side chain on an amino acid in the active site of an enzyme. The inhibitor chemically resembles a (one of the) substrate(s) and binds in the active site in the same way as the substrate(s) binds. The inhibitor, however, has a functional group, ususally a leaving group, that is replaced by a nucleophile in the enzyme active site. This covalent enzyme-inhibitor complex forms irreversibly, thereby irreversibly inactivating the enzyme. Therefore this type of inhibition is called "suicide inhibition" or affinity labeling and the inhibitor is called a "suicide inhibitor". This reaction with the suicide inhibitor removes active enzyme from the system; this removal is measured as inhibition. Since active enzyme is lost, the inhibition is not relieved at high substrate levels. The rate, at high substrate in the presence of the inhibitor,is still proportional to the amount of the enzyme-substrate complex. However, the maximum amount of that complex is limited by the remaining amount of active enzyme, not by the total enzyme added to the system. +I 1 / Vo Vo -I +I -I
So
1 / So
10
Suicide Inhibition
+I 1 / Vo Vo -I +I -I
So
1 / So
k2
P + E
The suicide inhibitor removes E so that the [ES] is lower, Vmax is lower, and inhibition cannot be overcome at high So
11
O CHY-HIS=N :
Cl CH2 C CH NH S CH2 O
irreversibly inactivated
Target Enzymes
(Box 21-1)
Aspirin
CO2H CH CH3
aspirin
CO2Na
Cyclooxygenases 1 and 2
O C CH 3 NH
CH CH3
O CH3
naproxen
OH
tylenol O CH3 CNHCHCO2 CH2 N-acetylcysteine antidote for SH tylenol poisoning
http://cti.itc.virginia.edu/~cmg/Demo/pdb/cycox/cycox_2.html
12
CO2H
+ Cox-1 O C CH 3 CH2OH
aspirin
CO2H
+
OH
salicylic acid
Cox-1 CH2O
C CH 3 O
http://www.scripps.edu/pub/goodsell/pdb/pdb17/pdb17_1.html
Tylenol and Vioxx, two other medications commonly used for arthritis, were similarly tested Both groups showed no competitive interaction with aspirin. http://www.pslgroup.com/dg/1e8fa2.htm
Two phases of inhibition: 1. Rapid competitive inhibition 2. Slower irreversible inhibition (covalent modification)
13
Cl
Cl O
Clorgyline N
Deprenyl N
Pargyline N
http://www.csusm.edu/DandB/AD.html
14