kinetics 4
TRANSCRIPT
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Biochemical Kinetics
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2 2
2
2
2
2
2
2 2
2 2 2
2 D
D
M + O M-O
[ ][ ]Dissociation Constant,
[ ]
[ ]Fraction of binding site occupied,
[ ] [ ]
[ ][ ][ ]
[ ][ ] [ ][ ]
When pO = K , =0.5:
K is the oxygen tenstion at
D
D
D D
D
M OK
MO
MO
O M
M OK O pO
M O O K pO K M
K
θ
θ
θ
=
=+
= = ≈+ ++
2
2 50
which myoglobin
is half saturated, pO
pO p
θ =
+
Kinetics Binding of Oxygen to Myoglobin
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Kinetics Binding of Oxygen to Myoglobin
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Transfer of Respiratory Gases
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Myoglobin Can Not Be Used For O2 Transport
Tissue Lung
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Hemoglobin Is Used For O2 Transport
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O2 Binding – Hb vs. Mb
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O2 Transport Capability, Hb vs Mb
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K = 1
n =1
n =2n =4
1
n
n
n
n
x
K y x
K
=
+
Behavior of Sigmoidal Binding Equation
Sharp and Faster kinetics
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1
n
n
n
n
x
K y x
K
=
+
K = 1
K = 3
n = 3
Behavior of Sigmoidal Binding Equation
Decrease in affinity
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2
0.5
2
0.5
2
0.5
1
1
n
n
n
n
n
n
pO
p
pO
p
pO p
θ
θ
θ
=
+
=−
This is called Hill Equation. Hill Coefficient = nH Hill Constant = KD
nH = 1: No cooperation; nH > 1: Positive cooperation
•For Hemoglobin n = 3 not 4
•n is represented as nH
• nH represent cooperativity
in binding , not the exact
stoichiometry
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Cooperativity Model (Allosteric Model) For Hemoglobin
Monod, Wyman, and Changeaux (MWC)
• Only T and R conformations exist • T has lower affinity for O 2 .• The two states are in equilibrium
• T
R transition involves shift in equilibrium constant
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Hemoglobin Has T and R States
T (Low Affinity) R (High Affinity)
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Buffer in Physiological System
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pKa = 6.86
pKa of Histidine side chain = 6
Buffer in Physiological System
Phosphate is the most common buffer
Proteins also act as buffer
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pKa = 6.3
Buffer in Physiological System
Carbonic acid also acts as buffer
Status of carbonic acid depends upon 2 other equilibriums
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Transport of Carbon Dioxide
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Bohr Effect
Low pH His146 of β chain is protonated Stabilizes T state
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Bohr Effect
• CO2 reacts with N terminal of all 4 chains of Hb.
• Release of H+, induces Bore Effect
• Carbamate also stabilizes T state
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Bohr Effect
Haldane effect: Deoxygenation of the blood increases its ability to carry carbon
dioxide;
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BPG Happens in RBC
BPG is formed only in RBC.
Production of BPG increases in Hypoxia.
BPG binds to T state of Hb and stabilizes that
Another Allosteric Control of Hemoglobin
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BPG helps to adopt to high altitude and in hypoxia
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Effect of Temperature on Hemoglobin
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Transport of Carbon Dioxide
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Fetal hemoglobin has 2 and 2 chains
• The γ chain is 72% identical to the β chain.
• The binding affinity of fetal hemoglobin for BPG is significantly lower thanthat of adult hemoglobin
• Thus, the O2 saturation capacity of fetal hemoglobin is greater than that
of adult hemoglobin
Fetal Hemoglobin
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Enzyme Kinetics
E
S P
E + Sk 1
<<<<−−−−−−−−>>>>
k −−−−1
ESk 2
<<<<−−−−−−−−>>>> P + E
An enzyme-catalyzed reaction of substrate S to product P, can bewritten as
In terms of mechanism the same reaction can be written as
k -2
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Assumptions
1.1. EquilibriumEquilibrium: the association and dissociation of the substrate and: the association and dissociation of the substrate and
enzyme is assumed to be a rapid equilibriumenzyme is assumed to be a rapid equilibrium
2.2. Steady stateSteady state: the enzyme substrate complex ES is at a constant: the enzyme substrate complex ES is at a constantvalue.value.
For this to happen [S] >> [E]For this to happen [S] >> [E]TT..
This is true if we only study theThis is true if we only study the initial velocityinitial velocity..
3. P ES not considered
4. Rate limiting step: ES P, (as the E + S ES is a rapid equilibrium)
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E + S
k 1
<<<<−−−−−−−−>>>>
k −−−−1
ES
k 2
−−−−−−−−>>>> P + E
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E + S
k 1
<<<<−−−−−−−−>>>>k −−−−1
ES
k 2
−−−−−−−−>>>> P + E
1 1 2[ ] .[ ].[ ] .[ ] .[ ]d ES k E S k ES k ESdt
−= − −
[ ]0 :
d ES
dt =
As we considered steady state for ES:
1 1 2
1 1 2 1 2
.[ ].[ ] .[ ] .[ ] 0;
.[ ].[ ] .[ ] .[ ] ( ).[ ];
k E S k ES k ES
k E S k ES k ES k k ES
−
− −
− − =
= + = +
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( [ ])[ ] [ ] .[ ]
[ ] .[ ][ ]
( [ ])
m T
T
m
K S ES E S
E S ES
K S
+ =
=+
1 1 2 1 2
1 2m
1
.[ ].[ ] .[ ] .[ ] ( ).[ ];
( )Define: K =
k E S k ES k ES k k ES
k k
k
− −
−
= + = +
+
1 1 2.[ ].[ ] ( ).[ ];
.[ ] [ ].[ ]
=([ ] [ ]).[ ]
m
T
k E S k k ES
K ES E S
E ES S
− = +
=
−
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As ES P is rate limiting step:
2Initial velocity of product formation, k .[ES]ov =
2 2 [ ] .[ ].[ ] .( [ ])
T o
m
E Sv k ES k K S
∴ = =+
max
max 2
Maximum initial velocity, v is achived when allenzymes are in use:
.[ ]T v k E =
max2
.[ ][ ] .[ ]. =( [ ]) ( [ ])
T o
m m
v S E Sv k
K S K S∴ =
+ +
Michaelis–Menten equation
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max
1
if [ ], 2m oK S v v= =
max.[ ]
( [ ])om
v Sv
K S= +
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LineweaverLineweaver--BurkBurk (double reciprocal plot)(double reciprocal plot)
max
max max
.[ ]
( [ ])
1 1 1.[ ]
om
m
o
v Sv
K S
K
v v S v
= +
= +
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E + S
k 1
<<<<−−−−−−−−>>>>k −−−−1
ES
k 2
−−−−−−−−>>>> P + E1 2
m
1
( )K =
k k
k −
+
2 1
1 2 1m
1 1
If :
( )K = D
k k
k k k K
k k
−
− −
<<+
= =
In this case, Km is the measure of affinity of the Substrate for Enzyme
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Measuring efficiency of an enzyme:
Turnover number, kcat = vmax /[E]T
(Rate normalized over amount used in assay;
Unit is 1/t; )
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Specificity Constant = kcat /Km
High affinity of substrate for enzyme Low KD Low Km
Require low amount of S to reach ½. Vmax
High turnover of reaction High kcat
+
High Specificity
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max
m
.[ ]
( [ ])
.[ ] .[ ]
( [ ])
for [S]<<K : [ ] .[ ]
o
m
cat T
m
cat
o T m
v Sv
K S
k E S
K S
k
v E SK
=+
=+
=
Kcat /Km is a second order rate constant. The upper limit of Kcat /Km is fixed by
the rate of diffusion of moleculesUpper limit is ~ 108 to 109 1/M.Sec
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•• KKmm relates to affinity ;relates to affinity ;
•• VVmaxmax relates to efficiencyrelates to efficiency::
•• KKmm tell how much substrate to use in an assaytell how much substrate to use in an assay
•• If more than one enzyme share the same substrate, KIf more than one enzyme share the same substrate, Kmm
determines how to decide which pathway the substrate will takedetermines how to decide which pathway the substrate will take
VVmaxmax helps to identify rhelps to identify rate limiting enzyme/step in pathwayate limiting enzyme/step in pathway
KKmm andand VVmaxmax can be used to determine effectiveness of inhibitorscan be used to determine effectiveness of inhibitors
and activators for enzymeand activators for enzyme
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Competitive Inhibition
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Competitive Inhibition
max
max max
.[ ]
( [ ])
1 1 1
.[ ]
Where,
[ ] [ ][ ]1 ;
[ ]
Apperent
o
m
m
o
I
I
m m
v Sv
K S
K
v v S v
I E I K
K EI
K K
α
α
α
α
=+
= +
= + =
=
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Competitive Inhibition
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Competitive Inhibition
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Uncompetitive Inhibition
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max max
maxmax
1 1 '.[ ]
Where,
[ ] [ ][ ]' 1 ; '
' [ ]
apperent'
m
o
I
I
K v v S v
I ES I K
K ESI
vv
α
α
α
= +
= + =
=
Uncompetitive Inhibition
Mixed Inhibition
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Mixed Inhibition
Mixed Inhibition
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Mixed Inhibition
max max
1 1 '.[ ]
Where,
[ ] [ ][ ]1 ;
[ ]
[ ] [ ][ ]' 1 ; '' [ ]
m
o
I
I
I
I
K
v v S v
I E I K
K EI
I ES I K K ESI
α α
α
α
= +
= + =
= + =
Irreversible Inhibition
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Irreversible Inhibition
Bacterial Cell wall is made up of
peptidoglycans
Tanspeptidase catalyze cross
linking of peptidoglycans
β-lactum antibiotics irreversibly
bind (react) to transpeptidase
and stops the reaction.
Allosteric Regulation
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Allosteric Regulation
Example: Aspartate
carbamoyltransferase (ATCase)
Catalyze one step in pyrimidine
biosynthesis
Made up of two polypeptides,catalytic (C) and regulatory (R):
ATCase C3R2
Allosteric Regulation
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Allosteric Regulation
CTP inhibit
ATP Stimulate
ATCase does not follow
MichaelisMichaelis--MentenMenten
kineticskinetics rather have
kinetics similar to Hill
kinetics