Allosteric Enzyme Model

Dyanmic Enzyme Kinetic Plots for Identical Data Sets
Dials
Hill Plot: Multivalen

MM = Michaelis-Menton or
Vmax = max. velocity & V
n = substrate binding sites &
Km (M)
20
50
n binding sites
3.0
V {(MM}
10
Allosteric model enzyme

Km (M)
1.0
20
50
V0/(Vmax-V0)
Vmax (M s-1)
V {h = 2.2}
2.0
0.0
Direct Linear Plots >
Michaelis-Menten enzyme
Rectangular Hyperbolic Plot: Multivalent Enzyme

Michaelis-Menton (MM) or Allosteric Enzyme
n = substrate binding sites & h = Hill coefficient
V0 (mM s-1)
max value
V/(Vmax - V) {MM}
(V/Vmax - V) {h = 2.2}
h - Hill coefficient
0.0
10.0
[S]0 20.0
(mM)
V0 = Vmax[S]0h/([S]0h+KMh)
30.0
40.0
h = Hill coefficient
2.2
[S
n binding sites
3
"Ya"/"Yd" = V0/(Vmax-V0) = [S]0h
Vmax (M s-1)
Double Reciprocal (Lineweaver-Burke) Plot

Michaelis-Menton (MM) or Allosteric Enzyme
n = Substrate binding sites; h = Hill coefficient
Scatchard Plot for a

Michaelis-Menton (MM
n = Substrate binding sit
1/V0 (mM-1 s)
5.0
0.15
3.0
2.0
1/V {MM}
1.0
-0.20
-0.10
0.0
0.00
0.10
0.20
0.30
0.40
0.50
1/[S]0 (mM-1)
04/27/2013 Page 1 of 20 Pages
Copyright, Duane W. Sears, 04/27/2013
n*(V0/Vmax)/[S]0 (mM-1)
4.0
0.10
0.05
0.00
0.0
Allosteric Model 142220727.xls
1.0 n

Vmax/V0 = KMh/[S]0h+1
r/[S]0h = (1/KMh)(n-r )
n = # of substrate binding sites
04/27/2013 Page 2 of 20 Pages
inear Plots >>

Direct Linear Plot: MM Enzyne
m = 1, 2, etc. (-[S]m, Vm) data pairs
ot: Multivalent Enzyme

s-Menton or Allosteric Enzyme
velocity & Vo = initial velocity
nding sites & h = Hill coefficient
x - V) {MM}
-S8,V8
x - V) {h = 2.2}
Linear
(S8,V8)
10
100
-20
rd Plot for a Multivalent Enzyme

Menton (MM) or Allosteric Enzyme
te binding sites; h = Hill coefficient
10
20 ) 30
[S]0 (mM
40
50
S8,V
8
Linea
r (S8,V
8)
3.0
V0 (mM s-1)
2.0
Linear ((n*V/Vmax)/[S])
04/27/2013 Page 3 of 20 Pages
0.0
Direct Linear Plot: Allosteric Enzyme

m = 1, 2, etc (-[S]m, Vm) data pairs
(n*V/Vmax)/[S]
2.0
-10
"Y" = Vmax = (V0/[S]0h)*KMh + V0 = a"X"h + b
1.0 n*V0/Vmax
1.0
-S6,V6
[S]0 (mM)
S]0h/KMh
V0 (mM s-1)
2.0
-20
1.0
-10
0.0
10[S]0 20
(mM )30
40
50

04/27/2013 Page 4 of 20 Pages
Kinetic Analysis of Multivalent Michaelis-Menten and a Model Allosteric Enzyme

Michaelis-Menton Kinetics
k1 ->
k3 ->
V0 = dP/dtt
[E]+ [S]0 < = > [ES] --> [E] + [P]0
= k3[ES]
= 0
Vmax = k3[E]total
<- k2
Steady state assumption: d[ES]/dt = 0

Product assumption: [P]t = 0 = 0 at t = 0
KM = (k2 + k3)/k1
V0 = Vmax[S]0/([S]0+KM)
k3 = kcat
Extended Michaelis-Menton Kinetics: n = # binding sites (valence) and h = Hill coefficient.

k1 ->
k3 ->
[E]+ n [S] < = > [ESn] --> [E] + n [P]

<- k2
V0 = dP/dtt = 0 = nk3[ESn]
Steady state assumption: d[ES]/dt = 0
Vmax = nk3[E]total
Product assumption: [P]t = 0 = 0 at t = 0
KM = (k2 + k3)/k1
V0 = Vmax[S]0h/([S]0h+KMh)
Y = V0 = f(X) = f([S]0)
Rectangular Hyperbolic Equation: h = Hill coefficient

V0/Vmax = " Ya" = [S]0h/([S]0h+KMh)
(h = 1 for classical Michaelis-Menton kinetics)
where KM = [S]50% = substrate concentration where V0 = 0.5 * Vmax

(Vmax-V0)/Vmax = "Yd" = KMh/([S]0h+KMh)
Double-Reciprocal Equation:
1/V0 = (KMh/Vmax)(1/[S]0h + 1/Vmax
Y = 1/V0 = f(X) = f(1/[S]0)
Y = aXh + b is the equation for a polynomial function where Y = 1/V0, X = 1/[S]0, a = KMh/Vmax, & b = 1/Vmax
Hill Equation:
V0/(Vmax-V0) = "Ya"/"Yd" =
[S]0h/KMh
Scatchard Equation:
n(V0/Vmax)/[S]0h = n(1/KMh)(1-V0/Vmax)
r/[S]0h
= (1/KM )(n-r)
Y = V0/(Vmax-V0) = f(X) = f([S]0)

r = n(V0/Vmax)
Y =
r/[S]0h
(When h = 1, this equation reverts to classical Scatchard Equation)
= f(X) = f(r)
r/[S]0 = ([S]0h-1/KMh)(n-r)
Direct Linear Equation
Vmax = (V0/[S]0h)*KMh + V0
04/27/2013 Page 5 of 20 Pages
Y = Vmax = f(X) = f(KM)
NOTE: Vmax & KM are pseudo-variables
Kinetic Analysis 142220727.xls

Y = aXh + b is the equation for a polynomial function where Y = Vmax, X = KM, a = V0/[S]0h, & b = V0
04/27/2013 Page 6 of 20 Pages
04/27/2013 Page 7 of 20 Pages
04/27/2013 Page 8 of 20 Pages
Reaction Velocity versus Substrate Concentration

(Rectangular Hyperbolic Plot)
max value
Velocity (V) of Product Formation for a Multivalent Enzyme with n Sustrate (S) Km
Binding
Sites
(M) -- MM enzyme
Michaelis-Menton (MM) or Allosteric Kinetics
20
50
(h = Hill Coefficient)
n binding sites
3
3
3.0
V (mM s-1)
V {(MM}
Vmax (M s-1) -- MM enzyme

3
3
-----------------------------------------Km (M) -- Allosteric enzyme
20
50
V {h = 2.2}
2.0
h (Hill coefficient)
2.2
n
1.0
0.0
0.0
n binding sites
3
3
10.0
20.0
[S] (mM)
30.0
CALCULATIONS
40.0
Vmax (M s-1) -- Allosteric enzyme

3
3
numeric
scientific
04/27/2013
Page 9 of 20 Page(s)
Copyright, Duane W. Sears, 1998
142220727.xls RectangularHyperbolic
Reaction Velocity versus Substrate Concentration

(Rectangular Hyperbolic Plot)
nzyme
04/27/2013
142220727.xls RectangularHyperbolic
Double-Reciprocal Plot
max value
Km (M) -- MM enzyme
20
50
Double Reciprocal (Lineweaver-Burke) Plot

MM = Michaelis-Menton Enzyme
or Allosteric Enzyme
n = Substrate binding sites; h = Hill coefficient
n binding sites
3
3
5.0
1 / V (mM-1 s)
1/V {MM}
Linear (1/V
{MM})

3
3
20
50
4.0
3.0
2.0
2.2
n
1.0
-0.20
-0.10
0.0
0.00
n binding sites
3
3
0.20
1 /0.10
[S] (mM-1)
0.30
CALCULATIONS
0.40
0.50

3
3
numeric
scientific
04/27/2013
Copyright, Duane W. Sears, Fall 1998
Double-Reciprocal 142220727.xls]
Double-Reciprocal Plot
- MM enzyme
s-1) -- MM enzyme
---------------------------- Allosteric enzyme
s-1) -- Allosteric enzyme
04/27/2013
Double-Reciprocal 142220727.xls]
Hill Plot
max value
Km (M) -- MM enzyme
20
50
Velocity (V) of Product Formation Relative to

(Vmax -V) where Vmax is the Maximum Velocity
MM = Michaelis-Menton; h = Hill coefficient
10.0
n binding sites
3
3
V/(Vmax - V) {MM}
1.0

3
3
20
50
V / (Vmax - V)
(V/Vmax - V) {h = 2.2}
1.0
100.0
10.0
2.2
n
n binding sites
3
3
0.1
[S] (mM)

3
3
CALCULATIONS
numeric
scientific
04/27/2013
Copyright Duane W. Sears
Hill Plot 142220727.xls
Hill Plot
- MM enzyme
s-1) -- MM enzyme
---------------------------- Allosteric enzyme
s-1) -- Allosteric enzyme
04/27/2013
Copyright Duane W. Sears
Hill Plot 142220727.xls
Scatchard Plot
max value
Km (M) -- MM enzyme
20
50
Scatchard Plot of Enzyme Kinetics

n *(V/Vmax)/[S] (mM-1)
0.15
n binding sites
3
3
(n*V/Vmax)/[S]
Linear ((n*V/Vmax)/[S])

3
3
20
50
n * (V/Vmax) / [S] {h = 2.2}
0.10
0.05
0.00
0.0
2.2
n
1.0
n * V / Vmax
2.0
3.0
n binding sites
3
3
3
3
CALCULATIONS
numeric
scientific
04/27/2013
Scatchard Plot 142220727.xls
Scatchard Plot
teric enzyme
04/27/2013
Scatchard Plot 142220727.xls
Direct Linear Plots

Km (M)
20
Km (M)
n binding sites
MM enzyme
20
Allosteric
n binding sites
enzyme
Direct Linear Plot of ( -[S]m, Vm) Data Pairs

m = 1, 2, etc -- MM Enzyme
-S8,V8
Linear
(S8,V8)
-S6,V6
Linear
(S6,V6)
-S4,V4
2.0
V (mM s-1)
V (mM s-1)
3.0
1.0
2.0
1.0
0.0
-20
-10
2.2
Direct Linear Plot of ( -[S]m, Vm) Data Pairs

m = 1, 2, etc -- Allosteric Enzyme
3.0
-S8,V8
Linear
(S8,V8)
-S6,V6
Linear
(S6,V6)
-S4,V4
0.0
0
10
20 ) 30
[S] (mM
40
50
-20
CALCULATIONS
-10
10 [S] 20
(mM ) 30
40
50
numeric
scientific
04/27/2013
Direct Linear 142220727.xls
Direct Linear Plots
oefficient)
50
04/27/2013
Direct Linear 142220727.xls
Enxyme Kinetic Data

Michaelis-Menton and Allosterically Regulated Enzymes
Vmax (M s-1) =
Michaelis-Menton Kinetics
KM (M) =
20
n=
V {(MM}
0.3333
0.6000
V/(Vmax - V) {MM}
n*V / Vmax
(n*V/Vmax)/[S]
2.5
5.0
pS
5.60
5.30
0.1250
0.2500
0.3333
0.6000
1.33E-01
1.20E-01
1/V {MM}
3.0000
1.6667
1 / [S]
4.00E-01
2.00E-01
-S1
-2.5
0.00
-S1,V1
0.0
0.33
7.5
10.0
12.5
5.12
5.00
4.90
0.8182
1.0000
1.1538
0.3750
0.5000
0.6250
0.8182
1.0000
1.1538
1.09E-01
1.00E-01
9.23E-02
1.2222
1.0000
0.8667
1.33E-01
1.00E-01
8.00E-02
'-S2
-5.0
0.00
-S2,V2
0.0
0.60
15.0
17.5
20.0
4.82
4.76
4.70
1.2857
1.4000
1.5000
0.7500
0.8750
1.0000
1.2857
1.4000
1.5000
8.57E-02
8.00E-02
7.50E-02
0.7778
0.7143
0.6667
6.67E-02
5.71E-02
5.00E-02
'-S3
-7.5
0.00
-S3,V3
0.0
0.82
22.5
25.0
27.5
4.65
4.60
4.56
1.5882
1.6667
1.7368
1.1250
1.2500
1.3750
1.5882
1.6667
1.7368
7.06E-02
6.67E-02
6.32E-02
0.6296
0.6000
0.5758
4.44E-02
4.00E-02
3.64E-02
'-S4
-10.0
0.00
-S4,V4
0.0
1.00
30.0
32.5
35.0
4.52
4.49
4.46
1.8000
1.8571
1.9091
1.5000
1.6250
1.7500
1.8000
1.8571
1.9091
6.00E-02
5.71E-02
5.45E-02
0.5556
0.5385
0.5238
3.33E-02
3.08E-02
2.86E-02
'-S5
-12.5
0.00
-S5,V5
0.0
1.15
37.5
4.43
1.9565
1.8750
1.9565
5.22E-02
0.5111
2.67E-02
KM (M) =
20
[S] (M)
2.5000
2.0000
1.5000
Chart Title
1.0000
Vmax (M s-1) = 3
Allosterially Regulated Enzyme
n=
0.5000
[S] (M)
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
35.0
37.5
pS
5.60
5.30
5.12
5.00
4.90
4.82
4.76
4.70
4.65
4.60
4.56
4.52
4.49
4.46
4.43
04/27/2013
V {h = 2.2}
0.0306
0.1357
0.3108
0.5362
0.7869
1.0406
1.2812
1.5000
1.6933
1.8610
2.0050
2.1279
2.2327
2.3221
2.3984
(V/Vmax - V) {h = 2.2}
0.0103
0.0474
0.1156
0.2176
0.3556
0.5310
0.7454
1.0000
1.2958
1.6338
2.0150
2.4401
2.9099
3.4252
3.9866
n * (V/Vmax) {h = 2.2}
0.0000 0.0306
0.00.1357
0.3108
0.5362
0.7869
1.0406
1.2812
1.5000
1.6933
1.8610
2.0050
2.1279
2.2327
2.3221
2.3984
5.0
n * (V/Vmax) / [S] {h = 2.2}

1.22E-02
10.0
15.0
20.0
2.71E-02
V/(Vmax
- V) {MM}
4.14E-02
V {(MM}
5.36E-02
Linear (V
{(MM})
6.30E-02
V/(Vmax
- V) {MM}
6.94E-02
7.32E-02
7.50E-02
7.53E-02
7.44E-02
7.29E-02
7.09E-02
6.87E-02
6.63E-02
6.40E-02
1/V {h = 2.2}
32.6686
25.0 7.3707
30.0
3.2174
1.8649
1.2708
0.9610
0.7805
0.6667
0.5906
0.5374
0.4988
0.4699
0.4479
0.4307
0.4169
1 / [S]
4.00E-01
35.0
2.00E-01
1.33E-01
1.00E-01
8.00E-02
6.67E-02
5.71E-02
5.00E-02
4.44E-02
4.00E-02
3.64E-02
3.33E-02
3.08E-02
2.86E-02
2.67E-02
-S1
-2.5
40.0
0.00
'-S2
-5.0
0.00
'-S3
-7.5
0.00
'-S4
-10.0
0.00
'-S5
-12.5
0.00
142220727.xls Kinetic Data
-S1,V1
0.0
0.03
-S2,V2
0.0
0.14
-S3,V3
0.0
0.31
-S4,V4
0.0
0.54
-S5,V5
0.0
0.79
Enxyme Kinetic Data

Michaelis-Menton and Allosterically Regulated Enzymes
'-S6
-15.0
0.00
-S6,V6
0.0
1.29
'-S11
-27.5
0.00
-S11,V11
0.0
1.74
'-S7
-17.5
0.00
-S7,V7
0.0
1.40
'-S12
-30.0
0.00
-S12,V12
0.0
1.80
'-S8
-20.0
0.00
-S8,V8
0.0
1.50
'-S13
-32.5
0.00
-S13,V13
0.0
1.86
'-S9
-22.5
0.00
-S9,V9
0.0
1.59
'-S14
-35.0
0.00
-S14,V14
0.0
1.91
'-S10
-25.0
0.00
-S10,V10
0.0
1.67
'-S15
-37.5
0.00
-S15,V15
0.0
1.96
Vmax (mM s-1) =

KM (mM) =
20
(valence) n =
Vmax (mM s-1) =
KM (mM) =
(valence) n =
20
3
max = n
integer
h=
'-S6
-15.0
0.00
'-S7
-17.5
0.00
'-S8
-20.0
0.00
'-S9
-22.5
0.00
'-S10
-25.0
0.00
2.2
22
2.2
-S6,V6
0.0
1.04
-S7,V7
0.0
1.28
-S8,V8
0.0
1.50
-S9,V9
0.0
1.69
-S10,V10
0.0
1.86
'-S11
-27.5
0.00
'-S12
-30.0
0.00
'-S13
-32.5
0.00
'-S14
-35.0
0.00
'-S15
-37.5
0.00
-S11,V11
0.0
2.00
-S12,V12
0.0
2.13
-S13,V13
0.0
2.23
-S14,V14
0.0
2.32
-S15,V15
0.0
2.40
04/27/2013
(Hill coefficient) h =
2.2
142220727.xls Kinetic Data

Allosteric Enzyme Model

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Allosteric Enzyme Model

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Dyanmic Enzyme Kinetic Plots for Identical Data Sets

Hill Plot: Multivalen

Allosteric model enzyme

Direct Linear Plots >

Rectangular Hyperbolic Plot: Multivalent Enzyme

"Ya"/"Yd" = V0/(Vmax-V0) = [S]0h

Double Reciprocal (Lineweaver-Burke) Plot

Scatchard Plot for a

04/27/2013 Page 1 of 20 Pages

Copyright, Duane W. Sears, 04/27/2013

Allosteric Model 142220727.xls

Dyanmic Enzyme Kinetic Plots for Identical Data Sets

04/27/2013 Page 2 of 20 Pages

Copyright, Duane W. Sears, 04/27/2013

Allosteric Model 142220727.xls

Dyanmic Enzyme Kinetic Plots for Identical Data Sets

inear Plots >>

ot: Multivalent Enzyme

rd Plot for a Multivalent Enzyme

04/27/2013 Page 3 of 20 Pages

Direct Linear Plot: Allosteric Enzyme

"Y" = Vmax = (V0/[S]0h)*KMh + V0 = a"X"h + b

Copyright, Duane W. Sears, 04/27/2013

Allosteric Model 142220727.xls

Dyanmic Enzyme Kinetic Plots for Identical Data Sets

04/27/2013 Page 4 of 20 Pages

Copyright, Duane W. Sears, 04/27/2013

Allosteric Model 142220727.xls

Kinetic Analysis of Multivalent Michaelis-Menten and a Model Allosteric Enzyme

[E]+ [S]0 < = > [ES] --> [E] + [P]0

Steady state assumption: d[ES]/dt = 0

Extended Michaelis-Menton Kinetics: n = # binding sites (valence) and h = Hill coefficient.

[E]+ n [S] < = > [ESn] --> [E] + n [P]

Steady state assumption: d[ES]/dt = 0

Product assumption: [P]t = 0 = 0 at t = 0

Rectangular Hyperbolic Equation: h = Hill coefficient

(h = 1 for classical Michaelis-Menton kinetics)

where KM = [S]50% = substrate concentration where V0 = 0.5 * Vmax

(h = 1 for classical Michaelis-Menton kinetics)

1/V0 = (KMh/Vmax)(1/[S]0h + 1/Vmax

Y = 1/V0 = f(X) = f(1/[S]0)

Y = V0/(Vmax-V0) = f(X) = f([S]0)

(When h = 1, this equation reverts to classical Scatchard Equation)

04/27/2013 Page 5 of 20 Pages

Y = Vmax = f(X) = f(KM)

Copyright, Duane W. Sears, 04/27/2013

NOTE: Vmax & KM are pseudo-variables

Kinetic Analysis 142220727.xls

Kinetic Analysis of Multivalent Michaelis-Menten and a Model Allosteric Enzyme

04/27/2013 Page 6 of 20 Pages

Copyright, Duane W. Sears, 04/27/2013

Kinetic Analysis 142220727.xls

Kinetic Analysis of Multivalent Michaelis-Menten and a Model Allosteric Enzyme

04/27/2013 Page 7 of 20 Pages

Copyright, Duane W. Sears, 04/27/2013

Kinetic Analysis 142220727.xls

Kinetic Analysis of Multivalent Michaelis-Menten and a Model Allosteric Enzyme

04/27/2013 Page 8 of 20 Pages

Copyright, Duane W. Sears, 04/27/2013

Kinetic Analysis 142220727.xls

Reaction Velocity versus Substrate Concentration

Vmax (M s-1) -- MM enzyme

Vmax (M s-1) -- Allosteric enzyme

Reaction Velocity versus Substrate Concentration