INGENIERA ENZIMTICA
PROBLEMARIO
1. ECUACINDE MICHAELIS-MENTEN
Considera una enzima industrial importante, la cual cataliza la conversin de un
sustrato para formar un producto con un valor agregado mayor. La enzima sigue el
siguiente mecanismo:
Un anlisis inicial de las tasas de reaccin para la reaccin en solucin, con E0 = 0.10
M y varias concentraciones de sustrato S0, produce los siguientes parmetros de la
ecuacin de Michaelis-Menten:
Vmax = 0.60 M/s; KM = 80 M.
Un tipo de experimento diferente indica que la constante de la tasa de asociacin, k1,
es
k1 = 2.0 x 106 M-1s-1 (2.0 M-1s-1).
Estima el valor de k2 y k-1.
Solucin:
De la definicin de Vmax tenemos:
k2 =
Vmax = k 2 [E 0 ]
) (
k 1 = K M k1 k 2 = (80 M ) 2 M 1s 1 6 s 1 = 154 s 1
Enzima A
Velocidad
[S]
(mM)
(M/min)
0.0002
0.3373
0.0002
0.3364
0.0005
0.6945
0.0005
0.6957
0.001
1.0764
0.001
1.1407
0.005
1.9636
0.005
1.7706
0.01
2.0641
0.01
2.0751
Enzima B
Velocidad
[S]
(mM)
(M/min)
0.005
0.3962
0.005
0.3857
0.01
0.6163
0.01
0.6413
0.05
1.5606
0.05
1.5689
0.1
1.9862
0.1
1.7909
0.25
2.2011
0.25
2.2047
Solucin:
3.5000
y = 0.5081x + 0.4224
R = 0.999
3.0000
2.5000
Vmax =
2.36719094
(M/min)
KM
1.20279143
(M)
Vmax/KM =
1.968
2.0000
1.5000
min
-1
1.0000
0.5000
0.0000
0
3.0000
y = 10.814x + 0.4323
R = 0.9967
2.5000
Vmax =
2.31346294
(M/min)
KM
25.0185663
(M)
Vmax/KM =
0.092
2.0000
1.5000
1.0000
0.5000
0.0000
0
0.05
0.1
0.15
0.2
0.25
min
-1
Sustrato
(M)
20.00
18.32
16.64
14.97
13.30
11.63
9.96
8.31
6.66
5.02
3.40
Utilizando la ecuacin
[S ]0 [S ] = K + V
t
M
max
[
S ]0
[
S]
ln
ln 0
[S ]
[S ]
y transformando los datos para obtener los ejes adecuados
(So-S)/[ln(So/S)]
ln(So/S)
x
y
0.087685 22.8089171 19.1482267
0.18369012 21.7758032 18.2705913
0.28974888 20.7075863 17.3632081
0.40819718 19.5983716 16.4210948
0.5422881 18.4403825 15.4376837
0.69674291 17.2229954 14.4040112
0.87878828 15.9310272 13.3072822
1.10030511 14.5414212
12.128099
1.38291271 13.0160059 10.8344243
1.77265446 11.2825147 9.36582479
25
20
15
10
y = 0.8488x - 0.2134
5
0
10
15
20
25
t/[ln(So/S)]
0.8488 M/min
0.2134 M
Vmax =
Km =
k cat =
1/Vmax
[S]
1
v / Vmax
0.75
vi =
b).
v0 =
17.61nM / min
100 = 1
100 = 86.96%
135nM / min
Vmax [S ]
(270nM / min )(4x10 5 nM )
=
= 3.72nM / min
[I ]
2 x10 6 nM
6
5
+ [S ] (2x10 nM )1 +
+ 4x10 nM
K M 1 +
5
K
1
.
5
x
10
nM
v
i% = 1 i
v0
c).
v0 =
vi =
Vmax [S ]
(270nM / min ) 2 x10 6 nM
=
= 17.61nM / min
[I ]
2x10 6 nM
6
6
+ [S ] 2x10 nM 1 +
+ 2 x10 nM
K M 1 +
5
1.5x10 nM
Ki
v
i% = 1 i
v0
vi =
3.72nM / min
100 = 1
100 = 91.74%
45nM / min
Vmax [S ]
(270nM / min ) 7.5x10 6 nM
=
= 210.21nM / min
[I ]
1x10 4 nM
6
6
+ [S ] 2x10 nM 1 +
+ 7.5x10 nM
K M 1 +
5
1.5x10 nM
Ki
v
i% = 1 i
v0
210.21nM / min
100 = 1
100 = 1.38%
213.16nM / min
[S ] + [I ]
K i 1 +
K M
Despejando [I]
= 3.7 x10 4 M
[I ] =
=
100 i%
(100 75)
b)
Sin inhibicin
v
Con inhibicin
(Ec. Michaelis)
v0
[S]0
(Inhibidor Competitivo)
vi
v0 = vi
[S]i
[S]
[I ] + [S ]
K M 1 +
i
KI
Despejando [S]i tenemos
4
KI
v0 =
)(
Vmax [S ]
5.15x10-7 M / min 6 x104 M
=
= 1.93x10-7 M / min
K M + [S ]
1x10-3 M + 6 x10 4 M
Vmax
[S ]
[I ]
1 +
K I
vi =
=
K M + [S ]
5.15x107 M
6 x10 4 M
4
2.5x10 M
1 +
3x105 M
v
2.07x10-8 M / min
100 = 89.29%
i% = 1 i 100 = 1
-7
v
1.93x10
M
/
min
[
I]
+ [S ]
K M 1 +
K i
1/Vmax
KM/Vmax
-1/KM
1
= 200 nM s 1
0.005
K M = 200 Vmax = 200 200 = 40,000 nM
Sabemos que Vmax = kcat [E]t, por lo tanto
V
200 nM s 1
k cat = max =
= 20 s 1
10 nM
[E ]t
El coeficiente de especificidad es:
k
20 s 1
= cat =
= 0.0005 s 1nM 1
K M 40,000 nM
En presencia del inhibidor tenemos
m = 260 s
b = 0.0065 s nM-1
1
app
Vmax
=
= 153.846 nM s 1
0.0065
K Mapp = 260 Vmax = 260 153.846 = 40,000 nM
Vmax =
1/[S]
Se observa que:
app
Vmax > Vmax
Vmax
[I ]
1 +
K i
Despejando Ki
Ki =
[I ]
V max
app
V max
30,000
= 100,000 nM
200
1
153.846
Para un inhibidor competitivo la relacin [S]0 a [S]i para observar una misma velocidad es:
[S ]i = [S ]0 1 + [I ]
Ki
Despejando Ki
Ki
[I ]
=
[S ]i 1 =
[S ]0
3.7 x10 M
= 2 x10 5 M
3
1.5x10 M
1
7.69x10 5 M
[S]
mM
0.200
0.250
0.333
0.500
1.000
2.000
2.500
3.330
4.000
5.000
Velocidad (nM/min)
Control
[I] = 6 M
16.67
6.25
20.00
7.69
24.98
10.00
33.33
14.29
50.00
25.00
66.67
40.00
71.40
45.45
76.92
52.63
80.00
57.14
83.33
62.50
Solucin:
Primero se determina qu tipo de inhibicin se tiene, por lo tanto
K M = 999,860 nM
K Mapp = 2,999,900 nM
<
[I ]
app
M
K
1
KM
6,000 nM
= 2,999.52 nM
2,999,900nM
1
999,860nM
i% =
100[I ]
[S ] + [I ]
K i 1 +
K M
se tiene:
[S ] = K M [I ] 100 1 1 = 333,500 nM
K i i%
KM/Vmax
1/Vmax
-KM
Vmax =
1
= 2.7144 M s 1
0.3684
Vmax 2.7144 M s 1
=
= 904.8 s 1
[E ]t
0.003 M
kcat
904.8 s 1
=
= 0.564 s 1M 1
K M 1,603.12 M
[S]