Lecture 6 - Metabolic Modeling

USC-MSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. Buque-Taboada

Metabolic Modeling**
Introduction
Basic types of reaction and related enzyme kinetics
Biochemical Networks
Thermodynamic description of chemical networks

** adaptation of the lectures by Prof. J.J. Heijnen, TUDelft, The Netherlands
Micro-organisms contain a network of
enzyme-catalyzed reactions:
Substrate, C
i
Product, C
j
X
1
X
3
X
2
X
4
E
1
E
3
E
6
E
5
E
4
E
2
C
i
,C
j
extra-cellular substrate or product concentrations
X
i
intra-cellular metabolite concentrations
E
i
intra-cellular enzyme concentrations
All reactions are coupled!
Basic types of reactions
Metabolic reactions belong to 2 types:
Uni-uni reaction: A P

Bi-bi reactions: A + B P + Q
Mixed forms can also occur:
Bi-uni reactions: A + B P

Uni-bi reactions: B P + Q
Rates
Rate of any enzyme-catalyzed reaction:

V
i
= [regulatory term] * [ mass-action term]
Consider the reversible M-M equation for the uni-uni reaction:
A P
(
(
-
+ +
=
eq
P
A
P
P
A A
i i
i
K
C
C
K
C
C K
E q
v
regulatory mass-action
term term
Terms
Mass-action term only contains the
direct reactants and products (A and P).

Regulatory term contains:
direct reactants and products (A, P)
modifier concentrations
enzyme concentration (linear)

Linear expressions
Uni-uni reaction:
P A i
C k C k
1 1
= v
Bi-bi reactions:
Q P B A i
C C k C C k
1 1
= v
Since
1
1
=
k
k
K
eq
|
|
.
|
\
|
=
eq
P
A i
K
C
C k
1
v
|
|
.
|
\
|
=
eq
Q P
B A i
K
C C
C C k
1
v
uni-uni reaction
bi-bi reactions
Structures in biochemical networks
X
1
X
2
X
3
E
1
E
2
Sequential linkage:
E
1
X
1
X
2
X
4
X
3
E
2
M MG
Group transfer
linkage:
donor couple:
X
1
and X
2
acceptor couple:
X
3
and X
4
Basic structures in metabolic systems
(Hofmeyer)
CHAIN
BRANCH
LOOP
MOEITY
CONSERVED
CYCLE
Metabolites in biochemical networks
A X
1
X
2
X
3
Q
E
1
E
2
E
3
E
4
Y
+
B P
Over-all rate equation
Rates = function of concentrations of:
terminal species
modifiers
sum of conserved moeities (enzyme, NAD
-
/NADH, etc.)
but, not the concentrations of intermediates,
which are not moeity conserved.
Concentration of intermediates = function of the concentrations of:
terminal species
modifiers
sum of conserved moeities (enzyme, NAD
-
, NADH, etc.)
Linear chain of enzymes
A X
1
X
2
X
3
P
k
1
k
2
k
3
k
4
k
-1
k
-2
k
-3
k
-4
E
1
E
2
E
3
E
4
( )
1 ) 1 ( 1 + + +
=
i i i i i i
X k X k E v
Rate of reaction:
K
i
= k
+i
/k
-i
Equilibrium constant:
Conversion rate of substrate A
( ) | |
4 4 3 2 1 3 3 2 1 2 2 1 1 1
4 3 2 1
1 1 1 1 1 1 1
/
E k K K K E k K K E k K E k
K K K K C C
r
P A
A
|
|
.
|
\
|
+
|
|
.
|
\
|
+
|
|
.
|
\
|
+
=
Here:
|
|
.
|
\
|
+ + +
|
|
.
|
\
|
=
4 4
1
3 2 1 3 3
1
2 1 2 2
1
1
1
1 1
1 1 1 1 1 1
1
E k
k
K K K E k
k
K K E k
k
K
E
K
C
C E k
r
eq
P
A
A
( ) ( ) ( )
eq
K k k k k / k k k k K K K K = =
4 3 2 1 4 3 2 1 4 3 2 1
Effect of enzyme concentration
On the reaction pathway, effect of enzyme concentration varies:
1
2
r
A
E
i
1. r
A
proportional to E
i
2. r
A
independent of E
i
Flux control coefficient
Flux control coefficient, C
Ei
= relative change in r
A
upon a
relative change in E
i
i
A
i
i
A
A
Ei
E
r
E
E
r
r
C
ln
ln
c
c
=
c
c
=
Flux
arginine
Enzyme activity
100%
100%
Control coefficient
By proper mathematical differentiation of the rate equation,
the four (4) control coefficients of the involved enzymes
are obtained:
1 2 2
2
1
K E Dk
C
E
=
2 1 3 3
3
1
K K E Dk
C
E
=
1 1
1
1
E Dk
C
E
=
3 2 1 4 4
4
1
K K K E Dk
C
E
=
E C
Ei
= 1
0 < C
Ei
< 1
* Focus on the slowest enzyme !
Why??
Branched pathway
supply
branch
A
Q
P
X
k
1
k
-1
k
3
k
2
k
-2
k
-3 E
1
E
2
E
3
two
output
branches
reversible branched pathway
Equilibrium constants:
2 1
2 1
k k
k k
K
AP
=
1
1
=
k
k
K
AX
1 3
3 1
=
k k
k k
K
AQ
2 3
3 2
=
k k
k k
K
PQ
2
2
=
k
k
K
PX
3
3
=
k
k
K
QX
Branched pathway: Rates
( ) ( )
( )( ) ( )( ) ( )( )
2 2 1 1 3 3 1 1 3 3 2 2
1 1 3 3
1 1 1
1 1
E k E k K E k E k K E k E k K
K
C
C
K E k K
C
C
K E k
r
QX PX AX
PQ
Q
P
QX AP
P
A
PX
P
+ +
|
|
.
|
\
|

|
|
.
|
\
|
=
( ) ( )
( )( ) ( )( ) ( )( )
2 2 1 1 3 3 1 1 3 3 2 2
1 1 2 2
1 1 1
1 1
K
C
C
K E k K
C
C
K E k
r
QX PX AX
PQ
Q
P
QX AQ
Q
A
QX
Q
+ +
|
|
.
|
\
|
+
|
|
.
|
\
|
=
( ) ( )
( )( ) ( )( ) ( )( )
2 2 1 1 3 3 1 1 3 3 2 2
2 2 3 3
1 1 1
1 1
K
C
C
K E k K
C
C
K E k
r
QX PX AX
AQ
Q
A
QX AP
P
A
PX
A
+ +
|
|
.
|
\
|
+
|
|
.
|
\
|
=
Branched pathway
Note:
r
A
= r
P
+ r
Q
For the branch flux ratio, R:
Q
P
r
r
R =
|
|
.
|
\
|
+
|
|
.
|
\
|
|
|
.
|
\
|

|
|
.
|
\
|
=
PQ
Q
P
QX AQ
Q
A
QX
PQ
Q
P
QX AP
P
A
PX
K
C
C
K E k K
C
C
K E k
K
C
C
K E k K
C
C
K E k
R
1 1 2 2
1 1 3 3
1 1
1 1
Finally, for the branched-point metabolite concentration C
X
,
( ) ( ) ( )
QX PX AX
Q P A
X
K
E k
K
E k
K
E k
C E k C E k C E k
C
3 3 2 2 1 1
3 3 2 2 1 1
+ +
+ +
=
Cyclic structures in the pathway
X
1
X
3
X
2
A P
X
1
X
3
X
1
X
2
X
3
X
4
X
5
E
3
E
2
E
1
cyclic structures
Cyclic structure, the parallel substrate loop
The parallel substrate loop
X
1
X
3
X
2
A P
E
4
E
3
E
2
k
-3
k
3
k-
2
k
2
k
-5
k
5
Equilibrium constants:
2
2
2
=
k
k
K
3
3
3
=
k
k
K
5
5
5
=
k
k
K
Equilibrium equation for X
1
and X
3
:
K
2
K
3
= K
5
The parallel loop
|
|
.
|
\
|
(
(
(
(
+
+ =
5
3
1
3 3 2 2 2 5 5
1 1 1
1
1
1
K
C
C
E k K E k E k
r
X
X
Proper derivation of the rate equation at steady-state
and 1
st
-order kinetics:
X
1
X
3
k+

k-
The rate equation shows that a parallel cyclic structure can be
lumped into an equivalent linear structure:
Moeity conserved cycle
A P
Q B
X
1
X
2
v
1
v
2
moeity
conserved
cycle
Coupling of two
different processes:
A to P and
B to Q
Rates ??
|
|
.
|
\
|
=
1
2
1 1 1
K
C C
C C k v
X P
X A
|
|
.
|
\
|
=
2
1
2 2 2
K
C C
C C k v
X Q
X B
Equilibrium constants?
K
1
= ?
K
2
= ?
Over-all reaction in the cycle:
A P
Q B
X
1
X
2
v
1
v
2
A + B P + Q
Rate of conversion of A, r
A
:
1
v
dt
dC
r
A
A
= =
A P
Q B
X
1
X
2
v
1
v
2
Rate equations for the intermediates:
0
2 1
1
= + = v v
dt
dC
X
0
2 1
2
= + = v v
dt
dC
X
In steady-state:
v
1
= v
2
Under all conditions: 0
2 1
= +
dt
dC
dt
dC
X X
C
X1
+ C
X2
= constant = T
conserved moeity sum
Moeity conserved cycles
Using the steady-state condition (v
1
= v
2
) and the
conserved moeity sum (C
X1
+ C
X2
= T), the following
rates are obtained:
|
|
.
|
\
|
+ +
|
|
.
|
\
|
+
|
|
.
|
\
|
+
=
2
2 2
1
1 1
2
1
1
1
K
C
k C k
K
C
k C k
C k
K
C
k
T
C
Q
B
P
A
B
P
X
|
|
.
|
\
|
+ +
|
|
.
|
\
|
+
|
|
.
|
\
|
+
=
2
2 2
1
1 1
2
2 1
2
K
C
k C k
K
C
k C k
K
C
k C k
T
C
Q
B
P
A
Q
A
X
|
|
.
|
\
|
+ +
|
|
.
|
\
|
+
|
|
.
|
\
|
=
2
2
1
1
2 1
2 1
K
C
C k
K
C
C k
K K
C C
C C T k k
r
Q
B
P
A
Q P
B A
A
( )
B A
B A
A
C k C k
C C T k k
r
2 1
2 1
+
=
Simplified to:
When??
Metabolite control coefficients
Metabolite control coefficient, C
X,ij
= relative change in
metabolite concentration X
i
with respect to
the relative change in each enzyme
concentration E
j
.

E C
X,ij
= 0 Why??
For example: for 3 enzymes

C
X1,1
+ C
X1,2
+ C
X1,3
= 0
( )
( )
j
i
i j
j i
j
j
i
i
ij X
E
X
X E
E X
E
E
X
X
C
ln
ln
,
o
o
o
o
o
o
= = =
Thermodynamic Driving Force
Consider a simple reaction:
A B
The kinetic driving force is:
|
.
|
\
|

K
B
A where K = equilibrium constant
Thermodynamic theory states that for AG
R
:
Gibbs energy of reaction
At reference (standard) state:
|
.
|
\
|
+ A = A
B
A
RT G G
o
R R
ln
( ) K RT G
o
R
ln = A
Relation of Kinetics & Thermodynamics
|
|
.
|
\
|
|
.
|
\
|
A
=
|
|
|
|
.
|
\
|
RT
G
K
A
B
R
exp 1 1
Combing kinetic and thermodynamic equations:
Close to equilibrium:
1 <<
A
RT
G
R
using for x << 1, the approximation
( ) ) ( ) exp( 1 x x ~
Thus,
( ) ' 1 G
RT
G
K
A
B
R
A =
|
.
|
\
|
A
=
|
|
|
|
.
|
\
|
( ) =
A
= A
RT
G
G
R
'
where
dimensionless
Consider an example: moeity cycle
A P
Q B
X
1
X
2
v
1
v
2
Using mass-action kinetics:
(
=
1
2
1 1 1
K
PX
AX k V
X AP
K K
X
X
A
P
K = =
1
2
1
Then, the rate expression is:
(
+
(
=
X AP AP
K
X
X
K
P k
K
P
A X k V
2
1
1
1 1 1
kinetic kinetic driving force
Transformation of driving forces
Transforming kinetic and thermodynamic driving forces:
( )
'
1
AP
AP
G
K
A
P
A =
|
|
|
|
.
|
\
|
( )
'
1
2
1
X
X
G
K
X
X
A =
|
|
|
|
.
|
\
|
Then, for V
1
and V
2
:
( )| | | |
'
1 1
'
1 1 1 X
AP
AP
G
K
P
X k G X k V A
|
|
.
|
\
|
+ A =
( )| | | |
'
1 2
'
2 2 2 X
BQ
X
BQ
G
K
K
Q X k G B X k V A
|
|
.
|
\
|
+ A =
Steady-state condition
At steady-state: V
1
= V
2
Then,
( ) ( )
BQ
X
AP
BQ AP
X
K
Q K
X k
K
P
X k
G B X k G A X k
G
1 2 1 1
'
2 2
'
1 1
'
+
A A
= A
Over-all rate equation of the coupled process:
( ) ( )
P
K
K k
Q
K
k
G K
Q
B
X G K
P
A
K X k k
r
AP
X
BQ
BQ BQ AP AP X
2 1
'
2
'
1 2 1
+
(
A + A
=
Equilibrium condition
Close to equilibrium:
1
1
2 1
~
~
~
BQ
AP
X
K
Q
B
K
P
A
X K X
At this condition, the rate equation is reduced to an expression
describing the sum driving force of the moiety cycle reactions:
A + B P + Q
( ) ( ) | |
P
K
K k
Q
K
k
G G X k k
r
AP
X
BQ
BQ AP
2 1
' '
2 2 1
+
A + A
=
(simplified form of the over-all rate equation close to equilibrium and at steady-state!!)
FINAL EXERCISE
Metabolic Modeling
Consider the pathway for penicillin production:
AA ACV IPN Pen
v
1
O
2
IPN
O ACV
ACV
X e k v
C X e k v
K
X
e k v
3 3 3
2 2 2 2
1
1 1 1
1
=
=
|
|
.
|
\
|
=
Rate kinetics:
2 . 0
30
150
1
1 . 0
2
3 3
2 2
1 1
1
=
=
=
=
=
O
o
o
o
C
e k
e k
e k
K
At reference steady-state ( =0.03 h
-1
):
mmol ACV per C-mol X
mmol ACV per C-mol X per h
L.mmol
-1
.h
-1
h
-1
mM


Lecture 6 - Metabolic Modeling

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Lecture 6 - Metabolic Modeling

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USC-MSChE Course: Bioprocess Technology

Metabolic Modeling Engr. Evelyn M. Buque-Taboada

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