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Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Microorganisms contain a network of
enzymecatalyzed 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
extracellular substrate or product concentrations
X
i
intracellular metabolite concentrations
E
i
intracellular enzyme concentrations
All reactions are coupled!
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Basic types of reactions
Metabolic reactions belong to 2 types:
Uniuni reaction: A P
Bibi reactions: A + B P + Q
Mixed forms can also occur:
Biuni reactions: A + B P
Unibi reactions: B P + Q
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Rates
Rate of any enzymecatalyzed reaction:
V
i
= [regulatory term] * [ massaction term]
Consider the reversible MM equation for the uniuni reaction:
A P
(
(
¸
(
¸
÷ 
+ +
=
eq
P
A
P
P
A A
i i
i
K
C
C
K
C
C K
E q
v
regulatory massaction
term term
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Terms
Massaction 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)
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Linear expressions
Uniuni reaction:
P A i
C k C k
1 1 ÷
÷ = v
Bibi 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
uniuni reaction
bibi reactions
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Basic structures in metabolic systems
(Hofmeyer)
CHAIN
BRANCH
LOOP
MOEITY
CONSERVED
CYCLE
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Metabolites in biochemical networks
A X
1
X
2
X
3
Q
E
1
E
2
E
3
E
4
Y
+
B P
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Overall 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.)
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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:
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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%
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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??
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
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 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
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
AQ
Q
A
QX AP
P
A
PX
A
+ +


.

\

÷ +


.

\

÷
=
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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 branchedpoint 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
+ +
+ +
=
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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 steadystate
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:
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
= ?
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Moeity conserved cycle
Overall 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
÷ = ÷ =
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Moeity conserved cycle
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 steadystate:
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”
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Moeity conserved cycles
Using the steadystate 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??
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
= = =
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Consider an example: moeity cycle
A P
Q B
X
1
X
2
v
1
v
2
Using massaction 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
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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 ÷ =
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
Steadystate condition
At steadystate: 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
Overall 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 ÷
=
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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 overall rate equation close to equilibrium and at steadystate!!)
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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 steadystate (µ =0.03 h
1
):
mmol ACV per Cmol X
mmol ACV per Cmol X per h
L.mmol
1
.h
1
h
1
mM
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
USCMSChE Course: Bioprocess Technology
Metabolic Modeling Engr. Evelyn M. BuqueTaboada
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