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

USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Micro-organisms contain a network of

enzyme-catalyzed reactions:

Substrate, Ci

E1

X1

E2

E3
X3

Ci,Cj
Xi
Ei

X2
Product, Cj

E5
E4

X4

E6

extra-cellular substrate or product concentrations

intra-cellular metabolite concentrations
intra-cellular enzyme concentrations
All reactions are coupled!

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Basic types of reactions

Metabolic reactions belong to 2 types:

Uni-uni reaction:

Bi-bi reactions:

A + B

P
P + Q

Mixed forms can also occur:

Bi-uni reactions:

A + B

Uni-bi reactions:

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

P
P + Q

Engr. Evelyn M. Buque-Taboada

Rates
Rate of any enzyme-catalyzed reaction:

Vi = [regulatory term] * [ mass-action term]

Consider the reversible M-M equation for the uni-uni reaction:

CP
i
C A

CP
K
eq

K A CA
KP
qi Ei

regulatory
term
USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

mass-action
term
Engr. Evelyn M. Buque-Taboada

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)

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Linear expressions
Uni-uni reaction:

i k1C A k 1C P

Bi-bi reactions:

i k1C AC B k 1C P CQ

Since

k1
K eq
k 1

C
P

i k1 C A

K
eq

CP CQ

i k1 C ACB

K
eq

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

uni-uni reaction

bi-bi reactions

Engr. Evelyn M. Buque-Taboada

Structures in biochemical networks

Sequential linkage:

E1

X1

E1

X1

Group transfer
linkage:

X2

X3

X2

donor couple:
X1 and X2

MG

X4

X3
E2

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

E2

acceptor couple:
X3 and X4

Engr. Evelyn M. Buque-Taboada

Basic structures in metabolic systems

(Hofmeyer)
CHAIN

BRANCH

LOOP
MOEITY
CONSERVED
CYCLE
USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Metabolites in biochemical networks

A
E1

X1

E2

X2

E3

X3

Q
E4

Y+

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

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.)

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Linear chain of enzymes

k1

k2

X1

k3

X2

k4

X3

k-1

k-2

k-3

k-4

E1

E2

E3

E4

Rate of reaction:

i Ei ki 1 X i k (i 1) X i 1

Equilibrium constant:

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Ki = k+i/k-i

Engr. Evelyn M. Buque-Taboada

Conversion rate of substrate A

rA

C A CP / K1K 2 K3 K 4

1
1 1
1 1
1
1

k1 E1 K1 k2 E2 K1 K 2 k3 E3 K1 K 2 K 3 k4 E4

Here:

K1K 2 K 3 K 4 k1k2 k3k4 / k1k2 k3k4 K eq

C
P

k1 E1 C A

K eq

rA
1 k1 1
1 k1 1
1
k1 1

1 E1

K1 k 2 E2 K1 K 2 k3 E3 K1 K 2 K 3 k 4 E4
USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Effect of enzyme concentration

On the reaction pathway, effect of enzyme concentration varies:

rA

2
1
1.
2.

rA proportional to Ei
rA independent of Ei

Ei
USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Flux control coefficient

Flux control coefficient, CEi = relative change in rA upon a
relative change in Ei

rA
ln rA
rA
C Ei

Ei ln Ei
Ei

100%

Flux
arginine

Enzyme activity

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

100%

Engr. Evelyn M. Buque-Taboada

Control coefficient
By proper mathematical differentiation of the rate equation,
the four (4) control coefficients of the involved enzymes
are obtained:
1
C E1
Dk1 E 1

CE 2

Dk 2 E2 K1

CE 3

Dk 3 E3 K1 K 2

CE 4

Dk 4 E4 K1 K 2 K 3

S CEi = 1
* Focus on the slowest enzyme !
0 < CEi < 1
USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

Why??
Engr. Evelyn M. Buque-Taboada

Branched pathway
reversible branched pathway

k2
supply
branch

k1

k-1
E1

k-2 E2

two
output
branches

k3 E3
k-3

Equilibrium constants:

K AP

k1k 2

k 1k 2

K AX

k1

k 1

K AQ

k1k 3

k3 k 1

K PX

k2

k2

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

K PQ

k 2 k 3

k3 k 2

K QX

k3

k 3

Engr. Evelyn M. Buque-Taboada

Branched pathway: Rates

CQ

CP
1

C A

CA

k3 E3 K PX
K AP k 2 E2 K QX
K AQ
rA
1
1
1

K AX k 2 E2 k3 E3 K PX k1 E1 k3 E3 K QX k1 E1 k 2 E2
1

CQ

CP
1

C A

CP
k3 E3 K PX
K AP k1 E1 K QX
K PQ
rP
1
1
1

K AX k 2 E2 k3 E3 K PX k1 E1 k3 E3 K QX k1 E1 k 2 E2
1

CQ
CQ
1
CA

CP

k2 E2 K QX
K AQ k1 E1 K QX
K PQ
rQ
1
1
1

K AX k 2 E2 k3 E3 K PX k1 E1 k3 E3 K QX k1 E1 k 2 E2
1

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Branched pathway
rA = r P + r Q

Note:

For the branch flux ratio, R:

1
k3 E3 K PX
1
k 2 E2 K QX

rP
rQ

C
1
CP Q
C A P
K AP k1 E1 K QX
K PQ

C
C
1
CA Q
CP Q

K AQ k1 E1 K QX
K PQ

Finally, for the branched-point metabolite concentration CX,

CX

k1E1 C A k2 E2 CP k3 E3 CQ
k1E1 k2 E2 k3 E3

K AX K PX K QX

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Cyclic structures in the pathway

X2
cyclic structures

X1

X1

E1

X2

X4

E3

X1

X3

X3

E2

X3
X5

Cyclic structure, the parallel substrate loop

USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

The parallel substrate loop

X2

E2

E3

k2

k-2

k-3
k5

X1

k-5

k3

X3

E4
Equilibrium constants:

K2

k2
k2

k3
K3
k 3

k5
K5
k 5

Equilibrium equation for X1 and X3:

K2K3 = K5
USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

The parallel loop

Proper derivation of the rate equation at steady-state
and 1st-order kinetics:

CX 3
1

C X 1
r

1
1
1
1
K5

k5 E5 k 2 E2 K 2 k3 E3

The rate equation shows that a parallel cyclic structure can be

lumped into an equivalent linear structure:
k+

X1

k-

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

X3

Engr. Evelyn M. Buque-Taboada

Moeity conserved cycle

A

moeity
conserved
cycle

X1

v2

Q
Rates ??

Coupling of two
different processes:
A to P and
B to Q

v1

C C
v1 k1 C AC X 1 P X 2
K1

CQC X 1

v2 k2 CBC X 2
K2

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

X2

Equilibrium constants?
K1 = ?
K2 = ?

Engr. Evelyn M. Buque-Taboada

Moeity conserved cycle

A

P
v1

X1

v2

X2

Over-all reaction in the cycle:

A +B

Rate of conversion of A, rA:

dC A
rA
v1
dt

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

P +Q

Engr. Evelyn M. Buque-Taboada

Moeity conserved cycle

A

P
v1

X1

X2

v2

Rate equations for the intermediates:

dCX 1
v1 v2 0
dt
dC X 2
v1 v2 0
dt

In steady-state:

v1 = v 2

Under all conditions:

dC X 1 dC X 2

0
dt
dt

CX1 + CX2 = constant = T

conserved moeity sum

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Moeity conserved cycles

Using the steady-state condition (v1 = v2) and the
conserved moeity sum (CX1 + CX2 = T), the following
rates are obtained:
CP

k1
k 2C B
K1
CX1

CQ
T

CP
k1C A k1
k 2C B k 2

K1
K2

C P CQ

k1k 2T C AC B
K1 K 2

rA
CQ

CP

k1 C A
k2 CB

K1
K2

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

CQ

k1C A k 2

K
CX 2
2

C
T

C
k1C A k1 P k 2C B k 2 Q
K1
K2

Simplified to:

rA

k1k 2T C AC B

k1C A k 2C B

When??

Engr. Evelyn M. Buque-Taboada

Metabolite control coefficients

Metabolite control coefficient, CX,ij = relative change in
metabolite concentration Xi with respect to
the relative change in each enzyme
concentration Ej.

X i

C X ,ij

X i E j ln X i
Xi

E j E j X i ln E j
Ej
For example: for 3 enzymes

S CX,ij = 0

Why??
CX1,1 + CX1,2 + CX1,3 = 0

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Thermodynamic Driving Force

Consider a simple reaction:

The kinetic driving force is: A B

where K = equilibrium constant

Thermodynamic theory states that for DGR:

A
DGR DGRo RT ln
B

Gibbs energy of reaction

At reference (standard) state:

DGRo RT ln K
USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Relation of Kinetics & Thermodynamics

Combing kinetic and thermodynamic equations:
B

DGR
A
1 1 exp

K
RT

Close to equilibrium: DGR 1

RT
Thus,

using for x << 1, the approximation

1 exp( x) ( x)

DG
R
A
1
DG '
K RT

DGR
dimensionless
where DG '
RT

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Consider an example: moeity cycle

A

P
v1
X1

v2

X2

Using mass-action kinetics:

PX 2
V1 k1 AX1

K
1

K1

P X2

K AP K X
A X1

Then, the rate expression is:

P k1P
V1 k1 X 1 A

K AP K AP

kinetic
USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

X2
X1

K
X

kinetic driving force

Engr. Evelyn M. Buque-Taboada

Transformation of driving forces

Transforming kinetic and thermodynamic driving forces:
X2

1 X 1
KX

'
1 A DGAP

K AP

DG '
X

Then, for V1 and V2:

V1 k1 X 1 DG

'
AP

V2 k 2 X 2 B DG

'
BQ

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

DGX'
k1 X 1
K AP

K X

k 2 X 1Q
DG X'

K BQ

Engr. Evelyn M. Buque-Taboada

Steady-state condition
At steady-state:
Then,

V1 = V2

'
'
k
X
A

D
G

k
X
B

D
G
1 1
AP
2 2
BQ
DGX'
P
K Q
k1 X 1
k2 X 1 X
K AP
K BQ

Over-all rate equation of the coupled process:

A
B
'
'
k1k 2 X 1 K X K AP DG AP X 2 K BQ DGBQ
P
Q

r
K BQ
K
k1
k 2 K X AP
Q
P

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

Equilibrium condition
Close to equilibrium:

X 1K X X 2
A
K AP 1
P
B
K BQ 1
Q

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

'
'
k1k2 X 2 DGAP
DGBQ
r
K BQ
K AP
k1
k2 K X
Q
P

(simplified form of the over-all rate equation close to equilibrium and at steady-state!!)
USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

FINAL EXERCISE
Metabolic Modeling
Consider the pathway for penicillin production:

AA

v1

ACV

IPN

Pen

O2
Rate kinetics:
X ACV

v1 k1e1 1
K1

v2 k2e2 X ACV CO 2
v3 k3e3 X IPN
USC-MSChE Course: Bioprocess Technology
Metabolic Modeling

At reference steady-state (m = 0.03 h-1):

K1 0.1 mmol ACV per C-mol X

k1e1o 1 mmol ACV per C-mol X per h
k 2 e2o 150 L.mmol-1.h-1
k3 e3o 30 h-1
CO 2 0.2 mM
Engr. Evelyn M. Buque-Taboada

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada

USC-MSChE Course: Bioprocess Technology

Metabolic Modeling

Engr. Evelyn M. Buque-Taboada