1

BIOCHEMICAL
REACTION NETWORKS
Metabolic Flux Analysis, MFA
Metabolic flux analysis
What is metabolic flux analysis?
What is the point of MFA?
Key concepts
Examples
2
Limitation of macroscopic models
Macroscopic models are very
useful, but there is a lot of
available information that is
not used.
No information about
intracellular reactions is
used. (i.e. the Biochemistry is
completely neglected!)
In Out
Cell
What is MFA?
Metabolic flux analysis is the calculation and analysis of
the flux distribution of the entire biochemical reaction
network in the system of study.
3
What is the point of MFA?
Known metabolic pathways impose constraints (in
addition to the elemental balances) which reduce the
degree of freedom of the system. This gives the
possibility to calculate fluxes from relatively few
measured fluxes.
In particular, intracellular fluxes which are difficult to
measure can thus be calculated. The intracellular flux
distribution is useful when identifying targets for
genetic engineering.
The carbonflux is dividedbetweendifferent
divergingbranches (or is combinedin
convergingbranches)
v1
X1
X3 X2
X4
X5 X6

v2
v3
v5 v4
v6
P1
v8
P3
v99 v7
P2
A

v1
X1
X3 X2
X4
X5
X6

v2
v3
v5 v4
v6
P1
v8
P3
v99 v7
P2
B
S
v10
v1
X1
X3
X2
X4
X5
X6

v2
v3
v5
v4
v6
P1
v8
P3
v99 v7
P2
C
S
v11
4
What information can be used?
Many metabolic pathways are well characterized
the glycolysis
the TCA-cycle
the PPP (pentose phosphate pathway)
Synthetic pathways for amino acids and nucleotides
S
1
S
2
.
.
.
S
N
P
1
P
2
.
.
.
P
M
v
1
-r
s,N
-r
s,1
r
p,1
r
p,M
X
1
X
2
.
.
.
X
K
v
J
v
2
.
Biomass

Figure 5.1 A general representationof reactions consideredinametabolic network. N substrates enter the
cell andareconvertedinto M metabolic productsviaatotal of K intracellular metabolites. Theconversions
occur via J intracellular reactions for which the rates are given by v
1
v
J
. Rates of substrate formation
(r
s,1
,,r
s,N
) andproductformation(r
p,1
,,r
p,M
) arealsoshown.
5
Example 1

A B
C
D
E
F
NADH
2 NADH
v
1

v
2
v
3
v
4
v
5
A hypothetical reaction scheme
Example 1
Assume pseudo-steady state for the intermediates B, D
and NADH 3 constraints on the system.
(1) v
1
- v
2
- v
3
= 0
(2) v
3
v
4
v
5
= 0
(3) v
2
2v
5
= 0
6
Example 1
Solving for v
2
, v
3
and v
4
in terms of v
1
and v
5
1. v
2
= 2v
5
2. v
3
= v
1
2v
5
3. v
4
= v
1
3v
5
All five fluxes could thus be calculated from
only two measured values, v
1
and v
5,
by
using the steady-state constraints.
Example 1
In this simple case, the substitutions could be made by
hand calculations, but this is not in general possible or to
be recommended.
Instead a matrix approach should be used, in which all
reactions are treated using linear algebra.
7
Key concepts
The matrix, T (the transpose of the stoichiometric
matrix) *
The steady-state reaction rates, v
The net rates of formation, r
*IMPORTANT NOTE: The nomenclature is different in the 2nd edition
of NV&L. In the 2nd edition, T denotes the stoichiometric matrix
0 =
|
|
|
.
|

\
|
O
X
p
s
Matrix containing all
coefficients in the reactions;
one row per reaction
substrates
products
intracellular biomass
components, metabolites
The reaction stoichiometries are
defined by the stoichiometric matrix O
*IMPORTANT NOTE: The nomenclature is different in the 2nd edition
of NV&L. In the 2nd edition, T denotes the stoichiometric matrix
8
Example2 (from NV&L, 2nd ed)
Substrate
Metabolite 2
Metabolite 1
Metabolite 3
C
B
A
v
1
v
2 v
3
v
4
v
6
v
5
v
1
=v
2
Balance for A
v
2
=v
3
+v
4
Balance for B
v
4
=v
5
+v
6
Balance for C
v
1
=v
2
Balance for A
v
1
=v
2
Balance for A
v
2
=v
3
+v
4
Balance for B
v
2
=v
3
+v
4
Balance for B
v
4
=v
5
+v
6
Balance for C
v
4
=v
5
+v
6
Balance for C
The stoichiometric matrix; O
|
|
|
|
|
|
|
|
.
|

\
|

= O
1 0 0 1 0 0 0
1 0 0 0 1 0 0
1 1 0 0 0 0 0
0 1 0 0 0 1 0
0 1 1 0 0 0 0
0 0 1 0 0 0 1
S P
1
P
2
P
3
A B C
9
The net formation rates, r, can be calculated
from v and T
v v
0
r
T = O =
|
|
.
|

\
|
T
Stoichiometric matrix
Vector of fluxes, i.e. steady-state
reaction rates
Vector of net formation rates
Metabolite steady-state net
formation rates
Example 2
|
|
|
|
|
|
|
|
|
.
|

\
|

=
|
|
|
|
|
|
|
|
.
|

\
|
|
|
|
|
|
|
|
|
|
.
|

\
|

=
|
|
|
|
|
|
|
|
|
.
|

\
|
6 5 4
4 3 2
2 1
6
5
3
1
6
5
4
3
2
1
3 ,
2 ,
1 ,
1 1 1 0 0 0
0 0 1 1 1 0
0 0 0 0 1 1
1 0 0 0 0 0
0 1 0 0 0 0
0 0 0 1 0 0
0 0 0 0 0 1
0
0
0
v v v
v v v
v v
v
v
v
v
v
v
v
v
v
v
r
r
r
r
p
p
p
s
The last three rows give the steady-state requirements
10
Example 3
4 metabolites
(A, B, C, D)
3 reactions
(A - B)
(B - C)
(B - D)
v
1
, v
2
, v
3
reaction rates (mol/m3 s)
v
1
v
2
v
3
Example 3
(
(
(

= O
1 0 1 0
0 1 1 0
0 0 1 1
1
st
reaction
2
nd
reaction
3
rd
reaction
A B C D
11
Example 3
v T r =
(
(
(

(
(
(
(

=
(
(
(
(

3
2
1
1 0 0
0 1 0
1 1 1
0 0 1
v
v
v
D
C
B
A
r
r
r
r
Example 3
(
(
(
(

=
(
(
(
(

3
2
3 2 1
1
v
v
v v v
v
D
C
B
A
r
r
r
r
seems reasonable..
Net rates of formation
Reaction rates fluxes
12
So if we know v, we can calculate r. However, normally the situation
is the opposite, i.e we want to calculate v from a (partially) know r
v
T
T
0
r
r
|
|
.
|

\
|
=
|
|
|
.
|

\
|
2
1
m
c
Net formation rates
to be calculated
Measured rates
Rates of formation of
intracellular metabolites =0
We want to calculate
this vector.
Dimensions of vectors involved
P =total number of compounds involved (i.e. the
dimension of the vector r)
J =number of fluxes (i.e. the dimension of the vector
v)
K =number of metabolites at steady-states
F =J K =(probably) equal to the degrees of
freedom in the system =number of needed
measured net formation rates, (i.e. the dimension of
the vector r
m
)
The dimension of the matrix T is thus (P x J )
13
(

=
2
1
T
T
T
Corresponding to the
non-measured part of r.
Dimensions (P-J ) x J
Corresponding to the
measured part of r and the
metabolites at steady-state.
Dimensions (J +(K-J ) x J , i.e.
J x J .
The matrix equation (1) can thus be divided
into two equations (2 and 3)
v T r =
v
1
T r
c
=
v
2
0
T
r
m
=
(

(2)
(3)
(1)
14
Equation 3 is now used to calculate v
v v =
(

=
(

0 0
1
2 2
1
2
1
2
m m
r
T T T
r
T
Multiply both sides from the left by the inverse of the matrix T
2
Example 1
revisited

A B
C
D
E
F
NADH
2 NADH
v
1

v
2
v
3
v
4
v
5
15
Example 1
Stoichiometry
|
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|
|
|
|
.
|

\
|
=
|
|
|
|
|
|
|
|
|
.
|

\
|
|
|
|
|
|
|
.
|

\
|

0
0
0
0
0

2 1 0 0 0 1 0
0 1 0 0 1 0 0
0 1 1 0 0 0 0
1 0 1 1 0 0 0
0 0 1 0 0 0 1
NADH
D
B
C
E
F
A
s
p
X
Example 1
v T =
|
|
|
.
|

\
|
0
m
c
r
r
Flux vector
Stoichiometric
matrix (transposed)
measured net
production rate
r to be calculated
assumed at steady
state ( r =0)
16
Example 1
Assume that r
A
and r
F
are measured
|
|
|
|
|
|
.
|

\
|
|
|
|
|
|
|
|
|
|
.
|

\
|

=
|
|
|
|
|
|
|
|
|
.
|

\
|
5
4
3
2
1

2 0 0 1 0
1 1 1 0 0
0 0 1 1 1
1 0 0 0 0
0 0 0 0 1
0 0 0 1 0
0 1 0 0 0
v
v
v
v
v
NADH
D
B
F
A
C
E
r
r
r
r
r
r
r
meas
r
calc
r
We get
v
2
0
T
r
m
=
(

|
|
|
|
|
|
.
|

\
|
(
(
(
(
(
(

=
(
(
(
(
(
(

5
4
3
2
1

2 0 0 1 0
1 1 1 0 0
0 0 1 1 1
1 0 0 0 0
0 0 0 0 1
0
0
0
v
v
v
v
v
F
A
r
r
17
The fluxes are obtained from
(

=

0
1
2
m
r
T v
To calculate the fluxes we need to invert T
2
. This is a major task to do
Manually, but trivial if using a suitable mathematical program like MATLAB.
We get:
(
(
(
(
(
(

0 0 0 1 0
1 1 1 3 1
1 0 1 2 1
1 0 0 2 0
0 0 0 0 1
1
2
T
In terms of the measured rates, we thus have
the fluxes:
(
(
(
(
(
(

=
(
(
(
(
(
(

F
F A
F A
F
A
r
r r
r r
r
r
3
2
2
5
4
3
2
1
v
v
v
v
v
Note. Remember
r
A
< 0, r
F
> 0
18
What about the non-measured formation
rates?
v T r
1
=
c
The non-measured product formation rates are obtained from
(
(
(
(
(
(

=
(

5
4
3
2
1
0 0 0 1 0
0 1 0 0 0
v
v
v
v
v
C
E
r
r
(

=
(

=
(

F
F A
C
E
C
E
r
r r
r
r v
r
r
2
3
2
4
v
We get:
Summary
MFA is a systematic way of analyzing fluxes in a
metabolic network
The degree of freedom (rank of) the matrix T determines
how many fluxes must be known to fully resolve the
metabolic fluxes in the network.
19
Recipe
Write down all known reactions and define
the stoichiometry
Write down the T-matrix
Determine the rank of T
Consider the observability of the system.
(What rates can and should be measured?)
Carry out the experiments and the analysis!
Fundamental equations
v
T
T
0
r
r
|
|
.
|

\
|
=
|
|
|
.
|

\
|
2
1
m
c
|
|
.
|

\
|
=

0
r
T v
m 1
2
v T r
1
=
c
Net formation rate
to be calculated
Measured rate
Intracellular metabolite, no
net formation
20
Simplification of metabolic networks
A
E
D
C B
F
v1 v2
v3
v5
v4
Consider
Full model
5 fluxes, 3 metabolites, 2 df
0 -1 0 1 0 0
0 0 0 -1 1 0
0 0 0 0 -1 1
0 0 1 0 0 -1
1 0 0 0 -1 0
O =
-1 0 0 0 0
-1 0 -1 0 0
0 1 0 0 1
0 1 0 0 0
-1 -1 -1 -1 -1
T
2
-1
=
|
|
|
|
|
|
.
|

\
|

=
|
|
|
|
|
|
.
|

\
|
F A
F
F
A
A
r r
r
r
r
r
5
4
3
2
1
v
v
v
v
v
Obviously, v1=v2 and v3=v4
21
The system can be reduced by only considering
branchpoints
A
E
C
F
v1
v3
v5
Consider the reduced system
-1 0 0
0 1 0
-1 -1 -1
T
2
-1
=
0 -1 0 1
0 0 1 -1
1 0 0 -1
O =
|
|
|
.
|

\
|

=
|
|
|
.
|

\
|
F A
F
A
r r
r
r
5
3
1
v
v
v
Back to the favourite example

A B
C
D
E
F
NADH
2 NADH
v
1
v
2
v
3
v
4
v
5
NAD+
2NAD+
22
0 0 -1 0 1 0 0 0
1 0 0 0 -1 0 1 -1
0 0 0 0 -1 1 0 0
0 1 0 0 0 1 0 0
0 0 0 1 0 1 -2 2
O =
0 0 0 0 1
1 -1 -1 0 0
0 0 1 1 1
0 1 0 0 -2
0 -1 0 0 2
T
2
=
But.. T2 is NOT invertible.
The rank of T2 is only 4 (not 5).
Applying the steady-state requirement also for NAD+should
give another constraint leaving only one rate to be measured.
Only one component in each co-factor pair (NADH/NAD+;
NADPH/NADP+; ATP/ADP etc.) should be included in the
stoichiometric matrix. If the steady state requirement is
met for one component it is automatically satisfied for the
other, since they are not independent.
23
Steady state approximation
A small complication.. (Note 5.1)
dt
dX
x X
dt
dx
dt
xX d
i
i
i
+ =
) (
chain rule
x T q
i i
v =
x
dt
dx
=
net volumetric acc. term of
compound i
volumetric formation
rate of biomass
Steady state approximation
i
i
i
X v T
dt
dX
=
Surprise!
Dilution term
Must be small for
PSS approx. to be
valid!
Intracellular acc. term
This is what is normally
assumed to be = 0!
Volumetric net formation term/ biomass conc.
(= q
i
/x)
24
Intracellular concentrations
Metabolite/co-factor Concentration
(M)
Metabolite/co-factor Concentration
(M)
Glucose (GLC)
Glucose-6-P (G6P)
Fructose-6-P (F6P)
Fructose-1,6-bisP (FDP)
Dihydroxyacetone P (DHAP)
Glyceraldehyde-3-P (GAP)
3-Phosphoglycerate (3PG)
5000
83
14
31
138
18.5
118
2-Phosphoglycerate (2PG)
Phosphoenolpyruvate (PEP)
Pyruvate (PYR)
ATP
ADP
P
i

29.5
23
51
1850
138
1000

Typically the specific growth rate is in the range 0.1 0.5 h
-1
. The
glycolytic flux for a dilution rate of 0.1 h
-1
is in the range 1 mmol
g
-1
h
-1
.
What if the system is
overdetermined? (i.e. the
dimension of r
m
is >F)
| X y =
Compare with linear regression using least squares
( )
T T
2
1
2 2
#
2
T T T T

=
|
|
.
|

\
|
=
0
r
T v
m #
2
where
( ) y X X X b
t t
1
=
Estimate of |
25
Whatif the system is underdetermined?
Sometimes the flux distribution can be calculated under
the assumption that metabolism is geared towards
maximizing some cellular objective function (e.g.
growth rate).
Linear programming can be used if the objective
function is a simple linear combination of the fluxes.