CHEM-E3170

Systems Biology
Spring 2017

Tero Eerikäinen 15.3.

MCA, Metabolic control analysis

Metabolic Control Analysis

• Theory
• Control and elasticity coefficients
• The methods for determining the coefficients
• Linear and branched pathways
• Examples
Metabolic Control Analysis
 Explains how the metabolic fluxes are controlled so that
the metabolite production and consumption are balanced
in the current growth conditions
 Repeals the terms "bottleneck enzyme" and "rate-limiting
enzyme"
 The analysis is based on elasticity coefficients (ε) and
control coefficients (FCC or CCC) values
 The lack of experimental data analysis may limit the
application of analysis
 The Control of Flux (H. KACSER and J. A. BURNS, 1973)
Metabolic Control Analysis
• The most important goals of metabolic engineering is
the flux adjustment.
• With MFA one can calculate and examine the flux
values​​, but it does not give a quantitative measure of
the flux control.
• Flux control is necessary to keep metabolite synthesis
and conversion rates balanced over a wide range of
conditions so that the intracellular concentrations of
the metabolites can not rise or lower to the
catastrophic levels.
• Understanding the internal control of fluxes is essential
for implementing rational metabolic engineering
Metabolic Control Analysis
• Enzyme control is often described only qualitatively, but the
comparison of the quantitative impact is missing.
• MCA is a linear perturbation theory of enzymatic control for the
internally non-linear metabolite network
• Despite its limitations, the MCA has proved to be useful in
• 1) showing the impact of individual reactions to the control of
metabolism,
• 2) presenting a concept of flux rate control in the network of enzyme
reactions
• 3) describing the effect of the enzyme activity to intracellular
concentrations of metabolites,
• 4) connecting the local enzyme kinetics to the metabolic behavior of
the entire system
Parameters and variables
• The description applies to the steady-state
• Enzyme activities Ei as well as the first substrate S0 and the final product P
concentrations are parameters here
• The fluxes through the pathway and the metabolite concentrations Si are system
variables
• The total steady state flux is described by the symbol J, the individual reaction
fluxes vi
• By changing the parameter values one gets different system variable
values​​, that is, for example, by changing enzyme activities different metabolite
concentrations and flux values can be obtained
• Intracellular compartmentalization is not a problem to the MCA. The transport
processes can be imported into the analysis as additional steps in the process
with their own reaction rates
• The spatial distribution of the metabolite concentrations can not be deduced
without a complex spatial model
 Control coefficients
• One of the MCA's purpose is to relate variables
(V) to the parameters (P)
• The sensitivity of the system variables in relation
to the parameters can be determined, for
example:
• the flux sensitivity in relation to the enzyme activities
• the sensitivity of intracellular metabolites in relation
to the enzyme activities, effector concentrations or
other system parameters
• Sensitivities are described in a combination of
factors, from which the most important are
control coefficients (CPV)
• The control coefficients are valid only in steady-
state mode (for this reason enzyme activities are
studied as parameters)

ln (activity of enzyme B) B0

The tumor cell proliferation control coefficient is computed as the

slope at the initial point A0 or B0: (A) Enzyme A shows a control
coefficient of 0.1 on the tumor cell proliferation; (B) Enzyme B
shows a control coefficient of 1 on the tumor cell proliferation
 Flux control coefficient
• Flux control coefficient (FCC) describes the effect of
small change in enzymatic activity to the metabolic flux
• The FCC is dimensionless and independent of the units
of flux and activity, and in linear pathways having
values ​between 0 ... 1, where Jk is the (steady-state)
flux of reaction k in the pathway and Ei the activity of
the i-th enzyme
• Various ways of describing the flux control coefficient :
 E  dJ  d ln J
CJ =    =
 J  dE  d ln E
 Ei  dJ k  d ln J k
CiJk
=   = i, k ∈ {1,2,..., L}
 Jk  dEi  d ln Ei
 Approximating flux control coefficient

• When the enzyme concentration change is very small

(or infitesimal), it can be approximated that:

∆ ln J k
CJk = i, k ∈ {1,2,..., L}
i
∆ ln E i
C iJk
J k = a ⋅ Ei a is a case by case constant
 Flux control coefficients
 In the figure, for example,
slope = (dJ / dE); E=0.5; J=0.65;
C=(dJ / dE)*(0.5/0.65)
 In even more generalized form in
place of the enzyme activity is
used the reaction rate vi of the
reaction i
 Another generalization is wherein
the parameter p exclusively
affects the rate (vi ) of the
reaction i. If the enzyme activity
is taken as the parameter the  v  dJ  d ln J k
C J =  i  k  =
k
i, k ∈ {1,2,..., L}
equation returns to the general  J k  dvi  d ln vi
i

formula of the FCC above -1

vi  dJ k  ∂vk 
C iJ k =    i, k ∈ {1,2,..., L}
Jk  dp  ∂p 
The effect of the flux control coefficient

• The enzyme having the highest flux control coefficient, has the
highest effect on flux control in that particular steady-state,
• Amplification of enzyme E2 increases the flux J fourfold
compared to amplification of enzyme E1

E1 ; v1 E2 ; v2
Flux-control summation theorem
 Due to the normalization the sum of control
coefficients is 1
 The FCCs are dependent on the structure of the
system and from their individual values ​can not draw
any conclusions without comparing to other FCCs
 In a long pathway control coefficients may have small
values ​and still a small value can be dominant when
compared to the other coefficient values.
 Therefore large number of mutations and selection
steps are often required when it is desired to improve
a metabolic pathway producing more e.g. certain
amino acid product.
L

∑ i =1
C Jk

i =1
k ∈ { 1,2 ,...,L}
Some remarks on flux-
control coefficients

 1) Enzyme activity is often regulated by the binding of the allosteric

activators or effectors. Enzyme concentrations should not be used,
but activity instead. Response coefficients (RJX ; described later
here) can be used to describe the influence of the effectors
 2) Flux’s sensitivity of the enzyme activity does not make a difference
whether it is a control or a regulatory enzyme and, therefore, the
control coefficient can be a misleading term. Paradoxically, the
regulatory enzyme, which is in the beginning of the pathway and is
subject to feedback inhibition may have a small FCC
 3) Flux control coefficients do not have predictability, because they
only work with a steady-state mode. MCA is just a tool to describe the
control of metabolism. Predictions and in vivo activities are separate
issues and require a variety of non-linear models
 S ←→
E1
X ←→
E2
P
CCC
 Concentration control coefficients (CCC) are used to
define the effect of system parameters on the
concentrations of the intracellular metabolites
 CCCs determine how the concentration ci of the
intermediate Xj changes relative to the activity of the
enzyme Ei
 Since any intermediate concentration remains
unchanged if all the enzyme activities are changed by
the same factor, this means that for each K metabolite
the sum of concentration control coefficients must be
zero
 From this results that for each metabolite concentration at
least one enzyme exerts negative control
 Thus, C2X is usually negative since the metabolite’s X
concentration (c) decreases as the enzyme activity E2
increases
E  dc j  d ln c j
Xj
= i   = i ∈ {1,2,..., L} j ∈ {1,2,..., K }  Ei
Ci
 c j
 dc j 
 dEi  d ln Ei   
or  c j  i 
dE
v  dc j  d ln c j
Xj
Ci = i   = i ∈ {1,2,..., L} j ∈ {1,2,..., K } L

∑C =0 j ∈ { 1,2 ,...,K}
Xj
 c j  dvi  d ln vi i
i =1
 Elasticity
 As the control coefficients are systemic properties, the elasticities
are local enzyme properties (of the enzyme i )
 The most common elasticity coefficients indicate how the reaction rate
changes when the metabolite concentration has certain change
 The partial derivatives in the elasticity coefficients indicate that other
variables must be constants
 Elasticies have a positive value for the reaction stimulating
metabolites (e.g., activators and substrates), and negative value for
the reaction decelerating metabolites (product or inhibitor)
 Elasticity coefficients can be used to quantify the impact of a number
of substrates and / or products on the reaction rate

cj  ∂ν i  ∂ lnν i
ε i
=   = i ∈ { 1,2 ,...,L} j ∈ { 1,2 ,...,K}
 ∂c 
ν i  j  ∂ ln c j
Xj
 Elasticity, Response coefficient
 More broadly, Xj can be thought as an
effector (which need not be an
intermediate metabolite) which only
affects the reaction rate v
 In this case, one can calculate so-
called response coefficient to the
enzyme by multiplying the flux control
coefficient with the elasticity.
 If a metabolite or effector is affected
by more than one enzyme i, the
overall response coefficient is
calculated from the sum of the
enzyme responses.
 Although the enzyme i
elasticity εiXj in relation to a
= i εXj i, k ∈ { 1,2 ,...,L} j ∈ { 1,2 ,...,K}
Jk Jk i
metabolite or effector can R Xj C
be large, the flux’s Jk L

∑
RXkj = CiJ k ε Xi j
Jk
response R Xj to a change J
k ∈ { 1,2 ,...,L} j ∈ { 1,2 ,...,K}
is significant only when the i =1
flux control coefficient CJki
to the corresponding flux is
non-zero
Elasticity and flux control S ←→
E1
X ←→
E2
P

 The FCCs are generally small with enzymes having high

elasticity coefficients and vice versa
 E.g. if the activity of the enzyme E2 drops due to specific
inhibition, increases the concentration of S.
 If the enzyme E2 has large elasticity coefficient in relation
to S, then the above-mentioned concentration increase
will compensate the enzyme activity change. Thus,
reduction in enzyme E2 activity has little effect on the
steady-state flux, i.e. the disturbed enzyme has a small
flux control coefficient.
 Similarly, if an enzyme with a small elasticity coefficient
relative its metabolites is perturbed, significant change in
the flux is caused at the same time.
 Elasticity and connectivity theorem
 The interaction between flux control coefficients and elasticity is
described with the flux–control connectivity theorem
 Connectivity theorem is widely regarded as the MCA's main
theorem, as it brings together and helps to understand the impact of
the local enzyme kinetics to the global flux control

∑ i εXj = 0
C Jk i

i =1
k ∈ {1,2,..., L} j ∈ { 1,2 ,...,K}

S ←→ X ←→ P
E1 E2

 For the previous two-phase 2

C1J ε X2
reaction the connectivity
theorem gives the solution: ∑C
i=1
i
J
ε i
Xj =C ε +C ε = 0 ⇒ J = − 1
1
J 1
X
J
2
2
X
C2 εX
 Elasticity and concentration control
 Reactions which are close to thermodynamic equilibrium are very
sensitive to changes in metabolite concentrations that is, their
elasticity is large (and the flux control coefficients small)
 When the concentration control coefficients and elasticities are
related corresponding formulas of connectivity theorem can be
formed
 In general, CCCs have perhaps less attention, but they are important
when the results of the MCA are to be used for enzyme amplifications
 Parameterp ∂ν
elasticity
∂ lnν
coefficient π, is used to describe enzymatic
π =
sensitivity
i l
= any other
toi
i ∈ { 1,2 ,...,L}
i
parameter than the pathway metabolite
pl
ν ∂p ∂ ln p
i l l

∑ i ε X j =0
C
i=1
Xm i
j , m∈{ 1,2 ,...,K} j ≠ m (different_metabolites)

or
L

∑C ε Xi = -1 j ∈{ 1,2 ,...,K}
Xj
i j
(same metabolites)
i=1
Generalization of MCA theorems

C = P + E⋅C
J X

• Enzyme activity changes affect to the flux control CJ, both directly (P)
and indirectly through changes in the concentrations (E * CX)
• Parameter elasticity matrix , i.e. matrix P, is ​often a unit matrix
• The formula above presents a complete general formula for all MCA-
theorems
• The compact representation, including both the summation theorem and
the connectivity theorem and is also suitable for the analysis of branched
metabolic pathways
• MCA theory may be difficult to identify from the general representation
Determination of flux control coefficients

• Large FCCs refer to a

limiting phase, while small
FCCs refer to distribution of
flux control among many
phases
• Determination the derivative
of a non-linear function (the
slope of the tangent) utilizing
finite differences
• The effect of measurement
error (y-axis) can be
emphasized, when the
difference in x-axis is small
Determination of flux control coefficients

1) Direct Method: The FCCs are determined directly

from the flux and activity measurements after small
activity changes
2) The indirect method: the FCCs are calculated after
defining the elasticity coefficients
3) FCCs are defined from transient metabolite
concentration measurements
4) Flux and activity measurements after large activity
perturbations
1. Direct methods

• Experimentation, by determining:
• metabolite flux as a function of enzyme activity
• function slopes
• Change in the expression level with genetic engineering
(up or down)
• Increasing the gene dosage or regulatory promoters to
modulate gene expression
 1. Titrations (direct methods)
•Titration of cell-free extract with the purified enzyme.
•This can be done e.g. by varying the activities of the studied enzyme(s) in an
environment having an excess of other enzymes in the same pathway (the effect of
which is not examined).
•For example, glycolysis with an excess of aldolase, triose phosphate isomerase and
glycerol-3-phosphate dehydrogenase titrated with HK, PFK and G6PI
•Titration with a specific inhibitor. − cI ,max dJ
•If the enzyme-inhibitor response relationship is C = i
J

found out, the FCC may be calculated J dcI cI =0

•Must also be able to calculate a situation in
which cI=0, i.e. there is no inhibitor.
•Titration of the inhibitor is the most widely used
method for the determination of the FCC, e.g., the
examination of the isolated cells and mitochondrial
respiration.
•The inhibitor should also be specific for the
studied target
•The problem is extrapolation of the flux versus
inhibitor curve (usually non-linear effect of the Comparison is made between the simulated dependence of the respiration
inhibitor) to zero concentration to determine the rate on the degree of inhibition of particular steps, and the titration curves
for these steps obtained experimentally by addition of increasing amounts
slope dJ/dcI at the beginning of the curve of specific inhibitors, Biochem. J. (1996) 319, 143-148
 2. Indirect methods, modulation
GPI
• Indirect: the elasticity is determined
experimentally first, and then the control reaction :... → G6P →F6P →...,
coefficients are calculated using MCA reaction rate depends on concentrations :
theory
• Double modulation: chemostat ∂vGPI ∂v
dJ = dcG 6 P + GPI dcF 6 P
experimental perturbations min. two ∂cG 6 P ∂cF 6 P
different external glucose level
perturbations and measure the scaled to steady state:
concentrations of the two metabolites
and the steady-state fluxes (J). d ln J = ε GGPI
6 P d ln cG 6 P + ε F 6 P d ln c F 6 P
GPI

• Then with the flux in relation to

concentrations can be calculated to
solve the elasticity coefficients following must be true in
• Linearly dependent pair of equations perturbation phases 1 and 2:
must not be formed
• If not successful, the pathway modulation above
and below the examined point may help
• Single-modulation, if one of the d ln cG1 6 P d ln cG2 6 P
elasticity coefficients is known ≠
d ln c1F 6 P d ln cF2 6 P
 2. Indirect methods, top-down method

 Reactions grouping, focusing the main

groups
group1 group2
 Division into groups (segments) that S → X → P
have one common intermediate. The
summation theory applies to flux J
Cgroup1 + C group 2 = 1
J
control coeffients
 Similarly, the elasticities can be cX ∂v group ,i
brought in relation to the metabolites ε group ,i
= i ∈ {1,2}
∂c X
X
to describe the rate of the reaction of v group ,i
the group
 Connectivity theory applies
J
Cgroup ε
1 X
group1
+ C J
ε
group 2 X
group 2
=0
 Group elasticities can be calculated
from the modulation tests if:
 X is a single effector
 Cross-effects between the groups does not
exist otherwise than via X (this can easily
cause problems)
 2. Indirect methods, kinetic models
 A mathematical model of the enzyme vmax ⋅ S
reaction rate: v=
 to calculate the elasticity in relation to effectors and
substrate Km + S
 For example, elasticity calculation from the
Michaelis-Menten equation:
 the rates of reaction are calculated in the vicinity of
substrate concentration S
 make the graph of log (S) vs. log (v)
 Slope of the graph at the point S is the elasticity
 The slope does not change even if the vmax
changes, so in fact, only knowledge of Km
values ​are needed to calculate the elasticity
 Uncertainty:
 Are the right effectors included?
 Comparing the in vitro measurement to the in vivo
conditions
2. Indirect methods, kinetic models
• If a complete kinetic model of a biochemical system is
known, MCA concept may not be needed
• However, it may be useful to know the MCA coefficients
calculated with the kinetic model. The coefficients can be
used to evaluate quantified changes in fluxes in various
situations
• Model robustness is desirable for predictions
• Also, only qualitatively correct description of biochemical
systems may be sufficient
 3. Transient measurements
L
 v (t ) 
• 4 assumptions:
1) External metabolite-pool does not affect the kinetics of ∑
i =1
C iJ  i  = 1
 Ji 
pathway
2) The linear approximation of the enzyme kinetics is vi (t) is transient flux in reaction i
adequate around the steady-state point over a wide
range of metabolite concentrations and J i is steady state flux.
3) Must be theoretically possible to determine the Same integrated :
transient fluxes for each reaction with the metabolite
concentrations measurements K
4) The metabolites are homogeneously distributed to the
system
∑α ((c (t) − c (0)) = t
j=1
j j j

• αj- factors can be calculated from the regression fit of the

metabolite transient concentration measurements, e.g. FCCs relate to α j values
after a pulse addition to a steady state chemostat
• αj coefficients are used to calculate the FCCs in a linear pathway :
• Least-square fit is not always possible if the metabolite ( C1J C J2 ...C LJ ) = (α1 α 2 ...α K )G * J
restrictions form some linear combinations
• A criticism has been against the assumption of linear in a branched pathway :
reaction kinetics, but the performance may be better if one
uses the logarithm of the metabolite concentrations C J = (α1 α 2 ...α K )G * J
 4. Theory of large deviations
• MCA theory calculations are intended for
very small changes, but on the other hand
monitoring the measurement errors from
the test results is difficult
• However, the practical experiments are
carried out using finite changes
• The flux control coefficient can be calculated
from the derivative of the flux as a function
of the activity
∂J ∆J
• When the stationary point is at the low ≠
∂E ∆E
activity the value of the flux control
coefficients is large, and when the activity is
added the relative change in flux rate and
flux control coefficients are reduced
• When given a finite change in enzyme
activity the calculated flux control coefficient
value is inaccurate
• Thus, one needs a way to approximate the
correct flux control coefficient for finite tests
• There is an expansion of MCA theory for this
purpose which is not described here
 MCA of linear pathways
• L = the number of enzymatic reactions
• L-1 = number of metabolites in the linear pathway
• Only one flux, and it is consistent with all reaction rates in the steady-state
• Also L pieces of FCCs which can be determined with L-1 connectivity theory for L-1
metabolite, which, together with the summation theory, enable the calculation of the
flux control coefficient from the elasticity coefficients
• Similarly, one obtains CCCs
• Both control coefficients (FCCs and CCCs) can be expressed in a matrix form (number
of equations = L2 )
• If the elasticity matrix is ​non-singular control coefficients can be calculated with
a matrix inversion FCC CCC

 1 1  1  C1J − C1X 1  − C1X L−1   1 0  0

 1    
 ε X1 ε X2  ε XL1  C2J − C2  − C2   0
X1 X L−1
1  0
=
1
             
 1   
ε X ε X2  ε X L−1  C LJ − C L  − C L   0
X L−1 
0  1 
L X1
 L−1 L −1
MCA of linear pathways
S ←→ X ←→
• Two-phase reaction in terms of X E1 E2
• Generally, the elasticity of the P
reaction is negative with respect to
the product and positive with respect
to the substrate (denominators of the
equation positive, so the FCCs  1 1  C1J − C1X   1 0 

positive here)  ε 1 ε 2  C J =
X  0 1
− C2  

• Sharing of the flux control will depend  X X  2 
on the elasticity coefficients
• high elasticity -> small flux control, and vice
solved :
versa
 ε X2 1 
• Only if the elasticity of the first  
reaction has absolute value of 0, flux
control coefficients values ​are 1
 C1J X
C1   ε X2 − ε 1X εX −εX
2 1 
 =
 CJ X  
(FCC1) and 0 (FCC2). C2   − ε X −1 
1
• However, in general, the reactions  2
 2 ε X2 − ε 1X 
 εX −εX
1
has always a little elasticity
  ε X2 1 
 
 C1J C1X   ε X2 −ε 1X ε X2 −ε 1X 
 J =
MCA of linear pathways 
 C2
X  
C2  −ε 1X
 2 1
−1 

 ε X −ε X ε X2 −ε 1X 

• If one assumes that the elasticity of the reaction 1 is

negative and the elasticity of the reaction 2 is positive in
relation to the product (X) one may deduce:
• CCC for the reaction 1 is positive: metabolite X concentration
increases as the rate of the reaction 1 increases
• CCC for the reaction 2 is negative: metabolite X concentration
decreases as the rate of the reaction 2 increases
• provided at least one of elasticity coefficient is high, the CCCs are
small (and vice versa). That is, if the reactions are elastic with respect
to the metabolite concentrations, the reaction rate changes has little
effect on the metabolite concentrations
 MCA and penicillin biosynthesis
• The elasticity coefficients of ACVS
and IPNS enzymes and ACV (the
inhibitor and the product of the first
and the substrate for the second
reaction):
1. ACV, ACVS and IPNS measurements
2. rACV and rIPN calculated from the kinetic
models
3. εACVACVS and εACVIPNS calculated from the
partial derivatives of the kinetic models
4. Flux control coefficients CJACVS and CJIPNS
calculated from the elasticities
• εACVACVS is negative (feedback
inhibition)
• A fed-batch experiment with pseudo-
steady-state assumption
MCA and penicillin biosynthesis
• ACV accumulation increases during the fed-batch cultivation
• ACV concentration growth decreases its synthesis (ACVS)
activity and the absolute value of the elasticity increases
• IPNS elasticity is positive, which means that increase of ACV
increases activity of synthesis, but activity growth decreases
with increasing concentration of ACV and IPNS saturates

100 150
MCA and penicillin biosynthesis
• Elasticity coefficients used to
calculate the flux control
coefficients (FCC)
• At the beginning the flux control
of ACVS is high (almost 1)
when the ACV has no inhibitory
effect
• As the ACV concentration
increases the flux control
moves to IPNS
• Neither of enzymes is a
reaction rate-limiting and the
FCCs are not constant in the
pathway under consideration
 MCA of branched pathways
• The control more complex
• Any enzyme reaction in the
network may affect the flux of
each branch, which needs its
own FCC matrix
• If one causes a perturbation
which changes J2 and J1 J1 = J 2 + J 3
remains constant, must J3 1 = f12 + f13
also change. in which
• This leads to the FCC J
f1k = k k ∈ ( 2 ,3 )
restriction: J1
− f12C3J1 + f13C2J1 = 0
 MCA of branched pathways
• Combined with the
summation and connectivity
theory the above-mentioned  C1J1   − f12ε X2 − (1 − f12 )ε X3 
restiction allows for the  J  1  
calculation of flux control  C2 1  = 1  f12ε X
1

 C J1  ε X − f12ε X − (1 − f12 )ε X
2 3

coefficients with the elasticity  (1 − f12 )ε 1X 

 3   
coefficients and one relative
flux rate
• Reference flux (constant)
was J1 , but corresponding (1 − f12 ) ⋅ C1J 2 + 1 ⋅ C3J 2 = 0
structural relations of the
FCC in other parts of flux f12 ⋅ C1J 3 + 1 ⋅ C2J 3 = 0
applies.
• A general matrix form can be
built for the determination of
control coefficients from  CinJ 
elasticity coefficients, ( LJF − E) X  = P
parameter elasticities and C 
partial fluxes through various
branches
Gluconeogenesis vs. glycolysis
• Groen et al. 1983, rat liver
cells, all relevant elasticity
coefficients measured
from gluconeogenesis, at
high lactate and pyruvate
concentrations.
• Elasticities were
determined by either
enzyme kinetics or
single/double- modulation
chemostat experiments
Gluconeogenesis vs. glycolysis
v f ,max cS / K S − vr ,max cP / K P
v = v f − vr =
• Enzyme kinetic determination 1 + cS / K S + c P / K P
was carried out with the inverse
cS ∂v 1 vf
of the Michaelis-Menten kinetics ε =
v
= −
v ∂cS 1 − Γ / K eq v f ,max
S
(f = forward, r = reverse)
• Partial differentiating gave the
cP ∂v 1 v
elasticities ε Pv = = − r
• Depending upon the ratio Γ/Keq, v ∂cP 1 − Γ / K eq vr ,max
elasticity terms form missä
• mainly due to saturation degree of the
enzyme with substrate and product c cP ,eq v f ,max K P
(Γ/Keq <<1) and the second term of the Γ= P ja K eq = =
equation is the dominant component or cS cS ,eq vr ,max K S
• when close to the equilibrium, the first
part of the (Γ/Keq ≈1) equation for the vr Γ
elasticity is defining
=
v f K eq
Gluconeogenesis vs. glycolysis
• Measurement of pyruvate in the cytosol and in mitochondria showed
that the Γ/Keq ≈ 0.86, thus, this step acts close to equilibrium
• Pyruvate carboxylase (PYR-> OAA) operates far from equilibrium
• The elasticity of oxaloacetate transport reaction was determined by
double-modulation. By varying the substrate (lactate) concentration or
by inhibiting PEP-carboxykinase (4) with the mercapto picolinic acid the
flux could be varied. The elasticity coefficients were determined from
oxaloacetate concentration variations
 Gluconeogenesis vs. glycolysis
• Flux control coefficients were calculated from elasticity coefficients
• Flux control coefficients are quite large in a combined route of PEP-> F1, 6BP, which was supposed to
be in equilibrium
• It is therefore not possible to draw a general conclusion that the balance of the reactions do not reflect
the flux control
• The highest FCC value is with pyruvate carboxylase reaction (note this has also the lowest elasticity),
and this reaction is certainly the key reaction of gluconeogenesis and this enzyme had a greatest
impact on flux control.
• PEP carboxy kinase has very low FCC (this has to be due to the tight linkage with pyruvate carboxylase
reaction having high FCC)
• Standard deviations ranged from 50-275%, respectively.
 Oxidative phosphorylation
 Analysis of oxidative
phosphorylation with the
isolated mitochondria
 The three reaction groups
connected by a proton motive
force ∆p (top-down approach
fits well)
 The perturbations with the
proton uncoupler (transports
protons across mitochondrial
membrane) and malonate
(which inhibits the respiratory
chain)
 Each group elasticities with
respect to the proton motive
force ∆p was determined by
single modulation
 Oxidative phosphorylation
 The elasticity coefficients in the non-
phosphorylating conditions for the
proton leakage (7.9) and the respiration
(-18.7) were obtained by linear ε ∆resp
p = −18.7 ε ∆leak
p = 7.9
regression of the experiment data (e,g,
respiratory flux vs. ∆p) and corresponding FCCs :
 The proton leak has a lot of importance
= 0.30 = 0.70
Jresp J
resp
under these circumstances and the Cresp Cleak
proton motive force affects more to flux
control than the transfer force
generation in respiration
 Flux control coefficients vary greatly
according to the circumstances
 In respiratory flux (Table 11.4, different
test), all of the flux controls are positive
and in non-phosphorylating conditions,
the flux control is mainly in the
processes that remove the proton
motive force. Phosphorylation and
leakage self-regulation are the largest
Summary
• The analysis is based on the elasticity coefficient (ε) and the
control coefficients (FCC or CCC) values.
• The effect of the individual reactions (as well as the entire pathway or
network) can be presented on the flux rate and the concentrations.
• Due to the normalization the sum of flux control coefficients is one.
• The sum of the concentration control coefficients must be zero.
• The control coefficients are systemic properties, while the elasticities
are local properties of an enzyme.
• The control coefficients are generally small for the enzymes with high
elasticity coefficients, and vice versa
• The connectivity theory is widely regarded as the MCA's main
theorem, as it combines (and helps to understand) local enzyme
kinetics impact to the global flux control