Download Control of metabolism

Engineering of Biological
Processes
Lecture 5: Control of metabolism
Mark Riley, Associate Professor
Department of Ag and Biosystems
Engineering
The University of Arizona, Tucson, AZ
2007
Objectives: Lecture 5
• Understand how metabolism is
controlled
• Model these reactions to shift carbon
and resources down certain paths
Control of overall rate of
metabolism
• Highly regulated process
• Controlled by
–
–
–
–
–
–
feedback mechanisms on enzymes
inhibited by products
stimulated by reactants
energy charge
oxygen concentration
environmental factors
• temperature, CO, some antibiotics
Metabolic processes
are controlled by
• The flow of metabolism is determined primarily by the
amount and activities of enzymes
– substrate amounts have a smaller effect
• Covalent modification
– regulatory enzymes are turned on or off by phosphorylation
(PO3)
– small triggering signals have a large effect on overall rates
• Reversible reactions are potential control sites
• Compartmentation
– glycolysis, fatty acid metabolism, and pentose phosphate
pathway in cytosol
– fatty acid oxidation, citric acid cycle, and oxidative
phosphorylation take place in mitochondria
Energy charge
1
[ATP]  [ADP]
2
Energy charge 
[ATP]  [ADP]  [AMP]
High energy charge means the cell has a lot of energy
Low energy charge means the cell has little energy
Control points
identification of enzymes
• Enzymes
– present at low enzymatic activity
• either low concentration or low intrinsic activity
– catalyze reactions that are not at equilibrium (under
normal conditions)
– usually catalyze slow reactions (rate-determining)
– often found at major branch points
• downstream end
– entryway into reaction that has the highest flux
Types of feedback control
1) Sequential feedback control
Inhibited by Y
D→E →Y
A→B →C
F→G→ Z
Inhibited by Z
Types of feedback control
2) Enzyme multiplicity
Inhibited by Y
D→E →Y
Inhibited by Y
A
B →C
Inhibited by Z
F→G→ Z
Inhibited by Z
Types of feedback control
3) Concerted feedback control
Inhibited by Y
Inhibited by Y+Z
D→E →Y
A→B →C
F→G→ Z
Inhibited by Z
Types of feedback control
4) Cumulative feedback control
Inhibited by Y
Inhibited by Y or Z
D→E →Y
A→B →C
F→G→ Z
Inhibited by Z
PFK = phosphofructokinase
Glucose
Glucose 6-Phosphate
Phosphogluconate
2-Keto-3-deoxy-6phosphogluconate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Lactate
GlyceraldehydeGlyceraldehyde
Glyceraldehyde 3-Phosphate
3-Phosphate
3-Phosphate
Phosphoenolpyruvate
+
Pyruvate
Acetaldehyde
Pyruvate
NADH
Ethanol
Acetate
Acetyl CoA
Citrate
Oxaloacetate
NADH
Isocitrate
CO2+NADH
a-Ketoglutarate
GTP
Malate
Fumarate
Succinate
FADH2
GDP+PiCO2+NADH
PFK = phosphofructokinase
Fructose 6-Phosphate + ATP
Fructose 1,6-Bisphosphate + ADP + Pi
Phosphofructokinase (PFK) allosteric enzyme
activated by ADP and Pi, but inhibited by ATP.
When [ATP] is high, PFK is turned off, effectively
shutting down glycolysis.
Allosteric = binding of one compound impacts the
binding of other compounds
Michaelis-Menten kinetics do not readily apply
Pasteur effect
• Rate of glycolysis under anaerobic (low O2)
conditions is higher then under aerobic (high O2).
• Carbohydrate consumption is 7x higher under
anaerobic conditions.
• Caused by inhibition of PFK by citrate and ATP
Glucose
Glucose 6-Phosphate
2-Keto-3-deoxy-6phosphogluconate
Phosphogluconate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
Glyceraldehyde
3-Phosphate
Phosphoenolpyruvate
Glyceraldehyde
3-Phosphate
+
Pyruvate
Acetaldehyde
Lactate
Pyruvate
NADH
Ethanol
Acetate
Pyruvate dehydrogenase
Acetyl CoA
Citrate
Oxaloacetate
NADH
Isocitrate
Malate
CO2+NADH
a-Ketoglutarate
Fumarate
GTP
Succinate
FADH2
GDP+Pi
CO2+NADH
Pyruvate dehydrogenase
Pyruvate + NAD+ + CoA
Acetyl CoA + CO2 + NADH
Pyruvate dehydrogenase (PDH) assemblage of 3 enzymes that each
catalyze one step in the overall reaction above.
PDH is inhibited by
products (acetyl CoA, NADH),
feedback regulation by nucleotides (ATP, GTP)
reversible phosphorylation (a PO3- is added to a serine residue).
phosphorylation is enhanced by a high energy charge.
Activated by AMP, ADP, NAD+
Flux vs. activity
• Activity – how quickly one enzyme
catalyzes one reaction
• Flux – overall rate of mass converted
forward and reverse reaction
E2
E1
A
B
C
E3
E4
D
Amplification of control signals
• Fluxes can be amplified, activities
cannot.
• Substrate cycles – separate enzymes
catalyze forward vs. reverse reactions
E2
E1
A
B
C
E3
E4
D
Flux
• Flux = rate of reaction
F = r = dC = vmax C
dt
Km + C
E2
E1
A
B
C
E4
D
E3
Fluxtot = F2 – F3
B to C
C to B
F2 = r2 = vmax2 B
F3 = r3 = vmax3 C
Km2 + B
Km3 + C
Amplification of control signals
PFK (phosphofructokinase) and
FBP (fructose 1,6 bisphosphatase)
ATP
ADP
PFK
Fructose 6-phosphate
Fructose 1,6-bisphosphate
FBP
Pi
Effect of AMP (adenosine
monophosphate)
• Activity of PFK is increased by AMP
• Activity of FBP is decreased by AMP
PFK
AMP concentration
Fractional saturation
(binding to PFK, FBP)
0
0
AMP
PFK
2.5
0.093
PFK
AMP
AMP
PFK
PFK
AMP
PFK
AMP
12.5
0.89
PFK
AMP
PFK
AMP
PFK
AMP
PFK
AMP
PFK
AMP
Enzyme activity as a function of
bound AMP
Enzyme activity mM / min
100
PFK activity
FPB activity
80
60
40
20
0
0
0.2
0.4
0.6
Fraction of AMP bound
0.8
1
100
Net Flux
Net flux mM / min
80
60
40
20
0
0
0.2
0.4
0.6
-20
Fraction of AMP bound
0.8
1
Effect of the substrate cycle
A 440-fold increase in flux (87.9 / 0.2)
results from
a 5-fold change in [AMP] (12.5 / 2.5).
This corresponds to 0.9 / 0.1 bound.
Design of an optimal catalyst
•
•
•
•
Which pathways are active?
Which is the slow step?
Which steps are highly regulated?
How do we funnel resources toward the
desired product?
Steps in metabolic analyses
• 1) Develop a model of metabolism
– Observe pathways
– Measure flux through key reactions
– Identify slow steps
• 2) Introduce perturbations
– Alter enzyme activity
• Changing substrate
• Vary concentrations of substrate
• Other activators / inhibitors
– Determine fluxes after relaxation
• New steady state
• 3) Analyze flux perturbation results
– Are branches rigid?
– Do changes in upstream flux impact split ratio or flux?
Basis of metabolic control
• Pacemaker Enzymes
– Regulation is accomplished by altering the activity of at least one
pacemaker enzyme (or rate-determining step) of the pathway.
• Identification of a Pacemaker Enzyme
– Normally it has a low activity overall,
– Is subject to control by metabolites other than its substrates,
– Often positioned as the first committed step of a pathway,
directly after major branch points, or at the last step of a “multiinput” pathway.
– Needs confirmation of the in vivo concentrations of the enzyme’s
substrate(s) and product(s).
Identify slow steps
• For fast reactions,
the concentration of
substrates and
products are
essentially at
equilibrium
• The role of “fast
reactions” in control
is low
Enzyme
Hexokinase
PFK
DPGP
Relaxation time
1100 sec
75 sec
34,000 sec
Pyruvate kinase
28 sec
Lactate
dehydrogenase
0.01 sec
Change enzymes
• Inhibit (destroy) a native enzyme
– Knockout
• Enhance the concentration of a native
enzyme
• Introduce a new enzyme
– Different species
– Used to permit utilization of new substrates
• C sources (5-ring sugars vs. 6-ring sugars)
Apparent Km values and their effect
S
Fluxtot
I
Flux1
P1
Flux2
Km1 < Km2
or,
P2
Fluxtot = F1 + F2
Flux1 = r1 = vmax1 S
Km1 + S
To funnel substrate
through branch 1, do
we want:
Km1 > Km2 ???
Flux2 = r2 = vmax2 S
Km2 + S
Some definitions
Total flux
Ftot
= vmax1 S
+ vmax2 S
Km1 + S
Km2 + S
Selectivity
vmax1 S
F1
F2
=
Km1 + S
vmax2 S
Km2 + S
Selectivity
r1 vmax 1  Km2  S 



r2 vmax 2  Km1  S 
So, to enhance r1, we want a small value of Km1
Michaelis Menten kinetics
20
r1 = vmax1 S
15
Km1 + S
r
Low Km will be the
path with the higher
flux (all other factors
being equal).
10
5
0
0
10
20
30
40
50
[S]
Low Km also means a
strong interaction
between substrate
and enzyme.
Low Km High Km
These two curves
have the same vmax,
but their Km values
differ by a factor of 2.