Engineering of Biological Processes Lecture 5: Control of metabolism

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Engineering of Biological ProcessesLecture 5
Control of metabolism

• Mark Riley, Associate Professor
• Department of Ag and Biosystems Engineering
• The University of Arizona, Tucson, AZ
• 2007

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Objectives Lecture 5

• Understand how metabolism is controlled
• Model these reactions to shift carbon and
  resources down certain paths

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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

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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

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Energy charge
High energy charge means the cell has a lot of
energy Low energy charge means the cell has
little energy
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Control pointsidentification 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

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Types of feedback control

• 1) Sequential feedback control

Inhibited by Y
Inhibited by Z
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Types of feedback control

• 2) Enzyme multiplicity

Inhibited by Y
D ? E ? Y
Inhibited by Y
A B ? C
F ? G ? Z
Inhibited by Z
Inhibited by Z
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Types of feedback control
3) Concerted feedback control
Inhibited by Y
Inhibited by YZ
Inhibited by Z
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Types of feedback control
4) Cumulative feedback control
Inhibited by Y
Inhibited by Y or Z
Inhibited by Z
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PFK phosphofructokinase
2-Keto-3-deoxy-6- phosphogluconate
Glucose
Glucose 6-Phosphate
Phosphogluconate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
Glyceraldehyde 3-Phosphate Pyruvate
Glyceraldehyde 3-Phosphate
Phosphoenolpyruvate
Acetaldehyde
Pyruvate
Lactate
Acetyl CoA
Acetate
Ethanol
Citrate
Oxaloacetate
Isocitrate
Malate
a-Ketoglutarate
Fumarate
Succinate
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PFK phosphofructokinase
Fructose 1,6-Bisphosphate ADP Pi
Fructose 6-Phosphate ATP
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
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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

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2-Keto-3-deoxy-6- phosphogluconate
Glucose
Glucose 6-Phosphate
Phosphogluconate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
Glyceraldehyde 3-Phosphate Pyruvate
Glyceraldehyde 3-Phosphate
Phosphoenolpyruvate
Acetaldehyde
Pyruvate
Lactate
Pyruvate dehydrogenase
Acetyl CoA
Acetate
Ethanol
Citrate
Oxaloacetate
Isocitrate
Malate
a-Ketoglutarate
Fumarate
Succinate
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Pyruvate dehydrogenase
Acetyl CoA CO2 NADH
Pyruvate NAD CoA
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
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Flux vs. activity

• Activity how quickly one enzyme catalyzes one
  reaction
• Flux overall rate of mass converted forward and
  reverse reaction

D
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Amplification of control signals

• Fluxes can be amplified, activities cannot.
• Substrate cycles separate enzymes catalyze
  forward vs. reverse reactions

D
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Flux

• Flux rate of reaction

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D
Fluxtot F2 F3
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Amplification of control signals
PFK (phosphofructokinase) and FBP (fructose 1,6
bisphosphatase)
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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
2.5 0.093
12.5 0.89
AMP
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Enzyme activity as a function of bound AMP
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(No Transcript)
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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.

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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?

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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?

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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 multi-input pathway.
• Needs confirmation of the in vivo concentrations
  of the enzymes substrate(s) and product(s).

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Identify slow steps
Enzyme Relaxation time
Hexokinase 1100 sec
PFK 75 sec
DPGP 34,000 sec
Pyruvate kinase 28 sec
Lactate dehydrogenase 0.01 sec

• For fast reactions, the concentration of
  substrates and products are essentially at
  equilibrium
• The role of fast reactions in control is low

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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)

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Apparent Km values and their effect
To funnel substrate through branch 1, do we
want Km1 lt Km2 or, Km1 gt Km2 ???
Fluxtot F1 F2
Flux1 r1 vmax1 S
Flux2 r2 vmax2 S
Km1 S
Km2 S
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Some definitions
Total flux
Selectivity
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Selectivity
So, to enhance r1, we want a small value of Km1
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Michaelis Menten kinetics
Low Km will be the path with the higher flux (all
other factors being equal). Low Km also means a
strong interaction between substrate and enzyme.
These two curves have the same vmax, but their Km
values differ by a factor of 2.