Engineering of Biological Processes Lecture 5: Control of metabolism Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University.

Engineering of Biological Processes

Lecture 5: Control of metabolism

Mark Riley, Associate ProfessorDepartment of Ag and Biosystems

EngineeringThe 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

[AMP][ADP][ATP]

[ADP]21

[ATP]chargeEnergy

High energy charge means the cell has a lot of energy

Low energy charge means the cell has little energy

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

Types of feedback control

1) Sequential feedback control

A → B → C

D → E → Y

F → G → Z

Inhibited by Y

Inhibited by Z

Types of feedback control

2) Enzyme multiplicity

A B → C

D → E → Y

F → G → Z

Inhibited by Y

Inhibited by Z

Inhibited by Z

Inhibited by Y

Types of feedback control

3) Concerted feedback control

A → B → C

D → E → Y

F → G → Z

Inhibited by Y

Inhibited by Z

Inhibited by Y+Z

Types of feedback control

4) Cumulative feedback control

A → B → C

D → E → Y

F → G → Z

Inhibited by Y

Inhibited by Z

Inhibited by Y or Z

Glucose Glucose 6-Phosphate

Fructose 6-Phosphate

Fructose 1,6-Bisphosphate

Glyceraldehyde 3-Phosphate

Pyruvate

Acetate Acetyl CoA

Citrate

-Ketoglutarate

Succinate

Fumarate

Oxaloacetate

Phosphogluconate

Glyceraldehyde 3-Phosphate

Acetaldehyde

2-Keto-3-deoxy-6-phosphogluconate

Glyceraldehyde 3-Phosphate

+Pyruvate

Lactate

Ethanol

Malate Isocitrate

CO2+NADHFADH2

CO2+NADH

NADH

NADH

GTP

GDP+Pi

Phosphoenolpyruvate

PFK = phosphofructokinase

Fructose 6-Phosphate + ATP Fructose 1,6-Bisphosphate + ADP + Pi

PFK = phosphofructokinase

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

Fructose 6-Phosphate

Fructose 1,6-Bisphosphate

Glyceraldehyde 3-Phosphate

Pyruvate

Acetate Acetyl CoA

Citrate

-Ketoglutarate

Succinate

Fumarate

Oxaloacetate

Phosphogluconate

Glyceraldehyde 3-Phosphate

Acetaldehyde

2-Keto-3-deoxy-6-phosphogluconate

Glyceraldehyde 3-Phosphate

+Pyruvate

Lactate

Ethanol

MalateIsocitrate

CO2+NADHFADH2

CO2+NADH

NADH

NADH

GTP

GDP+Pi

Phosphoenolpyruvate

Pyruvate dehydrogenase

Pyruvate + NAD+ + CoA Acetyl CoA + CO2 + NADH

Pyruvate dehydrogenase

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

A B CE1

E2

E3

E4D

Amplification of control signals• Fluxes can be amplified, activities

cannot.

• Substrate cycles – separate enzymes catalyze forward vs. reverse reactions

A B CE1

E2

E3

E4 D

Flux

• Flux = rate of reaction

F = r = dC = vmax C

dt Km + C

Fluxtot = F2 – F3

A B CE1

E2

E3

E4

F2 = r2 = vmax2 B

Km2 + B

B to C

F3 = r3 = vmax3 C

Km3 + C

C to B

D

Amplification of control signals

PFK (phosphofructokinase) and

FBP (fructose 1,6 bisphosphatase)

Fructose 6-phosphate Fructose 1,6-bisphosphatePFK

FBP

ATPADP

Pi

Effect of AMP (adenosine monophosphate)

• Activity of PFK is increased by AMP• Activity of FBP is decreased by AMP

AMP concentration Fractional saturation (binding to PFK, FBP)

0 0

2.5 0.093

12.5 0.89

PFK

AMP

PFK

AMP

PFK

AMP

PFK

AMP

PFK

AMP

PFK

PFK

AMP

PFK

AMP

PFK

AMP

PFK

AMP

PFK

AMP

Enzyme activity as a function of bound AMP

0

20

40

60

80

100

0 0.2 0.4 0.6 0.8 1

Fraction of AMP bound

En

zym

e ac

tivi

ty m

M /

min PFK activity

FPB activity

-20

0

20

40

60

80

100

0 0.2 0.4 0.6 0.8 1

Fraction of AMP bound

Net

flu

x m

M /

min

Net Flux

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 “multi-input” 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 Relaxation time

Hexokinase 1100 sec

PFK 75 sec

DPGP 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

P1 P2

I

S

Flux1 Flux2

Fluxtot

Fluxtot = F1 + F2

Flux1 = r1 = vmax1 S

Km1 + S

Flux2 = r2 = vmax2 S

Km2 + S

To funnel substrate through branch 1, do we want:

Km1 < Km2

or,

Km1 > Km2 ???

Some definitions

Ftot = vmax1 S

Km1 + S

+ vmax2 S

Km2 + S

Total flux

Selectivity

F1

F2 vmax2 S

Km2 + S

vmax1 S

Km1 + S=

Selectivity

SK

SK

vmax

vmax

r

r

m1

m2

2

1

2

1

So, to enhance r1, we want a small value of Km1

These two curves have the same vmax, but their Km values

differ by a factor of 2.

0

5

10

15

20

0 10 20 30 40 50

[S]

r

Low Km High Km

r1 = vmax1 S

Km1 + S

Low Km will be the path with the higher flux (all other factorsbeing equal).

Low Km also means a strong interaction between substrate and enzyme.

Michaelis Menten kinetics