Engineering of Biological Processes Lecture 5: Control of metabolism

1. 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

2. Objectives: Lecture 5 • Understand how metabolism is controlled • Model these reactions to shift carbon and resources down certain paths

3. 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

4. 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

5. Energy charge High energy charge means the cell has a lot of energy Low energy charge means the cell has little energy

6. 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

7. D → E → Y A →B → C F → G → Z Types of feedback control 1) Sequential feedback control Inhibited by Y Inhibited by Z

8. 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

9. D → E → Y A →B → C F → G → Z Types of feedback control 3) Concerted feedback control Inhibited by Y Inhibited by Y+Z Inhibited by Z

10. D → E → Y A →B → C F → G → Z Types of feedback control 4) Cumulative feedback control Inhibited by Y Inhibited by Y or Z Inhibited by Z

11. NADH NADH CO2+NADH GTP CO2+NADH GDP+Pi FADH2 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 Lactate Pyruvate Acetyl CoA Acetate Ethanol Citrate Oxaloacetate Isocitrate Malate a-Ketoglutarate Fumarate Succinate

12. 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

13. 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

14. NADH NADH CO2+NADH GTP CO2+NADH GDP+Pi FADH2 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 Lactate Pyruvate Pyruvate dehydrogenase Acetyl CoA Acetate Ethanol Citrate Oxaloacetate Isocitrate Malate a-Ketoglutarate Fumarate Succinate

15. 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+

16. E2 E1 E4 A B C E3 Flux vs. activity • Activity – how quickly one enzyme catalyzes one reaction • Flux – overall rate of mass converted forward and reverse reaction D

17. E2 E1 E4 A B C E3 Amplification of control signals • Fluxes can be amplified, activities cannot. • Substrate cycles – separate enzymes catalyze forward vs. reverse reactions D

18. F= r = dC = vmax C dt Km + C Flux • Flux = rate of reaction

19. E2 B to C C to B E1 E4 A B F2 = r2 = vmax2 B F3 = r3 = vmax3 C C Km2 + B Km3 + C E3 D Fluxtot = F2 – F3

20. ATP ADP PFK Fructose 6-phosphate Fructose 1,6-bisphosphate FBP Pi Amplification of control signals PFK (phosphofructokinase) and FBP (fructose 1,6 bisphosphatase)

21. PFK PFK PFK PFK PFK PFK PFK PFK PFK PFK AMP AMP AMP AMP AMP AMP AMP AMP AMP Effect of AMP (adenosine monophosphate) • Activity of PFK is increased by AMP • Activity of FBP is decreased by AMP PFK AMP

22. Enzyme activity as a function of bound AMP

24. 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.

25. 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?

26. 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?

27. 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).

28. 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

29. 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)

30. S Fluxtot I Flux1 Flux2 P1 P2 Apparent Km values and their effect To funnel substrate through branch 1, do we want: Km1 < Km2 or, Km1 > Km2 ??? Fluxtot = F1 + F2 Flux1 = r1 = vmax1 S Flux2 = r2 = vmax2 S Km1 + S Km2 + S

31. F1 F2 + vmax2 S vmax2 S = Km2 + S Km2 + S Ftot = vmax1 S vmax1 S Km1 + S Km1 + S Some definitions Total flux Selectivity

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

33. r1 = vmax1 S Low Km High Km Km1 + S 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.