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 (PO 3 ) –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 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 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 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

GlucoseGlucose 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 CO 2 +NADH FADH 2 CO 2 +NADH NADH GTP GDP+P i Phosphoenolpyruvate PFK = phosphofructokinase

Fructose 6-Phosphate + ATP Fructose 1,6-Bisphosphate + ADP + P i PFK = phosphofructokinase Phosphofructokinase (PFK) allosteric enzyme activated by ADP and P i, 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 O 2 ) conditions is higher then under aerobic (high O 2 ). Carbohydrate consumption is 7x higher under anaerobic conditions. Caused by inhibition of PFK by citrate and ATP

GlucoseGlucose 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 CO 2 +NADH FADH 2 CO 2 +NADH NADH GTP GDP+P i Phosphoenolpyruvate Pyruvate dehydrogenase

Pyruvate + NAD + + CoA Acetyl CoA + CO 2 + 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 PO 3- 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 ABC E1E1 E2E2 E3E3 E4E4 D

Amplification of control signals Fluxes can be amplified, activities cannot. Substrate cycles – separate enzymes catalyze forward vs. reverse reactions ABC E1E1 E2E2 E3E3 E4E4 D

Flux Flux = rate of reaction F = r = dC = v max C dt K m + C

Flux tot = F 2 – F 3 ABC E1E1 E2E2 E3E3 E4E4 F 2 = r 2 = vmax 2 B K m2 + B B to C F 3 = r 3 = vmax 3 C K m3 + C C to B D

Amplification of control signals PFK (phosphofructokinase) and FBP (fructose 1,6 bisphosphatase) Fructose 6-phosphateFructose 1,6-bisphosphate PFK FBP ATP ADP PiPi

Effect of AMP (adenosine monophosphate) Activity of PFK is increased by AMP Activity of FBP is decreased by AMP AMP concentrationFractional saturation (binding to PFK, FBP) PFK AMP PFK AMP PFK AMP PFK AMP PFK AMP PFK AMP PFK AMP PFK AMP PFK AMP PFK AMP

Enzyme activity as a function of bound AMP

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 enzymes 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 EnzymeRelaxation time Hexokinase1100 sec PFK75 sec DPGP34,000 sec Pyruvate kinase28 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 K m values and their effect P1P1 P2P2 I SFlux 1 Flux 2 Flux tot Flux tot = F 1 + F 2 Flux 1 = r 1 = vmax 1 S K m1 + S Flux 2 = r 2 = vmax 2 S K m2 + S To funnel substrate through branch 1, do we want: K m1 < K m2 or, K m1 > K m2 ???

Some definitions F tot = vmax 1 S K m1 + S + vmax 2 S K m2 + S Total flux Selectivity F1F1 F2F2 vmax 2 S K m2 + S vmax 1 S K m1 + S =

Selectivity So, to enhance r 1, we want a small value of K m1

These two curves have the same v max, but their K m values differ by a factor of 2. Low K m High K m r 1 = vmax 1 S K m1 + S Low K m will be the path with the higher flux (all other factors being equal). Low K m also means a strong interaction between substrate and enzyme. Michaelis Menten kinetics