Free energy

1. Important to recognize that metabolic need of individual cells is different from need of whole organism. Brain needs glucose even when body is starving. Liver will synthesize glucose via gluconeogenesis while brain uses glucose via glycolysis

3. Free energy A-B + C --- B-C + A If this reaction is displaced from equilibrium, some energy will be released as the reaction returns towards equilibrium. The energy released could be lost as heat or made to do work (ATP) Free energy (DG) of a chemical reaction is a measure of its capacity to do work. It is related to conc of substrate and product DG = -RTln[eqP]/[eqS]+RTln[initP]/[initS] If initial conc =1M, RTln[initP]/[initS]=0 You get DGo=-RTlnK At equilibrium free energy change is zero Higher the substrate conc, lower the product conc, greater the DG value.

4. Equilibrium At equilibrium, rate of forward and reverse reaction is identical A --- B No net flux in either direction A----- B---- At equilibrium No free energy change Change in free energy as a reaction proceeds towards equilibrium is the key driving force in biological processes. Equilibrium processes do not perform useful work (movement) Equilibrium processes can not be easily regulated Complex molecules (and processes) will not be made in large quantities under equilibrium conditions Biological systems obtain energy to prevent equilibriums from reaching.

5. Open and closed systems A-B + C --- B-C + A In a closed system, heat released will equal heat absorbed In an open system, heat released will be lost to environment, prevents the reaction from reaching equilibrium Furthermore, all free energy change is not lost as heat. Some is captured as chemical bond energy (ATP) A-B + C ---------- B-C + A / \ ADP+Pi ATP If all free energy change were captured as ATP, the reaction would be at equilibrium and there would be no net gain of ATP. However partial loss of energy as heat to the environment converts this reaction into a non equilibrium reaction allowing flux through the pathway towards ATP generation.

6. Dynamic steady state Living cells are NOT AT EQUILIBRIUM They maintain a DYNAMIC STEADY STATE Glucose enters cells, and CO2 leaves cell, but mass and composition of cell do not change appreciably. Cells appear to be but are not at equilibrium with surroundings. At molecular level each metabolic pathway is unidirectional and functioning. Rate of metabolic flow (flux) through the pathway is high, but concentration of substrate/intermediates/products remains constant. v1 v2 A------------S-------- P v1=v2 If this steady state is disrupted, by external change in energy etc, temporarily the fluxes through the pathway will change and regulatory mechanisms will be triggered and the organism will arrive at a new steady state to achieve homeostasis. Direction of flux in a pathway is dictated by position of equilibrium at each step of pathway

7. Steady state Steady State- all flows/fluxes are constant and unchanging. Living systems try and maintain a steady steady. An open system at steady state is at maximum thermodynamic efficiency. Changes in fluxes/flow require changes, which require energy. Changes in the environment result in perturbation of the steady state, and the organism responds and re-obtains new steady state. How are process maintained in non-equilibrium state? Exchange of matter and energy between organism and environment Substrates are derived from environment, products are returned to environment (Metabolic processes only attain equilibrium at death)

8. Flux and equilibrium 10/sec 20/s ec 110/sec A<-------------B <----------------C <--------------------D 0.1/sec 10/sec 100/sec Some reactions are close to equilibrium other are obviously far from equilibrium All the reactions are sufficiently away from equilibrium so that the process is not at equilibrium While in theory all enzymes are regulated, activity of only certain enzymes regulate flux through the pathway Reactions are usually limited by the substrate (intermediate) conc. Therefore: Function of enzyme- Catalysis AND Monitoring state of pathway Via conc of substrates/products)

10. In this simple pathway, the intermediate B has two alternative fates. To the extent that reaction B → E draws B away from the pathway A → D, it controls that pathway.

11. Enzymes and Equilibrium Almost all steps in a pathway lose heat energy and are therefore displaced slightly from equilibrium. Most enzyme catalyzed reactions in metabolism operate close to equilibrium. In a few cases, they are not close to equilibrium. (Non equilibrium enzymes possess low catalytic activity so substrates accumulate and they will limit flux through pathway) These reactions will generate the greatest free energy change and produce the most work River—dam—sluice gate—work turbines While to some extents all enzymes are regulated, the bottleneck enzymes are rate limiting and highly regulated. Product +ATP (equilibrium) Substrate energy Energy loss as reaction proceeds Product +ATP + heat (non-equilibrium) Product + heat Product energy

12. Enzymes working farthest from equilibrium are often used in regulation of a pathway because the reverse reaction is not easily attainable (would require a very great increase in conc of D to reverse the reaction) Natural bottlenecks! A ---- B ---- C -------- D ---- E ---- F ---- G

13. Factors that affect flux Flux through pathway are regulated by 1 Availability of substrate 2 Conc of enzymes responsible for rate limiting step 3 Allosteric regulation of enzyme (Feedback regulation) 4 Covalent modification of enzyme 5 Product removal Reduction in substrate will decrease activity of enzyme (provided enzyme is not saturated by substrate- most biological pathways operate at suboptimal conc of substrate) Removal of product enables reaction to proceed in a specific direction

14. Substrate concentration At substrate concentrations far below the Km, each increase in [S] produces a correspondingly large increase in the reaction velocity, v. For this region of the curve, the enzyme has an ε of about 1.0. At [S] >> Km, increasing [S] has little effect on v;ε here is close to 0.0.

15. Dependence of glycolytic flux in a rat liver homogenate on additional enzymes. Purified enzymes were added to an extract of liver carrying out glycolysis in vitro. The increase in flux through the pathway is shown on the yaxis.

16. Mechanism of gene regulation by the transcription factor FOXO1. Insulin activates a signaling cascade, leading to activation of protein kinase B (PKB). FOXO1 in the cytosol is phosphorylated by PKB, and the phosphorylated transcription factor is degraded by proteasomes. Unphosphorylated FOXO1 can enter the nucleus, bind to specific gene promoters, and trigger transcription of the associated genes. Insulin therefore has the effect of turning off the expression of these genes, which include glucose 6-phosphatase.

19. Regulation ofglucokinaseby sequestration in the nucleus. The protein inhibitor ofglucokinaseis a nuclear protein that draws glucokinaseinto the nucleus when the enzyme is not required but releases it to the cytosol when the glucose concentration is high and the enzyme is required.

20. Protein phosphorylation and dephosphorylation.

21. Reversal of entire pathways Reversal of entire pathway of glycolysis is difficult. DGo’ for glycolysis (glucose to pyruvate is -73 kJ/mol To do reverse reaction, you need to change product (pyruvate) conc many billion fold. Not feasible! Also reversing reaction leads to loss of ATP

22. Glycolysis Vs Gluconeogenesis Glu Regulation of three irreversible steps Cell uses some enzymes from glycolysis in gluconeogenesis Reactions with small DG are used by both pathways. Reactions with large DG- regulated Glucose 6-Phosphatase Hexokinase G6P F6P Fru 1,6 bisphosphatese1 PFK1 F1,6P2 PEP-carboxykinase PEP Oxaloacetate Pyruvate Kinase Pyruvatecarboxylase Pyruvate

23. Factors affecting enzymes

24. ATP requirement Effect of ATP concentration on the initial velocity of a typical ATP-dependent enzyme. These experimental data yield a Km for ATP of 5 mM. The concentration of ATP in animal tissues is ~5 mM.

25. Glucagon and insulin The three enzymes catalyze irreversible steps Concentration of these enzymes is regulated by hormones Insulin is secreted from pancreas (b-cells) when glucose levels in blood increase (Insulin promotes storage of energy). It induces transcription/translation of glucokinase, phosphofructokinase and pyruvatekinase (effects occur over hours) Glucagon is secreted from pancreas (a-cells) when blood glucose is low. It has opposite effects (induces release of glucose into blood)

26. Transporters Rate of entry of glucose into cells is regulated by transporters. Blood glucose level is 5mM Basal Glucose transporter in most cells has Km of 1mM. Less than blood glucose and so glucose is taken up easily. But in liver and pancreas glucose transporters have Km of ~15mM (close to blood glucose levels). This allows pancreatic cells to monitor glucose levels and thereby regulate insulin secretion. In liver cells, glucose is only taken up when it is very abundant. Then liver cells acquire glucose and convert it to glycogen and fatty acids.

27. Regulation of glucose

28. GlucokinaseHexokinase Kinetic parameter Km High (10mM) low (<0.1mM) low affinity high affinity Vmax high low Tissue distn Liver, pancreas muscle and other tissues Glucokinase and Hexokinase Glucokinase activity increases with increased glucose but is not inhibited by increased glu6PO4. The levels of the protein are regulated by insulin. Rate of reaction is driven by substrate-glucose not by demand for product-G6P. Allows all glu available to be converted to G6P and then if excess present, it is converted to glycogen and from there to triglycerides and fatty acids Hexokinase activity increases with increased glucose but activity is inhibited by increased G6P. The levels of enzyme are constitutive. It only generates ATP when energy is required. Glucokinase is not normally active because its Km is lower than normal blood glucose levels. Eating food increases glu in blood, activates glucokinase which converts glu to glycogen and fatty acids. Blood glucose is 5mM

29. Note the sigmoidicity forglucokinaseand the much lower Km for hexokinase I. When blood glucose rises above 5 mM,glucokinaseactivity increases, but hexokinase I is already operating near Vmax and cannot respond to an increase in glucose concentration.

31. Glycogen Pyruvate Kinase Hexokinase PFK1 Glu G6P F6P F1,6P2 PEP Pyruvate Pentose phosphate (nucleotides/cell div) Glycogen synthesis

32. Control of glycogen synthesis from blood glucose in muscle. Insulin affects three of the five steps in this pathway, but it is the effects on transport and hexokinase activity, not the change in glycogen synthase activity, that increase the flux toward glycogen.

33. Regulation of PFK1 Regulation of three irreversible steps PFK1 is rate limiting enzyme and primary site of regulation

34. PFK1 regulation by F2,6P2 • PFK-2 catalyzes • F6P + ATP -> F2,6P2 + ADP • PFK-2 allosterically activated by F6P and insulin (insulin induced dephosphorylation) • High Glu- high F6P • Therefore PFK2 active-- high F2,6P2 • F2,6P2 activates PFK1 and you get high glycolysis and fat synthesis FBPase1

35. PFK2/FBPase2 F2,6P2 is made and degraded during metabolic transition Its conc determines whether you get gylcolysis or gluconeogenesis It is a positive allostericeffector of PFK1 It is a negative (inhibitor) of FBPase1 F2,6P2 is made and degraded by a SINGLE enzyme with two distinct domains having two distinct activities -kinase (PFK2) synthesizes F2,6P2 -bisphosphatase (FBPase2) degrades F2,6P2 PFK2/FBPase2 is regulated by metabolic factors/hormones F6P activates PFK2 and inhibits FBPase2 thus regulating level of F2,6P2

36. In liver Lots of glucose, Lots of F6P made F6P activates PFK2 (also inhibits FBPase2) This makes more F2,6P2 F2,6P2 activates PFK1 (also inhibits FBPase1) This now makes more F1,6P2 When glucose levels drop and decrease F6P levels drop Inhibition of FBPase2 is reduced, F2,6P2 levels reduce. Lower levels of F2,6P2 reduced inhibition of FBPase1 and the reverse reaction now proceeds making more glucose. PFK2/FBPase2 is also regulated by phosphorylation by PKA and PP2A Phosphorylation- reduces PFK2 kinase activity and increases FBPase2 activity (generating more glucose) PKA is regulated by AMP. More AMP in cell means less ATP energy (i.e. less glucose) so liver makes more glucose to secrete into blood for other organs. G6P F6P PFK2/FBPase2 PFK1 F2,6 P2 FBPase1 F1,6 P2 PEP

38. Coordinated Regulation of PFK-1 and FBPase-1 Both are inducible, by opposite hormones (insulin and glucagon) Both are affected by F2,6P2, in opposite directions

39. Pyruvatedehydrogenase Pyruvate + CoA + NAD -> AcetylCoA + CO2 + NADH Amino acids Glucose Lactate PDH Acetyl CoA Oxaloacetate CO2+H2O Fatty acid Ketone TCA cycle Gluconeogenesis

40. Regulation of PDHMuscle • Resting (don’t need energy) • Hi energy state • Hi NADH & AcCoA & ATP • Inactivates PDH • Hi ATP & NADH & AcCoA • Inhibits PDH • Exercising (need energy) • Low NADH, ATP, AcCoA

41. Regulation of PDHLiver • Just been Fed (high blood glucose) • Need to convert glucose to Fatty acids • Hi energy • Insulin activates PDH • Starved (don’t need PDH) • No insulin • PDH inactive