Regulation of Metabolic Pathways

1. Regulation of Metabolic Pathways • Systems must respond to conditions • Homeostasis is not equilibrium • Dynamic Steady State • Flux - Rate of metabolic flow of material through pathways • Many ways to regulate – for example • At the protein level (e.g. allosteric control) • At the gene level • At transcription or translation • There are different time scales for regulation • < sec, seconds, hours, days • Based on situation that requires response

2. Maintaining ATP concentration is critical • Energy needed to sustain cellular processes • Typical cell • [ATP]  5 mM • ATP-using enzymes KM range 0.1 – 1 mM • Significant [ATP] drop would cause many reactions to decrease • Cells are sensitive to ratios ATP/ADP(or AMP) NADH/NAD+ NADPH/NADP+ ATP + glucose  ADP + glucose 6-phosphate • AMP is a very sensitive indicator – small changes make a big difference percentage-wise (normal conc. <0.1 mM)

3. -Fast response (sec or less) – usually allosteric control (faster response than synthesis or degradation of enzyme) -Covalent modification (also fast) most common: phosphorylation/dephosphorylation -Slower response (sec to hours) –exterior effects such as hormones, growth factors Overall regulatory networks will: 1. maximize efficience of energy source utilization by preventing futile cycles. 2. partition metabolites between alternative pathways (Ex: glycolysis and PPP). 3. use the best energy source for the immediate needs of the cell. 4. shut down biosynthetic pathways when their products accumulate. Vocabulary: Metabolic regulation – maintains homeostasis at the molecular level (e.g. hold concentrations of metabolites constant) Metabolic control – changes flux through a metabolic pathway

4. Coordinated Regulation of Glycolysis & Gluconeogenesis Futile (substrate) cycles are to be avoided cycles that recycle metabolites at the expense of ATP

5. Glycolysis Regulation • When ATP is needed, glycolysis is activated • When ATP levels are sufficient, glycolysis activity decreases Control points 1. Hexokinase 2. PFK-1 3. Pyruvate kinase • Hexokinase • Hexokinase reaction is metabolically irreversible • G6P (product) levels increase when glycolysis is inhibited at sites further along in the pathway Recall there are 4 isozymes • G6P inhibits hexokinase isozymes I, II and III • Glucokinase (hexokinase IV) forms G6P in the liver (for glycogen synthesis) when glucose is abundant (activity is modulated by fructose phosphates and a regulatory protein)

6. Isozymes I,II and II have similar KM(important in muscle) • Normally at saturation • Hexokinase IV has much higher KM (important in liver) • Important when blood glucose is high

7. Glucose enters mammalian cells by passive transport down a concentration gradient from blood to cells • GLUT is a family of six passive hexosetransporters • Glucose uptake into skeletal and heart muscle and adipocytes by GLUT 4 is stimulatedbyinsulin • Other GLUT transporters mediate glucose transport in and out of cells in the absence of insulin • GLUT2 is transporter for hepatocytes • Quick equilibrium of [glucose] with blood glucose in both cytosol and nucleus • Regulator protein – inside the nucleus • Binds Hexokinase IV and inhibits it • Protein has regulatory site • Competition between glucose and fructose 6-phosphate • Glucose stimulates release of hexokinase IV into cytoplasm • Fructose 6-phosphate inhibits this process • Hexokinase IV not affected by glucose 6-phosphate as the other isozymes are

8. Addition of a regulatory protein raises apparent KM for glucose (inhibits hexokinase IV)

9. Glucose 6-Phosphate Has a Pivotal Metabolic Role in Liver

10. 2. Regulation of Phosphofructokinase-1 • Important - this step commits glucose to glycolysis • PFK-1 has several regulatory sites • ATP is a substrate and an allostericinhibitor of PFK-1 (note that it’s an end-product of the pathway) • AMP allosterically activates PFK-1 by relieving the ATP inhibition (ADP is also an activator in mammalian systems) • Changes in AMP and ADP concentrations can control the flux through PFK-1 • AMP relieves ATP inhibition of PFK-1

11. Elevated levels of citrate (indicate ample substrates for citric acid cycle) also inhibit PFK-1 • Most important allosteric regulator is fructose 2,6-bisphosphate (later in the chapter) 3. Regulation of Pyruvate Kinase (PK) • At least 3 PK isozymes exist in vertebrates • Differ in distribution and modulators • Inhibited by high ATP, Acetyl-CoA, long-chain fatty acids (energy in good supply) • Liver form – low blood sugar glucagon  increased cAMP cAMP-dependent protein kinase  PK inactivation (is reversed by protein phosphatase)

12. Muscle form – epinephrine→increased cAMP → activates glycogen breakdown and glycolysis • PK is allosterically activated by Fructose 1,6 BP • PK inhibited by accumulation of alanine

13. Regulation of Gluconeogenesis • Fate of pyruvate • Go on to citric acid cycle – requires conversion to Acetyl Co-A by the pyruvate dehydrogenase complex • Gluconeogenesis – first step is conversion to oxaloacetate by pyruvate carboxylase • Acetyl Co-A accumulation • inhibits pyruvate dehydrogenase • activates pyruvate carboxylase

14. Coordinated regulation of PFK-1 and FBPase-1(1) Phosphofructokinase-1 (PFK-1) (glycolysis)(2) Fructose 1,6-bisphosphatase FBPase-1 (gluconeogenesis) • Modulating one enzyme in a substrate cycle will alter the flux through the two opposing pathways • Two coordinating modulators • AMP • Fructose 2,6-bisphosphate • Inhibiting PFK-1 stimulates gluconeogenesis • Inhibiting the phosphatase stimulates glycolysis • AMP concentration coordinates regulation • stimulates glycolysis • Inhibits gluconeogenesis

15. In the liver, the most important coordinating modulator is fructose 2,6-bisphophate (F2,6BP) • It is formed from F6P by the enzyme phosphofructokinase-2 (PFK-2) • It is broken down by the same enzyme, but at a different catalytic site in the enzyme – it’s a bifunctional protein • It is called fructose 2,6-bisphosphatase (FBPase-2) for this activity • Balance of PFK-2 to FBPase-2 activity controlled by • Glucagon • Insulin

16. F2,6BP stimulates glycolysis • F2,6BP inhibits gluconeogenesis

17. Effects of Glucagon and Insulin

18. The Pasteur Effect • Under anaerobicconditions the conversion of glucose to pyruvate is muchhigher than under aerobic conditions (yeast cells produce more ethanol and muscle cells accumulate lactate) • The Pasteur Effect is the slowingofglycolysis in the presence of oxygen • MoreATP is produced under aerobic conditions than under anaerobic conditions, therefore lessglucose is consumed aerobically

19. Regulation of Glycogen Metabolism • Muscleglycogen is fuel for muscle contraction • Liverglycogen is mostly converted to glucose for bloodstream transport to other tissues • Both mobilization and synthesis of glycogen are regulated by hormones and allosterically • Insulin, glucagon and epinephrine regulate mammalian glycogen metabolism (hormones) • Ca2+ and [AMP]/[ATP] (muscle glycogen phosphorylase) • [glucose] (liver glycogen phosphorylase) • [glucose 6-phosphate] (glycogen synthase) • Hormones • Insulin isproduced by -cells of the pancreas (high levels are associated with the fedstate) • -increases glucose transport into muscle, adipose tissue via GLUT 4 transporter • -stimulates glycogensynthesis in the liver

20. Glucagon is Secreted by the a cells of the pancreas in response to lowbloodglucose (elevated glucagon is associated with the fastedstate) • -Stimulates glycogendegradation to restore blood glucose to steady-state levels • -Only liver cells are rich in glucagon receptors • Epinephrine (adrenaline) Released from the adrenal glands in response to sudden energy requirement (“fight or flight”) • - Stimulates the breakdownofglycogen to G1P (which is converted to G6P) • -Increased G6P levels increase both the rate of glycolysis in muscle and glucoserelease to the bloodstream from the liver

21. Reciprocal Regulation of GlycogenPhosphorylase and Glycogen Synthase • Glycogen phosphorylase (GP) and glycogen synthase (GS) control glycogen metabolism in liver and muscle cells • GP and GS are reciprocallyregulated both covalently and allosterically (when one is active the other is inactive) • Covalent regulation by phosphorylation (-P) and dephosphorylation (-OH) • COVALENTMODIFICATION (Hormonal control) • Activeform “a” Inactiveform “b” • Glycogen phosphorylase -P -OH • Glycogen synthase -OH -P • Allosteric regulation of GP and GS • GP a (active form) - inhibited by GlucoseGP (muscle)- stimulated by Ca2+ and high [AMP] • GS b (inactive form) - activated by Glucose 6-Phosphate

23. Hormones initiate enzyme cascades • Catalyst activates a catalyst activates a catalyst, etc. • When blood glucose is low: epinephrine and glucagon activate proteinkinaseA • Glycogenolysis is increased (more blood glucose) • Glycogensynthesis is decreased