Principios de Regulación Metabólica

Transcription

1 Principios de Regulación Metabólica

2 15 Principles of Metabolic Regula7on 2013 W. H. Freeman and Company

3 Metabolic Pathways The biochemical reactions in the living cell metabolism are organized into metabolic pathways The pathways have dedicated purposes: Extraction of energy Storage of fuels Synthesis of important building blocks Elimination of waste materials The pathways can be represented as a map Follow the fate of metabolites and building blocks Identify enzymes that act on these metabolites Identify points and agents of regulation Identify sources of metabolic diseases

4 Map of Metabolic Pathways

5 Homeostasis Organisms maintain homeostasis by keeping the concentrations of most metabolites at steady state In steady state, the rate of synthesis of a metabolite equals the rate of breakdown of this metabolite Pathways are at steady state unless perturbed After perturbation a NEW steady state will be established La homeostasis es la capacidad del organismo para presentar una situación físico-química característica y constante dentro de ciertos límites, incluso frente a alteraciones o cambios impuestos por el medio ambiente.

6 Principles of Regulation The flow of metabolites through the pathways is regulated to maintain homeostasis Sometimes, the levels of required metabolites must be altered very rapidly Need to increase the capacity of glycolysis during action Need to reduce the capacity of glycolysis after the action Need to increase the capacity of gluconeogenesis after successful action

7 Feedback Inhibi7on In many cases, ul-mate products of metabolic pathways directly or indirectly inhibit their own biosynthe-c pathways ATP inhibits the commitment step of glycolysis

8 Rates of a Biochemical Reactions Rates of a biochemical reac-ons depend on many factors: Concentra-on of reactants vs. products Ac-vity of the catalyst Concentra-on of the enzyme Rate of transla-on vs. rate of degrada-on Intrinsic ac-vity of the enzyme Could depend on substrate, effectors or phosphoryla-on state Concentra-ons of effectors Allosteric regulators Compe-ng substrates ph, ionic environment Temperature

9 Factors that Determine the Ac7vity of Enzymes

10 Factores que determinan actividad enzimática

11 Rate of reaction depends on the concentration of substrates The rate is more sensitive to concentration at low concentrations Frequency of substrate meeting the enzyme matters The rate becomes insensitive at high substrate concentrations The enzyme is nearly saturated with substrate

12 Effect of [Substrate] on Enzyme Activity

13 Phosphoryla7on of enzymes affects their ac7vity Phosphoryla-on is catalyzed by protein kinases Dephosphoryla-on is catalyzed by protein phosphatases, or can be spontaneous Typically, proteins are phosphorylated on the hydroxyl groups of Ser, Thr or Tyr

14 Phosphoryla7on of enzymes affects their ac7vity Phosphorylation - Can alter electrostatic features of active site, alter enzyme s interaction with other proteins, or force conformational changes, which translates into changes in V max or K m.

15 Enzymes are also regulated by regulatory proteins Binding of regulatory protein subunits affects specificity.

16 Proteins have a finite lifespan Different proteins in the same tissue have very different half-lives Less than an hour to about a week for liver enzymes Stability correlates with the sequence at N-terminus Some proteins are as old as you are Crystallins in the eye lens

17 Reactions far from equilibrium are common points of regulation Within a metabolic pathway most reactions operate near equilibrium Key enzymes operate far from equilibrium These are the sites of regulation Control flow through the pathway To maintain steady state all enzymes operate at the same rate

18 Reactions far from equilibrium are common points of regulation The net flow of metabolites through steps 2 and 3 is the small difference between the rates of the forward and reverse reactions. Small changes in substrate or product concentration can produce large changes in the net rate, and can even change the direction of the net flow. We can identify these near equilibrium rxns. by comparing Q with K eq (1-2 orders of magnitude).

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20 Reactions far from equilibrium are common points of regulation For reactions far from equilibrium (for example, PFK-1) Keq is 1,000 but Q is is 0.1!!! Because the rxn is so far from equilibrium, the process is highly exergonic under cellular conditions and tends to go in the FWD direction. It is held far from equilibrium because of the particular cellular concentrations of substrates, products, allosteric effectors, enzyme activity, etc. However, the net forward rate of the reaction is equal to the net flow of other glycolytic intermediates and the reverse flow through PFK-1 remains near zero.

21 ATP and AMP are key cellular regulators A 10% decrease in [ATP] can greatly affect the activity of ATP utilizing enzymes A 10% decrease in [ATP] leads to a dramatic increase in [AMP] AMP can be a more potent allosteric regulator

22 AMP differentially affects pathways in different tissues via AMPK

23 Some enzymes in the pathway limit the flux of metabolites more than others Enzymes that are far from equilibrium (regulated) Not all regulated enzymes have the same affect on the entire pathway Some control flux through the pathway Others regulate steady state concentrations of metabolites in response to changes in flux Hexokinase and phosphofructokinase are appropriate targets for regulation of glycolytic flux Increased hexokinase activity enables activation of glucose Increased phosphofructokinase-1 activity enables catabolism of activated glucose via glycolysis

24 Hexokinase affects flux through glycolysis more than phosphofructokinase

25 Flow to glycogen synthase is controlled by glucose uptake and phosphorylation Insulin affects three steps

26 Glycolysis vs. Gluconeogenesis

27 There are four isozymes of hexokinase HK I is expressed in all tissues, to different levels HK IV is only expressed in the liver Has higher K m, so responsive to higher [glucose] Not inhibited by glucose-6-phosphate, so can function at higher [glucose] Functions to clear blood glucose at higher [glucose] for storage as glycogen Isozymes are different enzymes that catalyze the same reaction They typically share similar sequences May have different kinetic properties Can be regulated differently

28 Kine7c Proper7es of HK I vs. HK IV HKI is operating near Vmax when blood glucose rises above 5mM

29 Second, hexokinase IV is subject to inhibition by the reversible binding of a regulatory protein specific to liver. The binding is much tighter in the presence of the allosteric effector fructose 6-phosphate. Glucose competes with fructose 6-phosphate for binding and causes dissociation of the regulatory protein from hexokinase IV, relieving the inhibition. Immediately after a carbohydrate-rich meal, when blood glucose is high, glucose enters the hepatocyte via GLUT2 and activates hexokinase IV by this mechanism. During a fast, when blood glucose drops below 5 mm, fructose 6- phosphate triggers the inhibition of hexokinase IV by the regulatory protein, so the liver does not compete with other organs for the scarce glucose. The mechanism of inhibition by the regulatory protein is interesting: the protein anchors hexokinase IV inside the nucleus, where it is segregated from the other enzymes of glycolysis in the cytosol (Fig ). When the glucose concentration in the cell rises, it equilibrates with glucose in the nucleus by transport through the nuclear pores. Glucose causes dissociation of the regulatory protein, and hexokinase IV enters the cytosol and begins to phosphorylate glucose. Third, hexokinase IV is not inhibited by glucose 6-phosphate, and it can therefore continue to operate when the accumulation of glucose 6-phosphate completely inhibits hexokinases I III.

30 Hexokinase IV is regulated by sequestra7on (and transcrip7on)

31 Regulation of Phosphofructokinase-1 fructose-6-phosphate fructose 1,6-bisphosphate is the commitment step in glycolysis While ATP is a substrate, ATP is also a negative effector Do not spend glucose in glycolysis if there is plenty of ATP

32 Effect of ATP on Phosphofructokinase- 1 Allosteric regulation of muscle PFK-1 by ATP. At low [ATP], the K 0.5 for fructose 6-phosphate is relatively low, enabling the enzyme to function at a high rate at relatively low [fructose 6- phosphate]. (K 0.5 is the K m term for regulatory enzymes.) When [ATP] is high, K 0.5 for fructose 6- phosphate is greatly increased.

33 Regula7on of Phosphofructokinase 1 and Fructose 1,6- Bisphosphatase Go glycolysis if AMP is high and ATP is low Go gluconeogenesis if AMP is low

34 Fructose 2,6- Bisphosphate NOT a glycoly-c intermediate, only a regulator Produced specifically to regulate glycolysis and gluconeogenesis ac-vates phosphofructokinase (glycolysis) inhibits fructose 1,6- bisphosphatase (gluconeogenesis)

35 F- 2,6- bp is produced from fructose- 6- phosphate

36 Regulación de Gluconeogénesis Coordinada con regulación de glucólisis. 1) Ac.CoA [Ac.CoA] alta inhibe dehidrogenasa, a través de kinasa que inactiva la dehidrogenasa; y activa la carboxilasa. Piruvato no convertido a Ac.CoA está disponible para gluconeogénesis. Cuando status energético en la célula es rico: Fosforilación ox. se reduce, NADH se acumula, se inhibe el Ciclo de Krebs, y Ac.CoA se acumula.

37 2) Fructosa 1,6-Bifosfatasa - inhibida por AMP PFK-1 - activada por AMP y ADP, inhibida por ATP y citrato. Regulación recíproca. [AcCoA, citrato o ATP] altas - activan gluconeogénesis. Regulación alostérica (bien rápido)

38 Hígado - tiene controles adicionales: Fructosa 2,6 Bifosfatada - activa PFK-1 y glucólisis; inhibe Fructosa 1,6 Bifosfatasa y gluconeogénesis. Aumenta afinidad de PFK-1 por fructosa-6p. Fructosa 2,6- BiP relacionado estructuralmente a Fructosa 1-6 BiP pero no es intermediario de glucólisis o gluconeogénesis.

39 Regulación del nivel de Fructosa 2,6 Bifosfatada

40 Cuando baja nivel de glucosa en la sangre glucagon -indica que hay que producir glucosa (deg. de glucogeno o gluconeogénesis), regulando síntesis y deg. de Fructosa 2,6 BiP. La síntesis de Fructosa 2,6-BiP ocurre mediante la fosforilación de Fructosa 6-P por PFK-2. La degradación por Fructosa 2,6 Bifosfatasa 2 (FBPase2) Dos actividades enzimaticas que forman parte de la misma proteína. Glucagon activa producción de camp, una camp-dependent cinasa fosforila enzima y aumenta actividad de FBPase 2 e inhibe actividad de PFK-2. Se activa gluconeogénesis y se inhibe glucólisis.

41 Regulation of Pyruvate Kinase Allosterically activated by fructose-1,6-bisphosphate High flow through glycolysis Allosterically inhibited by signs of abundant energy supply (all tissues) ATP Acetyl-CoA and long-chain fatty acids Alanine (enough amino acids) Inactivated by phosphorylation in response to signs of glucose depletion (glucagon) (liver only) Glucose from liver is exported to brain and other vital organs

42 Regula7on of Pyruvate Kinase

43 Two Alternative Fates for Pyruvate Pyruvate can be a source of new glucose Store energy as glycogen Generate NADPH via pentose phosphate pathway Pyruvate can be a source of acetyl-coa Store energy as body fat Make ATP via citric acid cycle Acetyl-CoA stimulates glucose synthesis by activating pyruvate carboxylase

44 Two Alterna7ve Fates for Pyruvate

45 The amount of many metabolic enzymes is controlled by transcription

46 FOXO1 ac7vates transcrip7on in response to insulin (high bloood glucose)

47 The transcrip7on of many genes is controlled by many different factors

48 Metabolismo de Glucógeno Exceso de glucosa, se acumula en glucógeno o almidón (plantas), baja osmolaridad en la cel., pueden visualizarse los granos de glucógeno, prominentes en hepatocitos y músculo.

49 Reserva de glucógeno en músculo es suficiente para 1 hora si estás haciendo ejercicio vigoroso. Reserva de glucógeno en hígado es suficiente para horas en ayuno (pero no ejercicio vigoroso).

50 Glucose residues are removed from glycogen by glycogen phosphorylase

51 Dealing with Branch Points in Glycogen Glycogen phosphorylase works on non-reducing ends until it reaches four residues from an (α1 6) branch point Debranching enzyme transfers a block of three residues to the non-reducing end of the chain Debranching enzyme cleaves the single remaining (α1 6) linked glucose

52 Dealing with Branch Points in Glycogen

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54 Glucose- 1- phosphate must be isomerized to glucose- 6- phosphate for metabolism

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56 Glucose- 6- phosphate is dephosphorylated in the liver for transport out of the liver

57 Glycogen is synthesized by glycogen synthase

58 UDP- glucose is the substrate for glycogen synthase

59 Synthesis of branches in glycogen

60 Control of Glycogen Breakdown Glucagon/Epinephrine signaling pathway Starts phosphorylation cascade vis camp activates glycogen phosphorylase Glycogen phosphorylase cleaves glucose residues off glycogen, generating glucose-1-phosphate

61 Epinephrine and glucagon s7mulate breakdown of glycogen

62 Control of Glycogen Synthesis Insulin-signaling pathway increases glucose import into muscle stimulates the activity of muscle hexokinase activates glycogen synthase Increased hexokinase activity enables activation of glucose Glycogen synthase makes glycogen for energy storage

63 Glycogen synthase is controlled by phosphoryla7on

64 Control of Carbohydrate Metabolism in the Liver

65 Control of Carbohydrate Metabolism in the Liver vs. the Muscle

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