19b-Biochemistry-Lecture19b.pdf

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJ...

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJIMI DO NOT have a financial interest in commercial products or services. Lecture 19a & 19b Regulation of carbohydrates metabolism Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 14: p. 539-546 and chapter 15: p. 565-571 19b/ REGULATION OF CARBOHYDRATES METABOLISM GLYCOLYSIS AND GLUCONEOGENESIS NEED TO BE RECIPROCALLY REGULARED Opposing pathways of glycolysis (pink) and gluconeogenesis (blue) in rat liver. 7 steps are catalyzed by the same enzymes in the 2 pathways 3 steps are catalyzed by different enzymes in gluconeogenesis (the "bypass reactions") and glycolysis. Simultaneous operation of both pathways consumes ATP without performing chemical or biological work: Example: ATP + fructose 6-P à ADP + fructose 1,6-bisphosphate (PFK1 in glycolysis) fructose 1,6-bisphosphate + H2O à fructose 6-P + Pi (FBPase-1 in gluconeogenesis) SUM: ATP + H2O à ADP + Pi + heat (A futile cycle*) Thus, regulation of these pathways must be coordinated *Another futile cycle: PEP + ADP à Pyruvate + ATP (Glycolysis) Pyruvate + ATP à oxaloacetate + ADP Oxaloacetate + GTP à PEP + GDP (gluconeogenesis COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS 1/ Hexokinases and their regulation There are four isozymes of hexokinase (HK): HK I to III are expressed in all tissues (including muscle), to different levels HK IV (also called Glucokinase) is only expressed in the liver – Has higher Km, so responsive to higher [glucose] – Not inhibited by glucose-6-phosphate, so can function at higher [glucose]. All the other hexokinases are inhibited by glucose-6-P – 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 COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS 1/ Hexokinases and their regulation Hexokinases Isoenzymes of muscle (I to III) and liver (IV) are affected differently by their substrate glucose and product glucose 6-P Hexokinase IV is regulated by sequestration in the nucleus (and also by transcription) Liver Muscle Note the much lower Km for hexokinase I. When blood glucose rises above 5 mM, hexokinase IV activity increases, but hexokinase I is already operating near Vmax and cannot respond to an increase in glucose concentration. Hexokinases I, II, III have similar kinetic properties The protein inhibitor of hexokinase IV is a nuclear binding protein that draws hexokinase IV into the nucleus when the fructose 6-phosphate concentration in liver is high and releases it to the cytosol when the glucose concentration is high. COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS 2/ Regulation PhosphoFructoKinase-1 (PFK-1) fructose-6-phosphate à fructose 1,6-bisphosphate is the committed step in glycolysis While ATP is a substrate, ATP is also a negative effector – Meaning: do not spend glucose in glycolysis if there is plenty of ATP Allosteric regulation of muscle PFK-1 by ATP. - At low [ATP], the K0.5 for fructose 6phosphate is relatively low, enabling the enzyme to function at a high rate at relatively low [fructose 6-phosphate].) - When [ATP] is high, K0.5 for fructose 6phosphate is greatly increased. COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS 2/ Regulation of PhosphoFructoKinase-1 (PFK-1) and Fructose 1,6 Bisphosphatase-1 (FBPase-1) Go glycolysis if AMP is high and ATP is low Go gluconeogenesis if AMP is low F 2,6 BP X GLYCOLYSIS AND GLUCONEOGENESIS ARE DIFFERENTIALLY REGULATED BY A COMMON ALLOSTERIC EFFECTOR, FRUCTOSE 2,6 BISPHOSPHATE F2-6BP IS NOT a glycolytic intermediate, only a regulator of PFK-1 and FBPase-1 F26BP is produced specifically by PFK2 to regulate glycolysis and broken down by FBPase 2 to regulate gluconeogenesis Activates PFK-1 (glycolysis) Inhibits FBPase-1 (gluconeogenesis) Under hormonal control GLYCOLYSIS AND GLUCONEOGENESIS ARE DIFFERENTIALLY REGULATED BY A COMMON ALLOSTERIC EFFECTOR, FRUCTOSE 2,6 BISPHOSPHATE Fructose 2,6-bisphosphate (F26BP) has opposite effects on the enzymatic activities of phosphofructokinase-1 (PFK-1, a glycolytic enzyme) and fructose 1,6-bisphosphatase (FBPase-1, a gluconeogenic enzyme). PFK1 PFK-1 activity in the absence of F26BP (blue curve) is half-maximal when the concentration of fructose 6-phosphate is 2 mM (that is, K0.5 = 2 mM). When F26BP is present (red curve), the K0.5 for fructose 6phosphate is only 0.08 mM. Thus F26BP activates PFK-1 by increasing its apparent affinity for fructose 6-phosphate FBPase1 FBPase-1 activity in the absence of F26BP (blue curve) has a K0.5 of for fructose 1,6-bisphosphate of 5 μM, but in the presence of F26BP (red curve) the K0.5 is >70 μM. Thus F26BP inhibits FBPase-1 by decreasing its apparent affinity for fructose 1-6-bisphosphate COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS 2/ Regulation of 2,6-bisphosphate level by PFK-2 and FBPase-2 F26BP is produced from F6P: The cellular concentration of the regulator F26BP is determined by the rates of its synthesis by phosphofructokinase-2 (PFK2) and its breakdown by fructose 2,6bisphosphatase (FBPase-2). Regulation of F26BP levels: Both enzyme activities are part of the same polypeptide chain, and they are reciprocally regulated by insulin and glucagon. Insulin increases [F2,6BP] activating PFK-1(glycolysis) and inhibiting FBPase-1 (gluconeogenenesis) Glucagon does the opposite COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS 3/ Regulation of Pyruvate Kinase PK is involved in the formation of pyruvate from PEP Allosterically activated by fructose-1,6-bisphosphate – Feed-forward action à 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) in the liver only – Glucose from liver is exported to brain and other vital organs COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS 3/ Regulation of Pyruvate Kinase PK, which catalyzes the last step of glycolysis (PEP to Pyruvate) is allosterically inhibited by ATP, acetylCoA, and long-chain fatty acids (all signs of an abundant energy supply), and the accumulation of fructose 1,6-bisphosphate triggers its activation. Accumulation of alanine, which can be synthesized from pyruvate in one step, allosterically inhibits pyruvate kinase, slowing the production of pyruvate by glycolysis. The liver isozyme (L form) is also regulated hormonally. Glucagon activates cAMP-dependent protein kinase PKA, which phosphorylates the pyruvate kinase L isozyme, inactivating it. When the glucagon level drops, a protein phosphatase (PP) dephosphorylates pyruvate kinase, activating it. This mechanism prevents the liver from consuming glucose by glycolysis when blood glucose is low; instead, the liver exports glucose. Pyruvate Kinase deficiency leads to hemolytic anemia: Low rate of glycolysis à low ATP levels in RBCs (no mitochondria)à hemolysis The muscle isozyme (M form) is not affected by this phosphorylation mechanism. COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS 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 The fates of pyruvate are under the control of acetyl-CoA: àWhen acetyl CoA accumulates (after breakdown of excess fat in liver mitochondria), - it limits its own formation by inhibiting pyruvate dehydrogenase, - and activates pyruvate carboxylase making oxaloacetate and eventually glucose through gluconeogenesis COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS Regulation of the fates for pyruvate Pyruvate can be converted to glucose and glycogen via gluconeogenesis or oxidized to acetyl-CoA for energy production. Fatty Acids degradation The first enzyme in each path is regulated allosterically: acetyl-CoA, produced either by fatty acid oxidation or by the pyruvate dehydrogenase complex, stimulates pyruvate carboxylase and inhibits pyruvate dehydrogenase. COORDINATED REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS 1/ à 2/ à 3/ à THE AMOUNT OF MANY METABOLIC ENZYMES IS CONTROLLED BY TRANSCRIPTION Transcriptional regulation of glycolysis and gluconeogenesis changes the number of enzyme molecules For example, Insulin transcriptionally regulates more than 150 genes Change in gene expression Role in glucose metabolism Increased expression Hexokinase II (Muscle) Hexokinase IV (Liver) Phosphofructokinase-1 (PFK-1) PFK-2/FBPase-2 Pyruvate kinase Essential for glycolysis, which consumes glucose for energy Glucose 6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Malic enzyme Produce NADPH, which is essential for conversion of glucose to lipids ATP-citrate lyase Pyruvate dehydrogenase Produce acetyl-CoA, which is essential for conversion of glucose to lipids Acetyl-CoA carboxylase Fatty acid synthase complex Stearoyl-CoA dehydrogenase Acyl-CoA-glycerol transferases Essential for conversion of glucose to lipids Decreased expression PEP carboxykinase Glucose 6-phosphatase (catalytic subunit) Essential for glucose production by gluconeogenesis CONTROL OF GLYCOGEN BREAKDOWN Glycogen Phosphorylase is regulated by covalent modification, allosterically and hormonally (by insulin and glucagon) 1/ Allostery: Ca2+ (signal for muscle contraction) and AMP (accumulates in active muscle) are activators (stabilize the a form). ATP, glucose (in liver), G6P are inhibitors (stabilize the b form) 2/ Hormones: Glucagon/Epinephrine and Insulin signaling pathway 3/ covalent modification: The enzyme exists in two forms, a and b (phosphorylation) phosphorylase a which is more active phosphorylase b which is less active In the more active form of the enzyme, phosphorylase a, Ser14 residues, one on each subunit, are phosphorylated. Phosphorylase a is converted to the less active form, phosphorylase b,by dephosphorylation, catalyzed by phosphorylase a phosphatase (also known as phosphoprotein phosphatase 1, PP1) Phosphorylase b can be reconverted (reactivated) to phosphorylase a by phosphorylation through phosphorylase b kinase. Glucose (liver) Insulin (liver) CONTROL OF GLYCOGEN BREAKDOWN Glycogen Phosphorylase of liver is a glucose sensor After glucagon action, when glucose levels return to normal, glucose enters the hepatocytes and binds to an inhibitory allosteric site of phosphorylase a. Glucose binding to an allosteric site of phosphorylase a of liver induces a conformational change that exposes the phosphorylated serine residues to the action of phosphorylase a phosphatase 1 (PP1). Phosphorylase a phosphatase (PP1) converts phosphorylase a to phosphorylase b sharply reducing the activity of phosphorylase and slowing glycogen breakdown in response to high blood glucose levels. Insulin also acts indirectly to stimulate PP1 activity to slow down glycogen breakdown (see previous and next slides). CONTROL OF OF GLYCOGEN BREAKDOWN Epinephrine and glucagon stimulate breakdown of glycogen by a cascade mechanism to activate glycogen phosphorylase By binding to specific surface receptors, either epinephrine acting on a myocyte (left) or glucagon acting on a hepatocyte (right) activates a GTPbinding protein, Gsα. Active Gsα triggers a rise in [cAMP], activating PKA. This sets off a cascade of phosphorylations; PKA activates phosphorylase b kinase, which then activates glycogen phosphorylase. Such cascades effect a large amplification of the initial signal; the figures in pink boxes are probably low estimates of the actual increase in number of molecules at each stage of the cascade. The resulting breakdown of glycogen provides glucose, which in the myocyte can supply ATP (via glycolysis) for muscle contraction and in the hepatocyte is released into the blood to counter the low blood glucose. CONTROL OF GLYCOGEN SYNTHESIS FROM BLOOD GLUCOSE IN MUSCLE. Insulin-signaling pathway: – increases glucose import into muscle Transport – stimulates the activity of muscle hexokinase (enables activation of glucose) – activates glycogen synthase (makes glycogen for energy storage) Insulin affects 3 of the 5 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 (flow of metabolites) toward glycogen. CONTROL OF GLYCOGEN SYNTHESIS Glycogen Synthase is regulated by covalent modification, allosterically and hormonally Effects of Glycogen Synthase Kinase 3 (GSK3) on glycogen synthase activity Phosphorylation of Glycogen synthase a, the active form, (on three Ser residues). by glycogen synthase kinase 3 (GSK3) converts glycogen synthase to the inactive (b) form. GSK3 action requires prior phosphorylation (priming) by casein kinase (CKII). Insulin triggers activation of glycogen synthase b by blocking the activity of GSK3 and activating a phosphoprotein phosphatase (PP1). Epinephrine (muscle) and glucagon (liver) activates PKA, which phosphorylates the glycogen-targeting protein GM on a site that causes dissociation of PP1 from glycogen, thus inactivating glycogen synthase. Glucose 6-phosphate favors dephosphorylation of glycogen synthase by binding to it and promoting a conformation that is a good substrate for PP1 (thus leading to the activate glycogen synthase). CONTROL OF CARBOHYDRATE METABOLISM IN THE LIVER Allosteric and hormonal signals coordinate carbohydrate metabolism globally (Liver) Arrows indicate causal relationships between the changes they connect. For example, an arrow from ↓A to ↑B means that a decrease in A causes an increase in B. Pink arrows connect events that result from high blood glucose; blue arrows connect events that result from low blood glucose. CONTROL OF CARBOHYDRATE METABOLISM IN THE LIVER VS. MUSCLE Difference in the regulation of carbohydrate metabolism in liver and muscle In liver, either glucagon (indicating low blood glucose) or epinephrine (signaling the need to fight or flee) has the effect of maximizing the output of glucose into the bloodstream. In muscle, epinephrine increases glycogen breakdown and glycolysis, which together provide fuel to produce the ATP needed for muscle contraction SUMMARY Living organisms regulate the flux of metabolites through metabolic pathways by – increasing or decreasing enzyme concentrations – activating or inactivating key enzymes in the pathway The activity of key enzymes in glycolysis and gluconeogenesis is tightly and coordinately regulated via various activating and inhibiting metabolites Glycogen synthesis and degradation is regulated by hormones insulin, epinephrine, and glucagon that report on the levels of glucose in the body Remember to prepare for next lecture: Lehninger’s Biochemistry (8th ed), §chapter 16: p. 574-596