Chapter 12: Gluconeogenesis, Pentose Phosphate Pathway, & Glycogen Metabolism Glucose catabolism for the production of energy requires a source of Glc. Polysaccharides are degraded and the resulting Glc is stored as glycogen in muscle and liver. glycogen PPP Glc also syn from pyruvate (lactate and amino acids) Liver/kidney Glc needed in brain/muscle The pentose phosphate pathway (PPP) is the source of ribose (deoxyribose), and NADPH. NADPH is required for biosynthesis.

#1 #3 #10 Pathway Glycolysis Net Reaction: Glucose + 2 ADP + 2 NAD+ + 2 Pi  2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O #3 **Gluconeogenesis Net Reaction:** 2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 6 H2O  Glucose + 4 ADP + 2 GDP+ 2 NAD+ + 6 Pi Gluconeogenesis - glycolysis going backwards - 3 places differ- control points in glycolysis - 4 new enzymes (eukaryotes) - importance of near equilibrium reactions - ATP energy, NADH reducing equivalents consumed #10

Gluconeogenesis 6 ATP needed total 4 needed to overcome barrier of production of 2 mol of PEP

Gluconeogenesis: The Irreversible Steps Pyruvate  PEP; reversing the pyruvate kinase step of glycolysis. 4 subunits Biotin Allosteric + acetyl CoA Indicates CAC Backed-up No allosteric reg Hormonal induction Transcriptional regulation + glucagon (fasting) - Insulin (fed state)

Gluconeogenesis No ATP needed since Fru-1,6-bisP not high energy intermediate

Fru-1,6-biP  Fru-6-P; reversing the PFK-1 step of glycolysis. Large – DG and irreversible Allosteric modulation - AMP - 2,6-Fru bisP (opposing effect in glycolysis)

Glc-6  Glc; reversing the Glc hexokinase step of glycolysis. Irreversible Allosteric modulation - AMP Enzyme found only in liver, kidneys, small intestine. Bound to ER lumen…leads to release of Glc into bldstream Get to brain And muscle Most cases Glc-6-P is end product---used in other pathways (glycogen syn)

Gluconeogenesis: Precursors Major precurser in mammals: Lactate and Amino Acids, Since the body does not transfer pyruvate Lactate Amino Acids Pyruvate in tissues must go to liver First converted to alanine Cori cycle Major source of C for Glc syn during fasting Active muscle-- lactate Amino arise from muscle protein breakdown Lactate to pyruvate in liver Provide temporary and readily available supply of Glc to muscle (exercise)

Gluconeogenesis Gluconeogenesis -glucose biosynthesis found in all organisms Some tissues require glucose -brain, muscles After 16-24 hrs, glucose and glycogen reserves depleted Some tissues synthesis glucose from non-carbohydrate precursor -liver, kidney -lactate, alanine Easiest to start with pyruvate -converted from lactate or a.a.

Gluconeogenesis: Regulation Low [Glc]: glucagon increases protein kinase A (activates Fru-2,6-bisP phosphatase) lowering [Fru-2,6-bisP]. Activate Glc syn and Loss of glycolysis stim neg reg pyruvate kinase Substrate Cycle Dec the net flux of a pathway But allows a point for reg flux Modulate one enzyme effect 2 opposing pathways Inhibit PFK-1 ….. stim Glc syn

Regulation of Phosphofructokinase-1 Large oligomeric enzyme bacteria/mammals - tetramer yeast - octamer ATP - product of pathway - allosteric inhibitor AMP - allosteric activator - relieves inhibition by ATP Citrate - feedback inhibitor - regulates supply of pyruvate - links Glycolysis and CAC Fru-2,6-bisphosphate - strong activator - produced by PFK-2 when excess fru-6-phosphate - indirect means of substrate stimulation or feed forward activation

Regulation of Pyruvate Kinase + F 1,6 BP Inactivation by covalent modification -blood [Glc] drops, glucagon released -liver protein kinase A (PKA) turned on -PKA phosphorylates pyruvate kinase Allosteric (feed-forward) activation Fructose-1,6-bisphosphate -allosterically activates -produced in step three -links control steps together Allosteric inhibition by ATP -product of pathway and CAC High blood [Glc] Low blood [Glc]

Regulation of Phosphofructokinase-1 Produced in pancreas in response to low [Glc] Dual activities of PFK-2 reg steady-state conc of Fru-2,6-bisP Increased glycolysis Fruc-6P inc….inc F-2,6-bisP Stim PFK-1 Dec F-2,6-bisP PFK-1 less active…..dec glycolysis Activate Protein Kinase A PFK-1 and pyruvate kinase Dec glycolysis Inc glc syn Stimulate glycogen breakdown Figure 11-17

Pentose Phosphate Pathway Shunt glycogen PPP The pentose phosphate pathway (PPP) is the source of ribose (deoxyribose), and NADPH. NADPH is required for biosynthesis.

Pentose Phosphate Pathway Shunt Synthesize 3 pentose phosphates Ribulose 5-P Xylulose 5-P Ribose 5-P (DNA/RNA) And NADPH (for the reduction of RNA to DNA) Or NADPH and glycolytic intermediates Rapidly dividing cells need lots of NADPH and DNA High PPP activity

The Oxidation Stage of PPP Allosteric - NADPH Major reg step Loss of Carbon

The Non-Oxidation Stage of PPP All equilibruim rxns When cells need lot of NADPH and nucleotides - ribulose 5-phosphate  ribose 5-phosphate - end of pathway

The Non-Oxidation Stage of PPP Convert 5C sugars into glycolytic intermediates Can be used in glycolysis of Gluconeogenesis

Pentose Phosphate Pathway Thru PPP 3 Glc-6-P + 6 NADP+ + 3 H2O  2 Fru-6-P + G3P + 6 NADPH + 3 CO2 Allow sub regeneration via PPP and glyconeogenesis Recycle 6C sugar 6 ribulose 5-P 5 Glc 5-P 6 Glc-6-P + 12 NADP+  5 Glc-6-P + 12 NADPH + 6 CO2 + Pi Can be metabolized in Glycolysis or Glcneogenesis

Glycogen Metabolism Glycogen is the storage form of Glc found in muscles and liver. (Plants: stored as Starch) Glycogen complex: single glycogenin molecule (Tyr -OH) and >50,000 glucose residues Stores of Glc in time of plenty and supplies it in times of need Muscle: fuel for contraction Liver: produce Glc…released to Bldstream to other tissues All regulated by hormones: Glucagon, Epinephrin and Insulin

Glycogen Metabolism Synthesis: Different enzymes for syn and degradation Driven by PPi hydrolysis Major regulatory step

UDP-Glc synthases in protists, animals, and fungi. Key regulation by phosphorylation (hormonally regulated) Pre-existing Glycogenin primer UDP-Glc synthases in protists, animals, and fungi. ADP-Glc synthase in plants. Primer of 4 to 8 Glc on a Tyr (-OH) of glycogenin. 1st Glc from UDP-Glc via Glc transferase. Remaining Glc’s tranferred by glycogenin. Amylo-(1,4 1,6)-transglycolase catalyzes the branch point. (Alpha 1-6 link)

Degradation: Primary regulation Two subunits, two catalytic sites, allosteric sites. AMP- activator; ATP & Glc-6-P – inhibitor. Phosphorylation: active (phosphorylase a). Dephosphorylated: less active (phosphorylase b). Phosphorolysis rxn. Generates phosph-sugar not free glc Primary regulation

Energy yield from glycogen Higher than from glc Branching inc speed of syn and degradation phosphorolytic Reg by ATP and G-6-P Sequential removal of Glc From non-reducing end Stops 4 Glc from branch pt Primarily by phosphorylation hydrolytic Energy yield from glycogen Higher than from glc

Regulation of Glycogen Metabolism Hormonal Regulation: Fed state fasting Via cAMP phosphatase Decrease glycolysis Via PIP3 Insulin: secreted by pancreas when Glc high inc rate of transport into cell and glycogen syn GLUT4 Glucagon: secreted when Glc low Epi: released by adrenal gland in response to neural signal (flight or flight) Sudden energy response

Intracellular Regulation of Glycogen Metabolism by Interconvertible Enzymes: Low glc activate kinase and breakdown AMP phosphodiesterase cAMP Simultaneous effect Low [Glc]

Regulation of Phosphofructokinase-1 Produced in pancreas in response to low [Glc] Dual activities of PFK-2 reg steady-state conc of Fru-2,6-bisP Increased glycolysis Fruc-6P inc….inc F-2,6-bisP Stim PFK-1 Dec F-2,6-bisP PFK-1 less active…..dec glycolysis Activate Protein Kinase A PFK-1 and pyruvate kinase Dec glycolysis Inc glc syn Stimulate glycogen breakdown Figure 11-17

High [Glc]