Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 23 Gluconeogenesis. Glycogen Metabolism, and the Pentose Phosphate.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 23 Gluconeogenesis. Glycogen Metabolism, and the Pentose Phosphate Pathway to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Outline 23.1 Gluconeogenesis 23.2 Regulation of Gluconeogenesis 23.3 Glycogen Catabolism 23.4 Glycogen Synthesis 23.5 Control of Glycogen Metabolism 23.6 The Pentose Phosphate Pathway

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Gluconeogenesis Synthesis of "new glucose" from common metabolites Humans consume 160 g of glucose per day 75% of that is in the brain Body fluids contain only 20 g of glucose Glycogen stores yield 180-200 g of glucose So the body must be able to make its own glucose

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Substrates for Gluconeogenesis Pyruvate, lactate, glycerol, amino acids and all TCA intermediates can be utilized Fatty acids cannot! Why? Most fatty acids yield only acetyl-CoA Acetyl-CoA (through TCA cycle) cannot provide for net synthesis of sugars

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Gluconeogenesis I Occurs mainly in liver and kidneys Not the mere reversal of glycolysis for 2 reasons: –Energetics must change to make gluconeogenesis favorable (delta G of glycolysis = -74 kJ/mol –Reciprocal regulation must turn one on and the other off - this requires something new!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Gluconeogenesis II Something Borrowed, Something New Seven steps of glycolysis are retained: –Steps 2 and 4-9 Three steps are replaced: –Steps 1, 3, and 10 (the regulated steps!) The new reactions provide for a spontaneous pathway (  G negative in the direction of sugar synthesis), and they provide new mechanisms of regulation

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Pyruvate Carboxylase Pyruvate is converted to oxaloacetate The reaction requires ATP and bicarbonate as substrates That should make you think of biotin! Biotin is covalently linked to an active site lysine Acetyl-CoA is an allosteric activator The mechanism (Figure 23.4) is typical of biotin! Regulation: when ATP or acetyl-CoA are high, pyruvate enters gluconeogenesis Note the "conversion problem" in mitochondria

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company PEP Carboxykinase Conversion of oxaloacetate to PEP Lots of energy needed to drive this reaction! Energy is provided in 2 ways: –Decarboxylation is a favorable reaction –GTP is hydrolyzed GTP used here is equivalent to an ATP

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Fructose-1,6-bisphosphatase Hydrolysis of F-1,6-P to F-6-P Thermodynamically favorable -  G in liver is -8.6 kJ/mol Allosteric regulation: –citrate stimulates –fructose-2,6--bisphosphate inhibits –AMP inhibits

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Glucose-6-Phosphatase Conversion of Glucose-6-P to Glucose Presence of G-6-Pase in ER of liver and kidney cells makes gluconeogenesis possible Muscle and brain do not do gluconeogenesis G-6-P is hydrolyzed as it passes into the ER ER vesicles filled with glucose diffuse to the plasma membrane, fuse with it and open, releasing glucose into the bloodstream.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Lactate Recycling How your liver helps you during exercise.... Recall that vigorous exercise can lead to a buildup of lactate and NADH, due to oxygen shortage and the need for more glycolysis NADH can be reoxidized during the reduction of pyruvate to lactate Lactate is then returned to the liver, where it can be reoxidized to pyruvate by liver LDH Liver provides glucose to muscle for exercise and then reprocesses lactate into new glucose

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Regulation of Gluconeogenesis Reciprocal control with glycolysis When glycolysis is turned on, gluconeogenesis should be turned off When energy status of cell is high, glycolysis should be off and pyruvate, etc., should be used for synthesis and storage of glucose When energy status is low, glucose should be rapidly degraded to provide energy The regulated steps of glycolysis are the very steps that are regulated in the reverse direction!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Gluconeogenesis Regulation II Allosteric and Substrate-Level Control See Figure 23.11 Glucose-6-phosphatase is under substrate- level control, not allosteric control The fate of pyruvate depends on acetyl-CoA F-1,6-bisPase is inhibited by AMP, activated by citrate - the reverse of glycolysis Fructose-2,6-bisP is an allosteric inhibitor of F-1,6-bisPase

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 23.3 Glycogen Catabolism Getting glucose from storage (or diet)   -Amylase is an endoglycosidase It cleaves amylopectin or glycogen to maltose, maltotriose and other small oligosaccharides It is active on either side of a branch point, but activity is reduced near the branch points Debranching enzyme cleaves "limit dextrins" Note the 2 activities of the debranching enzyme

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Metabolism of Tissue Glycogen Digestive breakdown is unregulated - 100%! But tissue glycogen is an important energy reservoir - its breakdown is carefully controlled Glycogen consists of "granules" of high MW Glycogen phosphorylase cleaves glucose from the nonreducing ends of glycogen molecules This is a phosphorolysis, not a hydrolysis Metabolic advantage: product is a sugar-P - a "sort-of" glycolysis substrate

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Glycogen Phosphorylase A beautiful protein structure! A dimer of identical subunits (842 res. each) Each subunit contains a PLP, which participates in phosphorolysis, but not in the usual way! Note that NaBH 4 reduction does not affect activity See pages 473-479 to review glycogen phosphorylase

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 23.4 Glycogen Synthesis - I Glucose units are activated for transfer by formation of sugar nucleotides What are other examples of "activation"? –acetyl-CoA, biotin, THF, Leloir showed in the 1950s that glycogen synthesis depends on sugar nucleotides UDP-glucose pyrophosphorylase - Fig. 23.18 –a phosphoanhydride exchange –driven by pyrophosphate hydrolysis

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Glycogen Synthase Forms  -(1  4) glycosidic bonds in glycogen Glycogenin (a protein!) forms the core of a glycogen particle First glucose is linked to a tyrosine -OH Glycogen synthase transfers glucosyl units from UDP-glucose to C-4 hydroxyl at a nonreducing end of a glycogen strand. Note another oxonium ion intermediate (Fig. 23.19)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 23.5 Control of Glycogen Metabolism A highly regulated process, involving reciprocal control of glycogen phosphorylase and glycogen synthase GP allosterically activated by AMP and inhibited by ATP, glucose-6-P and caffeine GS is stimulated by glucose-6-P Both enzymes are regulated by covalent modification - phosphorylation

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Phosphorylation of GP and GS Covalent control Edwin Krebs and Edmond Fisher showed in 1956 that a "converting enzyme" converted phosphorylase b to phosphorylase a(P) Phosphorylation causes the amino terminus of the protein (res 10-22) to swing through 120 degrees, moving into the subunit interface and moving Ser-14 by more than 3.6 nm Nine Ser residues on GS are phosphorylated!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Enzyme Cascades and GP/GS Hormonal regulation Hormones (glucagon, epinephrine) activate adenylyl cyclase cAMP activates kinases and phosphatases that control the phosphorylation of GP and GS GTP-binding proteins (G proteins) mediate the communication between hormone receptor and adenylyl cyclase

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Hormonal Regulation of Glycogen Synthesis and Degradation Insulin is secreted from the pancreas (to liver) in response to an increase in blood glucose Note that the portal vein is the only vein in the body that feeds an organ! Insulin stimulates glycogen synthesis and inhibits glycogen breakdown Note other effects of insulin (Figure 23.22)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Hormonal Regulation II Glucagon and epinephrine Glucagon and epinephrine stimulate glycogen breakdown - opposite effect of insulin! Glucagon (29 res) is also secreted by pancreas Glucagon acts in liver and adipose tissue only! Epinephrine (adrenaline) is released from adrenal glands Epinephrine acts on liver and muscles The phosphorylase cascade amplifies the signal!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Epinephrine and Glucagon The difference.... Both are glycogenolytic but for different reasons! Epinephrine is the fight or flight hormone –rapidly mobilizes large amounts of energy Glucagon is for long-term maintenance of steady-state levels of glucose in the blood –activates glycogen breakdown –activates liver gluconeogenesis Relate these points to sites of action!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Pentose Phosphate Pathway aka hexose monophosphate shunt Provides NADPH for biosynthesis Produces ribose-5-P Two oxidative processes followed by five non-oxidative steps Operates mostly in cytoplasm of liver and adipose cells NADPH is used in cytosol for fatty acid synthesis

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Oxidative Steps of the Pentose Phosphate Pathway Glucose-6-P Dehydrogenase –Irreversible 1st step - highly regulated! Gluconolactonase –Uncatalyzed reaction happens too 6-Phosphogluconate Dehydrogenase –An oxidative decarboxylation (in that order!)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Nonoxidative Steps Five steps, only 4 types of reaction... Phosphopentose isomerase –converts ketose to aldose Phosphopentose Epimerase –epimerizes at C-3 Transketolase (TPP-dependent) –transfer of two-carbon units Transaldolase (Schiff base mechanism) –transfers a three-carbon unit

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Variations on the Pentose Phosphate Pathway 1) Both ribose-5-P and NADPH are needed 2) More ribose-5-P than NADPH is needed 3) More NADPH than ribose-5-P is needed 4) NADPH and ATP are needed, but ribose-5-P is not

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 23 Gluconeogenesis. Glycogen Metabolism, and the Pentose Phosphate.

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 23 Gluconeogenesis. Glycogen Metabolism, and the Pentose Phosphate.

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