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Biochemistry 2/e - Garrett & Grisham

Chapter 23
Gluconeogenesis. Glycogen Metabolism, and the Pentose Phosphate Pathway
to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham
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Biochemistry 2/e - Garrett & Grisham

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

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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

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Biochemistry 2/e - Garrett & Grisham

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!

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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


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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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.
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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!
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

Gluconeogenesis Regulation II
Allosteric and Substrate-Level Control See Figure 23.11 Glucose-6-phosphatase is under substratelevel 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

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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 NaBH4 reduction does not affect activity See pages 473-479 to review glycogen phosphorylase
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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
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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)
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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!
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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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)

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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!
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Biochemistry 2/e - Garrett & Grisham

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!


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Biochemistry 2/e - Garrett & Grisham

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

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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!)
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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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
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Biochemistry 2/e - Garrett & Grisham

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