Gluconeogenesis, glycogen metabolism

Garrett and Grisham, Biochemistry, Third Edition

Chapter 22

Gluconeogenesis, Glycogen Metabolism, and the Pentose

Phosphate Pathway

Biochemistryby

Reginald Garrett and Charles Grisham


• Nature of gluconeogenesis.• Pathway that synthesizes glucose from

noncarbohydrate precursors.• Synthesis of glycogen.• How are e- from glucose used in biosynthesis.


What Is Gluconeogenesis?

• 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.

Synthesis of "new glucose" from common metabolites


•Muscle: consume gluc via glycolysis pyr and lactate

» anaerobic conditions» gluc

»Pentose Phosphate pathway: NADPH»» Biosynthesis fatty acids/aa/ribose 5PO4» ATP, NAD+ FAD, CoA, DNA/RNA

The pathways of gluconeogenesis and glycolysis.

Animals can not synthesize gluc from fatty acids: acetyl-CoA cannot yield sugars!!!!

Except in plants when the glyoxylate cycle is active.

hexokinase

phosphofructokinase

Pyr kinase


Substrates for Gluconeogenesis

Pyruvate, lactate, glycerol, amino acids and all TCA intermediates can be utilized

• Fatty acids cannot! Most fatty acids yield only acetyl-CoA

• Acetyl-CoA (through TCA cycle) cannot provide for net synthesis of sugars

Acetyl-CoA can be a substrate for gluc synthesis only when the glyoxylate cycle is active.


Gluconeogenesis

• Occurs mainly in liver and kidneys

• Not the mere reversal of glycolysis for 2 reasons:

– Energetics must change to make gluconeogenesis favorable (∆G of glycolysis = -74 kJ/mol)

– Reciprocal regulation: one must turn on and the other off -this requires something new!

– Unique routes for each pathway!!!!


• 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

glucokinase

phosphofructokinase

Pyruvate kinase

ΔG°~0

ΔG°=-30 kj/mol


Pyruvate Carboxylase: biotin dependent enzyme

• Biotin is covalently linked to an active site lysine

• Acetyl-CoA is an allosteric activator: when ATP or acetyl-CoA are high, pyruvate enters gluconeogenesis

• Reaction occurs in mitochondria

A mechanism for the pyruvate carboxylase reaction. Bicarbonate must be activated for attack by the pyruvate carbanion. This activation is driven by ATP and involves formation of a carbonylphosphate intermediate—a mixed anhydride of carbonic and phosphoric acids. (Carbonylphosphate and carboxyphosphate are synonyms.)

Acetyl CoA activates the caboxylation of Biotin

carboxybiotin

oxaloacetate

carbonylphosphate

Pyr

Pyr dehydrogenaseTCA cycle

acetylCoA + NADH + CO2

carbanion

activated by acetyl-CoA

Pyruvate carboxylase is a compartmentalized reaction. Pyruvate is converted to oxaloacetate in the mitochondria. Because oxaloacetate cannot be transported across the mitochondrial membrane, it must be reduced to malate, transported to the cytosol, and then oxidized back to oxaloacetate before gluconeogenesis can continue. acetyl CoA TCAPyr carboxylase


PEP CarboxykinaseConversion 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

� ∆G pyr carbox/PEPcarboxykin = -22.6 kj/mol

– Phosphoglycerate mutase, – Phosphoglycerate kinase, – glyceraldehyde 3P dehydrogenase, – aldolase, – triose phosphate isomerase fructose 1,6 biphosphate


Fructose-1,6-bisphosphatase

• Thermodynamically favorable - ∆G in liver is -8.6 kJ/mol

• Allosteric regulation:– citrate stimulates – fructose-2,6-bisphosphate inhibits – AMP inhibits


Glucose-6-Phosphatase

• 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.

phosphohistidine intermediate


•Net reaction:

•2 pyr + 4ATP + 2 GTP + 2NADH + 2H+ + 6H2O» gluc + 4ADP + 2GDP + 6Pi + 2NAD+»ΔG°= -37.7 kj/mol (physiological conditions ΔG°= -15.6 kj/mol

•Reverse of glycolysis•2 pyr + 2ATP + 2NADH + 2H+ + 2H2O

» gluc + 2ADP + 2Pi + 2NAD+»ΔG°= +74 kj/mol


Lactate Recycling: Cori Cycle

Liver helps you during exercise....• Recall that vigorous exercise can

lead to a buildup of lactate and NADH. O2 shortage to regenerate NAD+ 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

lactate dehydrogenase


How Is Gluconeogenesis Regulated?

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!

The principal regulatory mechanisms in glycolysis and gluconeogenesis. Activators are indicated by plus signs and inhibitors by minus signs.

Allosteric and substrate level regulation!!!

Pyr dehydrogenase - acetylCoA


Allosteric and Substrate-Level Control

• 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

Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate in the (a) absence and (b) presence of 25 mM AMP. Effects of AMP and fructose-2,6-bisphosphate are synergetic.

In (a) and (b), enzyme activity is plotted against substrate (fructose-1,6-bisphosphate) concentration. Concentrations of fructose-2,6-bisphosphate (in mM) are indicated above each curve. (c) The effect of AMP (0, 10, and 25 mM) on the inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate. Activity was measured in the presence of 10 mM fructose-1,6-bisphosphate.

Effect AMP and F2-6BP are synergetic

Synthesis and degradation of fructose-2,6-bisphosphate are catalyzed by the same bifunctional enzyme.

Phosphofructokinase 2

Fructose 1,6biphosphatase

Phosphorylation inhibits PFK-2 activityand activates F2,6BPase

F2-6 biphosphatase

Phosphofructokinase 1


How Are Glycogen and Starch Catabolized in Animals?

Getting glucose from diet: • α-Amylase is an endoglycosidase,

it cleaves dietary amylopectin or glycogen (α1-4 linkages) 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“

• Two activities of the debranching enzyme:glucanotransferase and α1-6 glucosidase


The reactions of glycogen debranching enzyme. Transfer of a group of three α-(1 4)-linked glucose residues from a limit branch to another branch is followed by cleavage of the α -(1 6) bond of the residue that remains at the branch point.


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, liver and muscle

• 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


The glycogen phosphorylase reaction. ΔG°=3.1 kj/mol; ΔG in vivo=-6 kj/mol [Pi]/[glu1PO4]=100


How Is Glycogen Synthesized?Glucose units: activated for transfer

by formation of sugar nucleotides

• Acetyl-CoA: acetate• Biotin, THF activate one C-transfer• ATP: phosphate

• Leloir showed in the 1950s that glycogen synthesis depends on sugar nucleotides

• UDP-glucose pyrophosphorylase –– a phosphoanhydride exchange– driven by pyrophosphate

hydrolysis

ester linkage


The UDP-glucose pyrophosphorylase reaction is a phosphoanhydride exchange, with a phosphoryl oxygen of glucose-1-P attacking the α-phosphorus of UTP to form UDP-glucose and pyrophosphate.

pyrophosphate

Hydrolysis irreversible


Glycogen Synthase

Forms α-(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.

• oxonium ion intermediate is formed after cleavage of CO bond between gluc and β-PO4 of UDP-gluc. The intermediate is attacked by the C4-OH of terminal gluc of glycogen


The glycogen synthase reaction. Cleavage of the C-O bond of UDP-glucose yields an oxonium intermediate. Attack by the hydroxyl oxygen of the terminal residue of a glycogen molecule completes the reaction.

ΔG°= -13.3 kj/mol


Formation of glycogen branches by the branching enzyme (amylo 1,4 1,6 transglycosylase).

Increase solubility and increases points for degradation and synthesis.

Six- or seven-residue segments of a growing glycogen chain are transferred to the C-6 hydroxyl group of a glucose residue on the same or a nearby chain.


How Is Glycogen Metabolism Controlled?

A highly regulated process, involving reciprocal control of glycogen phosphorylase and glycogen synthase

• glycogen phosphorylase allosterically activated by AMP and inhibited by ATP, glucose-6-P and caffeine

• glycogen synthase is stimulated by glucose-6-P

• Both enzymes are regulated by covalent modification: phosphorylation


glycogen metabolism regulation phosphorylase regulation allosteric

phosphorylation (responsive to insulin, epinephrine, glucagon)

Muscle: uses glucose to produce energy Liver: maintains glucose levels

Muscle phosphorylase

active, relax inactive, tense

phosphorylase b phosphorylation Ser 14 phosphorylase a phosphorylase kinase

excitement, fear levels epinephrine Ser phosphorylation a form


phosphorylase b equilibrium favor T state phosphorylase a equilibrium favor R state

rotation around the dimer axis structural change

α helices move a loop out of active site!!!!

phosphoylase b: R form T form AMP vs ATP cell energy charge phosphoylase b is active with high [AMP] positive allosteric effector glucose 6 PO4 favor T state feedback inhibition

physiological conditions: glucose 6-PO4/ATP phosphoylase b inactive phosphoylase a is active Exercise AMP increase b active hormones more a form


Liver phosphorylase

-liver phosphorylase 90% identical muscle phosphorylase -liver: a form is more responsive than b form to T-R transition

glucose negative regulator

-no allosteric regulation by AMP no energy charge in liver!!! -isozymic forms tissue specific biochemical properties


phosphorylase kinase activate by phosphorylation and Ca++

signal transduction pathway!!!

phosphorylation by protein kinase A (PKA) active form

activated by cyclic AMP (second messenger)

epinephrine

calmodulin (Ca++ sensor)

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Gluconeogenesis, glycogen metabolism