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Garrett and Grisham, Biochemistry, Third Edition
Chapter 22
Gluconeogenesis, Glycogen Metabolism, and the Pentose
Phosphate Pathway
Biochemistryby
Reginald Garrett and Charles Grisham
Garrett and Grisham, Biochemistry, Third Edition
• Nature of gluconeogenesis.• Pathway that synthesizes glucose from
noncarbohydrate precursors.• Synthesis of glycogen.• How are e- from glucose used in biosynthesis.
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
•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
Garrett and Grisham, Biochemistry, Third Edition
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.
Garrett and Grisham, Biochemistry, Third Edition
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!!!!
Garrett and Grisham, Biochemistry, Third Edition
• 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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
•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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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.
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
The glycogen phosphorylase reaction. ΔG°=3.1 kj/mol; ΔG in vivo=-6 kj/mol [Pi]/[glu1PO4]=100
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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.
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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
Garrett and Grisham, Biochemistry, Third Edition
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)