Chapter 22

Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway

Biochemistry by 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

Garrett and Grisham, Biochemistry, Third Edition

hexokinase

phosphofructokinase

The pathways of gluconeogenesis and glycolysis.

Animals can not synthesize gluc from fatty acids: acetylCoA cannot yield sugars!!!! Except in plants when the glyoxylate cycle is active.
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.

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

glucokinase

Seven steps of glycolysis are retained: Steps 2 and 4-9.

phosphofructokinase

G=-30 kj/mol

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

G~0

Pyruvate kinase

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

Garrett and Grisham, Biochemistry, Third Edition

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 intermediatea mixed anhydride of carbonic and phosphoric acids. (Carbonylphosphate and carboxyphosphate are synonyms.)
Acetyl CoA activates the caboxylation of Biotin carbanion carbonylphosphate
activated by acetyl-CoA

carboxybiotin

Pyr Pyr dehydrogenase TCA cycle acetylCoA + NADH + CO2

oxaloacetate

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 Pyr carboxylase oxaloacetate before gluconeogenesis can continue.

acetyl CoA TCA

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
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+ G= -37.7 kj/mol (physiological conditions G= -15.6 kj/mol

Reverse of glycolysis 2 pyr + 2ATP + 2NADH + 2H+ + 2H2O gluc + 2ADP + 2Pi + 2NAD+ G= +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!

Garrett and Grisham, Biochemistry, Third Edition

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

Garrett and Grisham, Biochemistry, Third Edition

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-1,6bisphosphatase 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. Phosphorylation inhibits PFK-2 activity and activates F2,6BPase

Phosphofructokinase 2

F2-6 biphosphatase

Phosphofructokinase 1

Fructose 1,6biphosphatase

How Are Glycogen and Starch Catabolized in Animals?

Getting glucose from diet: -Amylase is an endoglycosidase, it cleaves dietary amylopectin or glycogen (1-4 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 1-6 glucosidase

Garrett and Grisham, Biochemistry, Third Edition

The reactions of glycogen debranching enzyme. Transfer of a group of three -(1 4)linked glucose residues from a limit branch to another branch is followed by cleavage of the -(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.

G=3.1 kj/mol;
G 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
ester linkage

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

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 phosphorus of UTP to form UDP-glucose and pyrophosphate.

Hydrolysis irreversible pyrophosphate

Garrett and Grisham, Biochemistry, Third Edition

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. oxonium ion intermediate is formed after cleavage of CO bond between gluc and -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. G= -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
phosphorylation Ser 14

phosphorylase b excitement, fear

phosphorylase a Ser phosphorylation a form

phosphorylase kinase levels epinephrine

Garrett and Grisham, Biochemistry, Third Edition

phosphorylase b phosphorylase a

equilibrium favor T state equilibrium favor R state

rotation around the dimer axis structural change 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 Garrett and Grisham, Biochemistry, Third Edition 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 -isozymic forms

no energy charge in liver!!!

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)

Garrett and Grisham, Biochemistry, Third Edition