Carbohydrate

metabolism III

Outline

• Glycogen catabolism

• Glycogen cynthesis

• Control of glycogen metabolism

• Metabolism of fructose, galactose and

mannose

• Biosynthesys of glucuronic acid

• Disorders of carbohydrate metabolism

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

Glycogen is a polymer of glucose residues linked by

(14) glycosidic bonds, mainly

(16) glycosidic bonds, at branch points.

Glycogen chains and branches are longer than shown.

Glucose is stored as glycogen predominantly in liverand muscle cells.

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

Glycogen catabolismbreakdown

• Glycogen Phosphorylase catalyzes phosphorolytic cleavage of the (14) glycosidic linkages of glycogen, releasing glucose-1-phosphate as reaction product.

glycogen(n residues) + Pi

glycogen(n–1 residues) + glucose-1-P

• This phosphorolysis may be compared tohydrolysis:

• Hydrolysis: R-O-R' + HOH R-OH + R'-OH

• Phosphorolysis: R-O-R' + HO-PO32- R-OH + R'-O-PO3

2-

Glycogen Phosphorylase (GP)

• Pyridoxal phosphate (PLP), a

derivative of vitamin B6, serves

as prosthetic group for GP.

• PLP is held at the active site by a

Schiff base linkage, formed by

reaction of the aldehyde of PLP

with the -amino group of a

lysine residue.

• In contrast to its role in other

enzymes, the phosphate of PLP

is involved in acid/base catalysis

by Phosphorylase.

• A glycogen storage site on the surface of

the Phosphorylase enzyme binds the

glycogen particle.

• Given the distance between storage and

active sites, Phosphorylase can cleave

(14) linkages only to within 4 residues of

an (16) branch point.

• This is called a "limit branch".

Glycogen Phosphorylase

Debranching enzyme

• Debranching enzyme has 2 independent active sites, consisting of residues in different segments of a single polypeptide chain:

• The transferase of the debranching enzyme transfers 3 glucose residues from a 4-residue limit branch to the end of another branch, diminishing the limit branch to a single glucose residue.

• The (16) glucosidase moiety of the debranching enzyme then catalyzes hydrolysis of the (16) linkage, yielding free glucose. This is a minor fraction of glucose released from glycogen.

• The major product of glycogen breakdown is glucose-1-phosphate, from Phosphorylase activity.

Phosphoglucomutase

• catalyzes the reversible reaction:

glucose-1-phosphate glucose-6-phosphate

• serine -OH at the active site donates and accepts Pi.

• the bisphosphate is not released

• Glucose-6-P may enter Glycolysis or (mainly in liver) be dephosphorylated for release to the blood.

• Liver Glucose-6-phosphatase catalyzes the following, essential to the liver's role in maintaining blood glucose: glucose-6-P + H2O glucose + Pi

• Most other tissues lack this enzyme.

Glycogen Synthesis

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

– a phosphoanhydride exchange

– driven by pyrophosphate hydrolysis

• Uridine diphosphate glucose (UDP-glucose) is the immediate precursor for glycogen synthesis.

• As glucose residues are added to glycogen, UDP-glucose is the substrate and UDP is released as a reaction product.

• Nucleotide diphosphate sugars are precursors also for synthesis of other complex carbohydrates, including oligosaccharide chains of glycoproteins, etc.

Glycogen Synthesis

UDP-Glucose pyrophosphorylase

• UDP-glucose is formed from glucose-1-phosphate:

• glucose-1-phosphate + UTP UDP-glucose + PPi

• PPi + H2O 2 Pi

Overall:

glucose-1-phosphate + UTP UDP-glucose + 2 Pi

• Spontaneous hydrolysis of the ~P bond in PPi

(P~P) drives the overall reaction.

• Cleavage of PPi is the only energy cost for glycogen synthesis (one ~P bond per glucose residue).

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.

• A glycosidic bond is formed between the anomeric C1 of the glucose moiety derived from UDP-glucose and the hydroxyl oxygen of a tyrosine side-chain of Glycogenin.

• UDP is released as a product.

• Glycogen Synthase then catalyzes elongation

of glycogen chains initiated by Glycogenin.

• Question: Where would you expect to find

Glycogenin within a cell?

• Answer: Most of the Glycogenin is found

associated with glycogen particles (branched

glycogen chains) in the cytoplasm.

Glycogen Synthase

Glycogen Synthase

• Glycogen Synthase catalyzes transfer of the glucose moiety of UDP-glucose to the hydroxyl at C4 of the terminal residue of a glycogen chain to form an

(1 4) glycosidic linkage:

glycogen(n residues) + UDP-glucose

glycogen(n +1 residues) + UDP

• A branching enzyme transfers a segment from the end of a glycogen chain to the C6 hydroxyl of a glucose residue of glycogen to yield a branch with an

(1 6) linkage.

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

Regulation of glycogene metabolism

• Both synthesis and breakdown of glycogen are spontaneous.

• If both pathways were active simultaneously in a cell, there would be a "futile cycle" with cleavage of one ~P bond per cycle (in forming UDP-glucose).

• To prevent such a futile cycle, Glycogen Synthase and Glycogen Phosphorylase are reciprocally regulated, by allosteric effectors and by phosphorylation.

Regulation of GP

• Glycogen Synthase is allosterically activated by glucose-6-P (opposite of effect on GP).

• Thus Glycogen Synthase is active when high blood glucose leads to elevated intracellular glucose-6-P.

• It is useful to a cell to store glucose as glycogen when the input to Glycolysis (glucose-6-P), and the main product of Glycolysis (ATP), are adequate.

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)

• Nine Ser residues on GS are

phosphorylated!

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

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!

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

• Signal

cascade by

which

Glycogen

Phosphorylase

is activated.

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

Sources of Sugars

• Glucose: lactose (dairy products) and

sucrose (table sugar)

• Fructose: fruits and sucrose

• Galactose: lactose

• Mannose: polysaccharides and

glycoproteins

Other Substrates for Glycolysis

Fructose, galactose, and mannose

• Fructose and mannose are routed into glycolysis by fairly conventional means.

• Galactose is more interesting - the Leloir pathway "converts" galactose to glucose

Metabolism of fructose

• Source - food – (saccharose, free)

• Synthese in cels – reduction of glucose

sorbitol oxidation fructose

• Metabolism occures in the liver (faster

than glucose – fructokinase have higher

activity)

• Target – glycolyse

Sucrose(Table Sugar)

O- -D-Glucopyranosyl-(1—>2)- -D-Fructofuranoside

Sucrose -D-glucose + -D-fructosea-Glucosidase(Invertase)

Glycolysis

Mutarotation

-D-fructose

All Tissues

Muscle Metabolism of Fructose(Anaerobic Glycolysis)

Large Amounts of Hexokinase

• Formation fructose-6-P (muscle) alternative wayphosphorylation - limited, because activity ofhexokinase is 1/20 from activity for glucose

• Minority alternative way – reduction fructose to sorbitolproduce glucose

Liver Metabolism of Fructose I(Little Hexokinase)

• Obvious way transformation is fosforylation in the liver (partially in intestine epithel and in kidney)

• Fosforylation can not effect hunger or insulinecompensate glucose for diabetics

Liver Metabolism of Fructose II

Liver Metabolism of Fructose III

Fructose Intolerance

• Too Much Fructose

–Fructose-1-P Aldolase is rate-limiting

–Depletion of Pi

–Reduction in [ATP]

– Increase in glycolysis

–Accumulation of lactate (acid) in blood

• Fructose-1-P Aldolase Deficiency (Genetic Disease)

Metabolism of galactose

• Source - lactose

• Occures in the liver

• Galactose is necessary for synthesis of lactose, glycolipids, protheoglycans, glycoprotheins or can converted to glucose

• UDP-galactose is high energy molecules, galactose have ability bounded to other saccharides(-OH compound) and formed O-glycosidic bound (syntheseglycosaminoglycanes, glycoproteines, glycolipides)

Lactose Metabolism(Dairy Products)

• Mutarotation of β-D-Galactose

• Glycolytic Enzymes are specific and do not

recognize galactose!

Phosphorylation of Galactose

Activation of Galactose

Epimerization of UDP-Galactose

• Glycoproteins

• Glycolipids

Why UDP-Galactose?

Metabolism of manose

• Minority part of diet

• Important part of glycoproteines

The Role of Glucuronic acid

• Uridine-1-diphospho glucose can be used for:

• biosynthesis of glycogen

• biosynthesis of galactose

• after the oxidation at the 6th carbon atom is formed UDP-

glucuronic acid, which is used for:

• conjugation (bilirubine etc.)

• biosynthesis of glycosaminoglycanes (hialuronic acid,

heparin)

• biosynthesis of several pentoses (xylose, L-xylulose)

• biosynthesis of ascorbic acid

• Products capable be transformed in phosphopentose

pathway:

Glucuronate pathway Phosphopentose pathway

Synthesis of

glucuronic acid

Transformation of glucose-6-P

Glc-6-P Glucose (55 %)

Fru-6-PGlucosamine

(aminosaccharides)

Glycolysis (25 %)Glc-1-P

Glycogene (18 %)Glucuronate

Gulonate (vit. C)

6-P-Gluconate (2 %)

Penthose cycle

fosfatase

isomerase

mutasedehydrogenase

UTP