Pentose phosphate pathway-glycogen metabolism

Outlines of the PPP • Functions of the pentose phosphate pathway • Oxidative reactions: forming ribose 5-phosphate from glucose 6-phosphate • Non-oxidative reactions: forming glycolysi

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Outlines of the PPP

•  Functions of the pentose phosphate pathway

•   Oxidative reactions: forming ribose 5-phosphate from glucose 6-phosphate

•   Non-oxidative reactions: forming glycolysis

intermediates

•   Transition of carbon skeleton

•   Control of the PPP

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Functions of pentose phosphate pathway

•  To produce NADPH, the 2nd

cellular currency NADPH is

used for reductive biosynthesis

of fatty acids and cholesterol

•  To produce ribose 5-phosphate

used for nucleic acid synthesis

•  Despite their close chemical

structures, NADPH & NADH

are not metabolically

interchangeable:

-  NADH uses free energy of metabolite oxidation for ATP synthesis

-  NADPH uses free energy of metabolite oxidation for reductive biosynthesis

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Functions of pentose phosphate pathway

Biochemistry, Tymoczko, Berge, Strayer

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The pentose phosphate pathway (PPP)

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1.  Oxidative phase: oxidation reaction

Glucose 6-phosphate 6-Phosphoglucono-δ lactone

Ribulose 5-phosphate 6-Phosphogluconate

Overall: Formation of 2 reducing power NADPH & 1 ribulose 6-phosphate from the 1st phase (oxidative phase) of the PPP

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2 Non-oxidative phase: isomerization reactions

Ribulose 5- phosphate (R5P) Xylulose 5-phosphate (X5P) R5P + X5P

Erythose 4-phosphate

Fructose 6-phosphate +

Glyceraldehyde 3-phosphate

Sedoheptulose 7-phosphate +

Xylulose 5-phosphate

+

Fructose 6-phosphate

+

Glyceraldehyde

3-phosphate

Overall: generation of G3P & F6P from 2 R5P & 1

X5P in the 2st phase (non-oxidative phase)

Ribulose 5-phosphate Ribose 5- phosphate (R5P)

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Outlines of the PPP

•  Oxidative reactions: forming ribose 5-phosphate from glucose 6-phosphate

•   Non-oxidative reactions: forming glycolysis

intermediates

•   Transition of carbon skeleton

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Oxidative phase of the PPP

•  Overall reaction:

–  Conversion of glucose phosphate (6C) into ribulose

6-phosphate (5C) –  Generation of 2 NADPH molecules

Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

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–  This metabolite may arise through glycogen breakdown

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•  Reaction 1:

–  G6P, a cyclic hemiacetal with C1 in the aldehyde oxidation state,

is thereby oxidized to a cyclic ester (lactone) –  The enzyme is specific for NADP and is strongly inhibited by

NADPH –  The 1 st NADPH is generated

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•  Reaction 2: ring opening

–  Hydrolysis of phosphoglucono-δ lactone to form

6-phosphogluconate

–  Enzyme: gluconolactonase

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•  Reaction 3: formation of ribulose 6-phosphate

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Outlines of the PPP

•  Oxidative reactions: forming ribulose

5-phosphate from glucose 6-5-phosphate

•  Non-oxidative reactions: forming glycolysis

intermediates

•  Transition of carbon skeleton

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Non-oxidative phase of the PPP

•  Reaction 4: Isomerization of ribulose 5- phosphate to

form ribose 5-phosphate

–  Enzyme: ribulose 5-phosphate isomerase

–  Mechanism: similar to triose phosphate isomerase (reaction 5 in glycolysis, isomerization of dihydroxyacetone to glyceraldehyde 3-phosphate)

Biochemistry by Garrett and Grisham

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•  Reaction 5: Interconversion of dihydroxyacetone

phosphate to glyceraldehyde-3-phosphate:

First stage of glycolysis

to oxygen in C2

4 Glutamic acid now

is acting as an acid by donating proton to C2

3 His removes a proton from OH group

of C1

5 The product is

formed, Glu and His

are returned to their

ionized and neutral

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•  Reaction 5: Epimerization of ribulose 5-phosphate to

form xylulose 5-phosphate

–  Enzyme: ribulose 5-phosphate epimerase

–  Mechanism: reaction proceeds by an enediol intermediate and

an inversion at C3

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•  Reaction 6: Conversion of xylulose 5-phosphate (5C)

and ribose 5-phosphate (5C) glyceraldehyde

3-phosphate (3C) and sepdoheptulose 7-3-phosphate (7C)

–  Enzyme: transketolase (transfers a ketone)

–  Mechanism: requires coenzyme thiamine pyrophosphate

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•  Transfer a 2C unit from X5P to TPP

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•  Transfer a 2C unit from TPP to R5P

Biochemistry by Garrett and Grisham 20

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•  Reaction 7: Conversion of sedoheptulose 7-phosphate (7C) and glycerladehyde 3-phosphate (3C) to form

erythrose 4-phosphate (4C) and fructose 6-phosphate (6C)

–  Enzyme: transaldolase (transfers a 3C dihydroxyacetone unit )

–  Mechanism: does not require a prosthetic group

Biochemistry by Garrett and Grisham 21

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•  Reaction 8: Conversion of xylulose 5-phosphate (5C)

and erythrose 4-phosphate (4C) to form glycerladehyde 3-phosphate (3C) and fructose 6-phosphate (6C)

–  Enzyme: transketolase (transfers a 2C unit )

–  Mechanism: require a prosthetic group TPP

Biochemistry by Garrett and Grisham Biochemistry by Garrett and Grisham

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•  Overall reaction in nonoxidative phase:

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Outlines of the PPP

•  Oxidative reactions: forming ribulose

5-phosphate from glucose 6-5-phosphate

•  Non-oxidative reactions: forming glycolysis

intermediates

•  Transition of carbon skeleton

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Regulation of the PPP

Relationship between glycolysis and the PPP

1 The PPP, which begins with G6P produced in of glycolysis,

generates NADPH for use in reductive biosynthesis reactions and R5P for nucleotide synthesis

2 Excess R5P is converted to glycolytic intermediates (F6P & GAP) by a sequence of reactions that can operate in reverse to

generate additional R5P, if needed

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Utilization of Glucose 6-phosphate

•  Glucose 6-phosphate can be used as a substrate either for glycolysis or for the pentose phosphate pathway

depending on the cell’s need for biosynthesis (PPP) or for energy from metabolism (glycolysis)

•  The fate of glucose 6-phosphate is determined to a large extent by the relative activities of phosphofructokinase (glycolysis) and glucose 6-P dehydrogenase (PPP)

•  PFK is inhibited when the ATP/AMP ratio increases, it is inhibited by citrate but activated by fructose-2,6-

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1.  Both Ribose 5-P and NADPH Are Needed by the Cell:

→ the first 4 reactions of the PPP predominate

2 More Ribose 5-P Than NADPH Is Needed by the Cell:

→ oxidative steps of the PPP are bypassed

usage of 2 fructose 6-P and 1 glyceraldehyde 3-P

from glycolysis to produce 3 ribose 5-P

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3 More NADPH Than Ribose 5-P Is Needed by the Cell:

→  Ribose 5-P produced in the PPP is recycled to produce

glycolytic intermediates

→  transketolase and transaldolase reactions to convert

ribulose 5-P to fructose 6-P and glyceraldehyde3-P,

which can be recycled to glucose 6-P via

gluconeogenesis

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4 Both NADPH and ATP Are Needed by the Cell, but

Ribose 5-P is Not

→ accomplished in a series of reactions similar to case 3

if the fructose 6-P and glyceraldehyde3-P produced in proceed through glycolysis to produce ATP and pyruvate, which itself can yield even more ATP by continuing on to the TCA cycle

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G6P dehydrogenase protects cell from ROS

•  NADPH generated in the PPP by glucose 6-phosphate dehydrogenase plays a vital role in protecting the cells from ROS generated in oxidative metabolism

•  Reduced glutathione (GSH), a tripeptide with a free

sulfhydryl group, combats oxidative stress by reducing ROS to harmless forms Its task accomplished, the

glutathione is now in the oxidized form (GSSG) and must

be reduced to regenerate GSH:

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Glycogen

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Review of glycogen

•  Storage of polysaccharides in animals

•  Present in all cells but most prevalent in skeletal muscle and in liver as cytoplasmic granules

•  The primary structure of glycogen resembles that of amylopectin but more highly branched, with branch points occurring every 8 to 14 glucose residues

Branches every 25-30 units Branches every 8-14 units

Biochemistry lecture note from Prof Hrycyna, Purdue University

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Review of glycogen

•  Sugars react with oxidizing agents → reducing sugars

•  Saccharides bearing anomeric carbons that have not

formed glycosides are termed reducing sugars

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Overview of glucose metabolism

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Outlines of glycogen metabolism

•  Glycogen degradation

•  Regulation of glycogen degradation

reciprocally regulated

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Glycogen degradation or glycogenolysis

Glycogen is degraded at its nonreducing ends to release glucose units

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3 enzymes required in glycogen degradation:

1.  Glycogen phosphorylase (or phosphorylase): catalyzes

phospholysis of glycogen to yield glucose 1-phosphate (G1P)

2.  Glycogen debranching enzyme: removes branches of

glycogen, making more glucose residues accessible to

phosphorylase

3.  Phosphoglucomutase: converts G1P to G6P

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1 Glycogen phosphorylase (or phosphorylase): a dimer of

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1.  Glycogen phosphorylase (or phosphorylase): a dimer of

2 identical 97kD subunits

-  Each subunit has a glycogen binding site and a catalytic site

-  The orthophosphate substrate is bound to the catalytic site

-  The cofactor PLP is linked to Lys 680 of the enzyme

-  The separation of the binding site and catalytic site allows the

catalytic site to phosphorolyze several glucose units before the enzyme must rebind the glycogen substrate

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1.  Glycogen phosphorylase (or phosphorylase):

- PLP cofactor, a vitamin B6 derivative

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+

+ +

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2 Glycogen debranching enzyme

→  2 additional enzymes,

transferase and α-1,6-glucosidase:

•  Transferase shifts a block of 3

glucosyl residues from one outer branch to another

•  α-1,6-glucosidase hydrolyzes the

exposed single glucose residue joined by an a-1,6-glycosidic linkage

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3 Phosphoglucomutase converts glucose 1-phosphate to glucose 6-phosphate

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3 Phosphoglucomutase:

Its mechanism is similar to phosphoglycerate mutase

Remember: Phosphoglycerate mutase catalyzes the interconversion

of 3-phosphoglycerate into 2-phosphoglycerate in glycolysis

Phospho-Ser hydroxyl group residue

Phosphoglucomutase Phosphoglycerate mutase

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Glucose 6-phosphatase generates glucose in liver

•  Glucose 6-phosphate released from glycogen

breakdown can continue the glycolysis or the PPP

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Glucose 6-phosphatase generates glucose in liver

•  Glucose 6-phosphate can be converted into glucose by glucose 6-phosphatase presenting in liver:

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•  Regulation of glycogen degradation

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Regulation of glycogen phosphorylase

•  Phosphorylase is regulated by several allosteric effectors that signal the energy state of the cell as well as by

reversible phosphorylation, which is responsive to

hormones such as insulin, epinephrine, and glucagon

•  There are differences in the control of glycogen

degradation in skeletal muscle and liver, due to the fact

that the muscle uses glucose to produce energy for itself,

whereas the liver maintains glucose homeostasis of the organism as a whole

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Structure of phosphorylase: dimer composing of 2 subunits

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1.  A) Muscle phosphorylase is regulated by intracellular

energy charge:

Default state of muscle

phosphorylase is b form (b form

when resting)

Contracting muscle, ATP → AMP, muscle

phosphorylase b is activated → active R state → glycogen degradation

Low energy charge (high AMP conc.) favors the transition to R- state, signaled glycogen degradation

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1 B) Muscle phosphorylase is regulated by hormones

(covalent modification)

Fear or excitement of exercise increases the

hormone epinephrine level, b form is converted

into a form by phoshorylation of a single serine

residue in each subunit

Phosphorylase kinase

Phosphorylation

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2 Liver phosphorylase produces glucose for the use by

other tissues

When glucose is sufficient, no

need to degrade glycogen, the

enzyme reverts to the less active

T state

The default state of liver phoshorylase

is in a form (active R state) to form glucose to export to other tissues when blood-glucose is low

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3 Phosphorylase kinase is activated by

phosphorylation and Ca2+

Phosphorylation kinase A (PKA)

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4 Epinephrine & glucagon signal the need for

•  Glucagon and epinephrine trigger

cAMP cascade → initiate glycogen

degradation

•  When energy needs have been

met, phosphorylase kinase &

phosphorylase are inactivated→

glycogen degradation is shut off

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•  Glycogen synthesis

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Step 1: Uridine diphosphate glucose (UDP-glucose)

•  UDP-glucose is an activated form of glucose which acts as a glucose donor

•  UDP-glucose is synthesized from glucose phosphate and UTP The reaction is catalyzed by UDP glucose pyrophosphorylase:

1-•  Pyrophosphate PPi is rapidly hydrolysed to form 2 orthophosphate This irreversible reaction drives the synthesis of UDP-glucose:

Biochemistry, Tymoczko, Berge, Strayer Fundamentals of biochemistry-Life at the

molecular level, Voet, Voet, Pratt

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