Chapter 4 Carbohydrates Metabolism The biochemistry and molecular biology department of CMU.

Chapter 4

Carbohydrates Metabolism

The biochemistry and molecular biology department of CMU

§ 1 Overview

• Carbohydrates in general are polyhydroxy aldehydes or ketones or compounds which yield these on hydrolysis.

Biosignificance of Carbohydrates

• The major source of carbon atoms and energy for living organisms.

• Supplying a huge array of metabolic intermediates for biosynthetic reactions.

• The structural elements in cell coat or connective tissues.

Glucose transporters (GLUT)

• GLUT1~5

GLUT1: RBC

GLUT4: adipose tissue, muscle

The metabolism of glucose

• glycolysis• aerobic oxidation• pentose phosphate pathway• glycogen synthesis and

catabolism• gluconeogenesis

glycogen

Glycogenesis Glycogenolysis

Pentose phosphate pathway

Ribose, NADPH

Glycolysis lactate

H2O+CO2

aerobic oxidation

Digestion absorption

starch

Lactate, amino acids, glycerol

glucose

Gluconeo-

genesis

§2 Glycolysis

Glycolysis

• The anaerobic catabolic pathway by

which a molecule of glucose is broken

down into two molecules of lactate.

glucose →2lactic acid (lack of O2)

• All of the enzymes of glycolysis locate

in cytosol.

1. The procedure of glycolysis

G

pyruvate

lactic acid

glycolytic pathway

1) Glycolytic pathway :

G → pyruvate

including 10 reactions.

• Phosphorylated G cannot get out of cell

• Hexokinase , HK (4 isoenzymes) ,

glucokinase, GK in liver ;

• Irreversible .

(1) G phosphorylated into glucose 6-phosphate

OH

OH

H

OHH

OHH

OH

CH2

H

HOOH

OH

H

OHH

OHH

OH

CH2

H

OPATP ADP

Hexokinase

Mg2+

G G-6-P

hexokinase

glucokinase

occurrence in all tissues only in liver

Km value 0.1mmol/L 10mmol/L

Substrate G, fructose, glucose mannose

Regulation G-6-P Insulin

Comparison of hexokinase and glucokinase

(2) G-6-P → fructose 6-phosphate

OH

OH

H

OHH

OHH

OH

CH2

H

OP

G-6-P

isomerase OH

CH2OH

H

CH2

OH H

H OH

OOP

F-6-P

(3) F-6-P → fructose 1,6-bisphosphate

• The second phosphorylation • phosphofructokinase-1, PFK-1

OH

CH2OH

H

CH2

OH H

H OH

OOP

F-1,6-BP

OH

CH2

H

CH2

OH H

H OH

OOP O P

ATP ADP

Mg2+

F-6-P

PFK-1

(4) F-1,6-BP → 2 Triose phosphates

• Reversible

F-1,6-BP

CH2

C O

C HHO

C OHH

C OHH

CH2

O P

O P

CH2

C O

O P CHO

CHOH

CH2 O PCH2OH

+aldolase

dihydroxyacetone phosphate,

DHAP

glyceraldehyde 3-phosphate,

GAP

(5) Triose phosphate isomerization

G→2 molecule glyceraldehyde-3-phosphate, consume 2 ATP .

CH2

C O

O P CHO

CHOH

CH2 O PCH2OH

DHAP GAP

phosphotriose isomerase

(6) Glyceraldehyde 3-phosphate → glycerate 1,3-bisphosphate

CHO

CHOH

CH2 O P

NAD+ NADH+H +Pi

glyceraldehyde 3-phosphate

dehydrogenase,GAPDH

C

CHOH

CH2 O P

O O~ P

glycerate1,3-bisphosphate,

1,3-BPG

glyceraldehyde 3-phosphate

(7) 1,3-BPG → glycerate 3-phosphate

• Substrate level phosphorylation

COO-

CHOH

CH2 O P

C

CHOH

CH2 O P

O O~ PADP ATP

glycerate 1,3-bisphosphate

glycerate3-phosphate

Phosphoglyceratekinase

(8) Glycerate 3-phosphate → glycerate 2-phosphate

COO-

CHOH

CH2 O P

COO-

CH

CH2OH

O P

glycerate3-phosphate

glycerate 2-phosphate

Mutase

(9) Glycerate 2-phosphate → phosphoenol pyruvate

COO-

CH

CH2OH

O P

COO-

C

CH2

O

PEP

~ P + H2O

enolase

glycerate 2-phosphate

(10) PEP →pyruvate

• Second substrate level phosphorylation• irreversible

COO-

C

CH3

ADP ATPCOO-

C

CH2

O

PEP

~ Ppyruvate kinase

O

Pyruvate

2) Pyruvate → lactate

COO

C

CH3

NAD+NADH + H+

O

Pyr

COO

CHOH

CH3Lactate dehydrogenase,

LDHLactic acid

Summary of Glycolysis

ATP ADPMg2+

PFK-1

GAP DHAP

glycerate 1,3-bisphosphate

NADH+H+

glyceraldehyde 3-phosphatedehydrogenase

H3PO4

NADH+H+

NAD+

ADP

ATP glycerate3-phosphate

glycerate 2-phosphate

H2O

PEP

ATP

ADP

pyruvate kinase

lactate

pyruvate

G G-6-P F- 6-P F- 1,6-BP

NAD+

Phosphoglyceratekinase

IsomeraseAldolase

MutaseEnolase

LDH

HK

ATP ADPMg2+

Total reaction:

C6H12O6 + 2ADP + 2Pi

2CH3CHOHCOOH + 2ATP + 2H2O

Formation of ATP:

The net yield is 2 ~P or 2 molecules of

ATP per glucose.

2. Regulation of Glycolysis

• Three key enzymes catalyze

irreversible reactions : Hexokinase,

Phosphofructokinase & Pyruvate

Kinase.

1) PFK-1

The reaction catalyzed by PFK-1 is

usually the rate-limiting step of the

Glycolysis pathway.

This enzyme is regulated by covalent

modification, allosteric regulation.

bifunctional enzyme

2) Pyruvate kinase

• Allosteric regulation:

F-1,6-BP acts as allosteric activator ；

ATP and Ala in liver act as allosteric inhibitors;

• Covalent modification:

phosphorylated by Glucagon

through cAMP and PKA and inhibited.

ATP ADP

PKA

Glucagon

Pyruvate Kinase (active)

Pyruvate Kinase- P (inactive)

cAMP

3) Hexokinase and glucokinase

• This enzyme is regulated by covalent modification, allosteric regulation and isoenzyme regulation.

• Inhibited by its product G-6-P.

• Insulin induces synthesis of glucokinase.

3. Significance of glycolysis

1) Glycolysis is the emergency energy-yielding pathway.

2) Glycolysis is the main way to produce ATP in some tissues, even though the oxygen supply is sufficient, such as red blood cells, retina, testis, skin, medulla of kidney.

• In glycolysis, 1mol G produces 2mol lactic acid and 2mol ATP.

§ 3 Aerobic Oxidation of Glucose

• The process of complete

oxidation of glucose to CO2 and water with liberation of energy as the form of ATP is named aerobic oxidation.

• The main pathway of G oxidation.

1. Process of aerobic oxidation

G Pyr

cytosol Mitochodria

glycolyticpathway

secondstage

thirdstage

CO2 + H2O+ATPPyr CH3CO~SCoA

firststage

TAC

1) Oxidative decarboxylation of Pyruvate to Acetyl CoA

• irreversible;• in mitochodria.

COO-

C

CH3

NAD+ NADH + H +

O

pyruvate

CH3CPyruvate

dehydrogenasecomplex

Acetyl CoA

O

~SCoA+ HSCoA + CO2

Pyruvate dehydrogenase complex:

E1 pyruvate dehydrogenase

Es E2 dihydrolipoyl transacetylase

E3 dihydrolipoyl dehydrogenase

thiamine pyrophosphate, TPP (VB1)

HSCoA (pantothenic acid)

cofactors lipoic Acid

NAD+ (Vpp)

FAD (VB2)

HSCoA

NAD+

Pyruvate dehydrogenase complex:

The structure of pyruvate dehydrogenase complex

S S

CH

H2C

H2C (CH2)4 COOH

SH SH

CH

H2C

H2C (CH2)4 COOH+2H

-2H

lipoic acid dihydrolipoic acid

C

C

NH2HC

NCH2

SC

C

NC

NCH

CH3

CH2CH2H3C O P O

O-

O

P

O

O-

O-

+

TPP

HSCoA

HS CH2 CH2 NH C CH2

O

CH2 NH C C

O

OH

H

C CH2

CH3

CH3

O P O

OH

O

P

OH

O

O

3'AMP

¦Â-alanine pantoic acid pyrophosphate

pantothenic acid

4'-phosphopantotheine

¦Â-mercapto-ethylamine

CO2

CoASHNAD+

NADH+H+

2) Tricarboxylic acid cycle, TCAC

• The cycle comprises the combination of a

molecule of acetyl-CoA with oxaloacetate,

resulting in the formation of a six-carbon

tricarboxylic acid, citrate. There follows a

series of reactions in the course of which

two molecules of CO2 are released and

oxaloacetate is regenerated.

• Also called citrate cycle or Krebs cycle.

(1) Process of reactions

Citrate cycle

CO

CH2

COO

COO

CH3CO~SCoA

C

CH2

COO

COO

CH2

HO

COO

C

CH

COO

COO

CH2 COO

CH

CH

COO

COO

CH2 COO

H2O

H2O

HO

CO2

CH2

CH2

COCOO

COOCH2

CH2

COO

CO~ SCoA CO2

NAD+NADH+H+

CH2

CH2

COO

COO GDP+PiGTP

CH

CH2

COO

COO

OOC CH

C COOH

HONAD+

NADH+H+

FAD

FADH2

H2O

acetyl CoA

H2Ooxaloacetate

citrate synthase

citrate

aconitase

cis-aconitate

aconitase

isocitrate

NAD+

NADH+H+

isocitrate dehydrogenase

¦Á-keto-glutarate

¦Á-ketoglutaratedehydrogenase

complex

succinyl-CoA

ADP ATP

CoASH

succinyl CoA syntetase

succinate dehydrogenase

fumarate

succinate

fumarase

malate

malate dehydrogenase

HSCoA

HSCoA

Summary of Krebs Cycle ①

Reducing equivalents

② The net reaction of the TCAC:

acetylCoA+3NAD++FAD+GDP+Pi+2H2O

→ 2CO2+3NADH+3H++FADH2+GTP+

HSCoA

③ Irreversible and aerobic reaction

④ The enzymes are located in the

mitochondrial matrix.

⑤ Anaplerotic reaction of oxaloacetate

pyruvate carboxylase

Biotin

ATP ADP + Pi

+ CO2C

CH3

COOH

OC

C

COOH

COOH

O

H2

NAD+ NADH+H+

malic acid DH+ CO2

malic enzymeC

CH3

COOH

O

NADPH+H+ NADP+

CHOH

C

COOH

COOH

C

C

COOH

COOH

O

H2H2

(2) Bio-significance of TCAC

① Acts as the final common pathway for

the oxidation of carbohydrates, lipids,

and proteins.

② Serves as the crossroad for the

interconversion among carbohydrates,

lipids, and non-essential amino acids,

and as a source of biosynthetic

intermediates.

Krebs Cycle is at the hinge of metabolism.

2. ATP produced in the aerobic oxidation

• acetyl CoA → TCAC : 3 (NADH+H+) + FADH2 + 1GTP → 12 ATP.

• pyruvate →acetyl CoA: NADH+H+ → 3 ATP

• 1 G → 2 pyruvate : 2(NADH+H+) → 6 or 8ATP

1mol G ： 36 or 38mol ATP

（ 12 ＋ 3 ） ×2 ＋ 6 （ 8 ）＝ 36 （ 38 ）

3. The regulation of aerobic oxidation

• The Key Enzymes of aerobic oxidation

The Key Enzymes of glycolysis

Pyruvate Dehydrogenase Complex

Citrate synthase

Isocitrate dehydrogenase (rate-limiting )

-Ketoglutarate dehydrogenase

(1) Pyruvate dehydrogenase complex

Pyruvate dehydrogenase(active form)

allosteric inhibitors:

ATP, acetyl CoA,NADH, FA

allosteric activators:AMP, CoA, NAD+,Ca2+

pyruvate dehydrogenase (inactive form)

P

pyruvate dehydrogenase kinase

pyruvate dehydrogenase phosphatase

ATP

ADPH2O

Pi

Ca2+,insulin acetyl CoA,NADH

ADP,NAD+

(2) Citrate synthase

• Allosteric activator: ADP

• Allosteric inhibitor: NADH, succinyl CoA, citrate, ATP

(3) Isocitrate dehydrogenase

• Allosteric activator: ADP, Ca2+

• Allosteric inhibitor: ATP

(4) -Ketoglutarate dehydrogenase

• Similar with Pyruvate dehydrogenase complex

Oxidative phosphorylation→TCAC↑

• ATP/ADP↑ inhibit TCAC,

Oxidative phosphorylation ↓

• ATP/ADP↓ ， promote TCAC ， Oxidative phosphorylation ↑

4. Pasteur Effect

• Under aerobic conditions, glycolysis is inhibited and this inhibitory effect of oxygen on glycolysis is known as Pasteur effect.

• The key point is NADH ： NADH mitochondria

Pyr TCAC CO2 ＋ H2O

Pyr can’t produce to lactate.

§4 Pentose Phosphate Pathway

1. The procedure of pentose phosphate pathway/shunt

In cytosol

1) Oxidative Phase

NADP+NADPH+H+

H2O

CO2

G-6-P

Xylulose 5-P

Ribulose 5-P

Ribose 5-P

G-6-P dehydrogenase

6-Phosphogluconate

6-phosphogluconate dehydrogenase

6-Phosphogluconolactonase

6-phosphogluco-nolactone

Epimerase

Isomerase

NADP+

NADPH+H+

2) Non-Oxidative PhaseRibose 5-p

Xylulose 5-p

Xylulose 5-p

Fructose 6-p

Glyceraldehyde 3-p

Fructose 6-p

• Transketolase: requires TPP• Transaldolase

Glycolysis

The net reation:

3G-6-P + 6NADP+ →

2F-6-P + GAP + 6NADPH + H+ + 3CO2

2. Regulation of pentose phosphate pathway

Glucose-6-phosphate Dehydrogenase is the rate-limiting enzyme.

NADPH/NADP+↑, inhibit;

NADPH/NADP+↓, activate.

3. Significance of pentose Phosphate pathway

1) To supply ribose 5-phosphate for bio-synthesis of nucleic acid;

2) To supply NADPH as H-donor in metabolism;

NADPH is very important “reducing power” for the synthesis of fatty acids and cholesterol, and amino acids, etc.

NADPH is the coenzyme of glutathione

reductase to keep the normal level of

reduced glutathione;

So, NADPH, glutathione and glutathione reductase together will preserved the integrity of RBC membrane.

2GSH

G-S-S-G NADPH + H+

glutathione reductase

NADP+H2O2

2H2O

Deficiency of glucose 6-phosphate dehydrogenase results in hemolytic anemia.

favism

NADPH serves as the coenzyme of mixed function oxidases (mono-oxygenases). In liver this enzyme participates in biotransformation.

§5 Glycogen synthesis and catabolism

Glycogen is a polymer of glucose residues linked by (1→4) glycosidic bonds, mainly (1→6) glycosidic bonds, at

branch points.

• The process of glycogenesis

occurs in cytosol of liver and

skeletal muscle mainly.

1. Glycogen synthesis (Glycogenesis)

• UDPG: G active pattern, G active donor.• In glycogen anabolism, 1 G consumes

2~P. • Glycogen synthase: key E.

GHK or GK

G-6-P

ATP ADP

G-1-PUDPG

pyrophosphorylase

UDPG

UTP PPi Gn UDP

Gn+1glycogensynthase

O O

OHOH

HH

H

CH2

H

HN

N

O

O

OP

O

O

P

O

O

H O

OH

H

OHH

OH

CH2OH

H

O

H

UDPG

Branching enzyme

Phosphorylase: key E;

The end products: 85% of G-1-P and 15% of free G;

There is no the activity of glucose 6-phosphatase (G-6-Pase) in skeletal muscle.

Gn

Pi Gn-1

G-1-P G-6-P G-6-Pase

H2O Pi

GPhosphorylase

2. Glycogen catabolism (glycogenolysis)

Debranching enzyme:

glucan transferase

-1,6-glucosidase

Nonreducing ends(1→6) linkage

Glycogen phosphorylase

(1→6) glucosidase activity of debranching enzyme Glucose

Transferase activity of debranching enzyme

3. Regulation of glycogenesis and

glycogenolysis

1) Allosteric regulation

In liver:

G phosphorylase

glycogenolysis

In muscle:AMP phosphorylase-b

ATPG-6-P

phosphorylase-a

glycogenolysisCa2+

2) Covalent modification

Glucagonepinephrine

Adenylyl cyclase

cAMP

G proteinreceptor

PKA

glycogenolysis

Phosphorylase

Glycogen synthase

glycogenesisBlood sugar

glucagon, epinephrine

inactiveadenylate cyclase

activeadenylate cyclase

ATP cAMP

inactivePKA

activePKA

phosphorylase bkinase

phosphorylase bkinase

P

ATP

ADP

H2O

Pi

phosphorylase b

P

P

ATP ADP

Pi

H2OATP ADP

glycogen synthase

glycogen synthase

P

H2OPi protein phosphatase-1

(active) (inactive)

inhibitor-1 (active)

inhibitor-1 (inactive)

phosphorylase a

ATP

§6 Gluconeogenesis

• Concept:

The process of transformation of non-carbohydrates to glucose or glycogen is termed as gluconeogenesis.

• Materials: lactate, glycerol, pyruvate and glucogenic amino acid.

• Site: mainly liver, kidney.

1. Gluconeogenic pathway

• The main pathway for gluconeogenesis

is essentially a reversal of glycolysis,

but there are three energy barriers

obstructing a simple reversal of

glycolysis.

1) The shunt of carboxylation of Pyr

PEP

ADP

ATP

oxaloacetic acid

Pyr carboxylase

ADP+Pi ATP CO2Biotin

GTP

GDPCO2

PEP carboxykinase

Pyr kinase

COO-

C

CH3

COO-

CH

CH2

O~ P

O

pyruvate

COO-

C

CH2

O

COOH £¨ Mt.£©

£¨ 1/3Mt. 2/3cytosal£©.

2) F-1, 6-BP →F-6-P

F-6-P F-1,6-BP

ATP ADP

Pi H2O

PFK-1

Fructose-bisphosphatase

3) G-6-P →G

• 2 lactic acid G consume ATP?

G G-6-P

ATP ADP

Pi H2O

Glucose-6-phosphatase

HK

gluconeogenesisglucose

G-6-P

glycogen

F-1,6BP

glyceral-dehyde 3-P

glycerol1.3-bisphospho- glycerate

glycerate 3-P

glycerate 2-P

lactate

G-1-P

malic acid

phosphoenol pyruvate

pyruvate

GTP

GDP

CO22/3

malic acid

pyruvate

phosphoenol pyruvate

GTP

GDP

CO21/3

CO2

CYTOSOL MITOCHONDRIA

NAD+ NADH+H+

NAD+

NADH+H+

NAD+

NADH+H+

glutamate

¦Á-ketoglutarate ¦Á-ketoglutarate

glutamate

OAAAspAspOAADHAP

ATP

ADP

ATP

ADP

PKADP

ATP

F-6-P

2. Regulation of gluconeogenesis

• Substrate cycle:

The interconversion of two substrates

catalyzed by different enzymes for

singly direction reactions is called

“substrate cycle”.

• The substrate cycle produces net

hydrolysis of ATP or GTP.------futile

cycle

Key enzymes of gluconeogenesis

PEP carboxykinase

Pyr carboxylase

Fructose-bisphosphatase

Glucose-6-phosphatase

F-1,6-BP

ATP

ADP

Pi

H2O

PFK-1FBPase-1

F-6-P

F-2,6-BP

AMP

glycolysis

gluconeogenesis:

F-1,6-BP

ATP

ADPF-2,6-BP

PEP

Pyr

acetyl CoA

glucagon

insulinglucagonAla in liverOAA

3. Significance of gluconeogenesis

(1) Replenishment of Glucose by Gluconeogenesis and Maintaining Normal Blood Sugar Level.

(2) Replenishment of Liver Glycogen.

(3) Regulation of Acid-base Balance.

First stages(cytosol)

Second stages(Mt.)

Third stages(Mt.)

Lactic acid (Cori) cycle• Lactate, formed by the oxidation of

glucose in skeletal muscle and by blood, is transported to the liver where it re-forms glucose, which again becomes available via the circulation for oxidation in the tissues. This process is known as the lactic acid cycle or Cori cycle.

• prevent acidosis ； reused lactate

muscle

glucose

pyruvate

lactate

glucose

blood

pyruvate

lactate

glycolytic pathway

glucose

liver

lactate

NAD+

NADH+H+

NADH+H+

NAD+

gluconeo-genesis

Lactic acid cycle

§6 Blood Sugar and Its

Regulation

1. The source and fate of blood sugar

blood sugar3.89¡« 6.11mmol/L

dietary supply

liver glycogen

(gluconeogenesis)

other saccharides

CO2 + H2O + energy

glycogen

other saccharides

non-carbohydrates

>8.89¡«10.00mmol/L(threshold of kidney)

non-carbohydrate

(lipids and some amino acids)

urine glucose

origin (income) fate (outcome)

Blood sugar level must be maintained within a limited range to ensure the supply of glucose to brain.

The blood glucose concentration is 3.89 ～ 6.11mmol/L normally.

2. Regulation of blood sugar level

1 ） insulin ： for decreasing blood sugar levels.

2 ） glucagon ： for increasing blood sugar levels.

3 ） glucocorticoid: for increasing blood sugar levels.

4 ） adrenaline ： for increasing blood sugar levels.

3. Abnormal Blood Sugar Level

• Hyperglycemia: > 7.22 ～ 7.78 mmol/L

• The renal threshold for glucose: 8.89

～ 10.00mmol/L

• Hypoglycemia: < 3.33 ～ 3.89mmol/L

Pyruvate as a junction point