Metabolism of carbohydrates

Sources of glucose (Glc)● from food (4 hours after meal)● from glycogen (from 4 to 24 hours after meal)● from gluconeogenesis (days after meal, during starvation)

Figure was assumed from Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley‑Liss, Inc., New York, 1997

Glycemia

• glucose concentration in the blood

• physiological range of fasting glycemia 3,3 – 5,6 mmol/L

• is regulated by hormones (insulin, glucagon, epinephrine, kortisol, …)

Glucose can enter into cells:

a) by facilitative diffusion (GLUT 1 – 7)• GLUT 1 – blood-brain barrier, erythrocytes

• GLUT 2 – liver, β-cells in pancreas

• GLUT 3 – neurons

• GLUT 4 – skeletal muscles, heart muscle, adipose tissue

b) by cotransport with Na+ ion (SGLT-1 and 2)

Figure was assumed from textbook: Devlin, T. M. (editor): Textbook of Biochemistry with Clinical Correlations, 4th ed. Wiley‑Liss, Inc., New York, 1997.

small intestine, kidneys

An effect of insulin on insulin-sensitive cells

Transport of Glc into cells is dependent on insulin effect (GLUT-4) in the following tissues: skeletal and heart muscle and adipose tissue

Figure is found on http://www.mfi.ku.dk/ppaulev/chapter27/Chapter%2027.htm

Metabolic pathways included in utilization of Glc – glycolysis, pentose cycle, glycogen

synthesis

Phosphorylation of glucose after enter into cell Glc is always phosphorylated to

form Glc-6-P enzyme hexokinase catalyzes esterification of Glc ATP is a donor of phosphate group! enzyme is inhibited by excess of Glc-6-P 2 isoenzymes of hexokinase exist: hexokinase and

glucokinase hexokinase has a higher affinity to glucose than

glucokinase

Hexokinase vs. glucokinase

Figure is found on http://web.indstate.edu/thcme/mwking/glycolysis.html

KM hexokinase = 0,1 mMKM glucokinase = 10 mM

Glycolysis

• substrate: Glc-6-P• product: pyruvate • function: source of ATP• subcellular location: cytosol• organ location: all tissues• regulatory enzymes: hexokinase/glucokinase, 6-phosphofructokinase-1 (main regulatory

enzyme), pyruvatekinase

Regulatory enzymes are activated by hormone insulin!

Glucose

Glucose in blood:Breakdown of polysaccharides

Synthesis from noncarbohydrate precursors (gluconeogenesis)

Glucose Utilization

(Nucleic acid and NAD synthesis)

(ATP and intermediates)

Glycolysis

“Sweet splitting”

Catabolism of 1 mol glucose to form 2 mol pyruvate

Sequence of 10 enzyme-catalyzed reactions

Occurs in almost every living cell

Two stages (phases):

Hexose/preparatory (stage 1, consumes 2 ATP)

Triose/payoff (stage 2, generates 4 ATP and 2NADH)

Anaerobic process (no oxygen required)

Overall chemical reaction:

D-glucose + 2ADP + 2Pi + 2NAD+ → 2 pyruvate + 2ATP + 2NADH + 2H+ + 2H2O

Provides significant portion of free energy used by most organisms

1

2

3

4

5

6

7

8

9

10(McKee and McKee, Biochemistry, 3rd ed.)

The GlycolyticPathway

Glycolysis

Figure is found on http://web.indstate.edu/thcme/mwking/glycolysis.html

1. Synthesis of glucose-6-phosphate (G6P)

OH

OH

H

OH

H

OHH

OH

CH2

H

OH

OH

H

OH

H

OHH

OH

CH2

H

OO3P

HO

2-

ATP

ADP

Glucose is phosphorylated immediately after entering cell

Prevent transport out of cellIncrease activity of phosphate ester oxygen

Enzyme = hexokinaseCatalyze phosphorylation of hexosesRemember: kinase = transfer phosphoryl group between ATP and metabolite

ATP-Mg2+ complex is cosubstrateSource of phosphoryl groupMetal shields negative charge, making P more accessible and electrophilic

Reaction is irreversibleProduct not accommodated by enzyme active siteG’° = -16.7 kJ/mol

Conformational change of Hexokinase

Active site engulfed when glucose bindsBrings ATP-Mg2+ closer to glucose C6

Proximity

Excludes water from active sitePrevents competing phosphoryl group transfer to water

Less polar environment increases rate of nucleophilic reaction

2. Conversion of G6P to fructose-6-phosphate (F6P)

OH

OH

H

OH

H

OHH

OH

CH2

H

OH

H2C

H

CH2

OH H

H OHO

OO3P2-

2-OHOO3P

Aldose converted to Ketose

Enzyme = phosphoglucose isomerase (PGI)

aka phosphohexose isomerase

G’° = 1.7 kJ/mol

Reaction occurs on linear form of G6PSubstrate binds to enzyme

Ring opening catalyzed by Lys or His residue

Proton transfer to/from Glu residue achieves isomerism

Ring closes, is released from PGI

C1 of F6P now available for phosphorylation

Mechanism of the Phosphohexose Isomerase Reaction

3. Phosphorylation of F6P to form fructose-1,6-bisphosphate (FBP)

OH

H2C

H

CH2

OH H

H OHO

2- 2-

2-

ATP

ADP

OHOO3P

OH

H2C

H

CH2

OH H

H OHO

OOO3P PO3

Irreversible reactionG’° = -14.2 kJ/mol

Catalyzed by phosphofructokinase (PFK-1)

Regulatory enzyme

Inhibited by high levels of ATP and citrate (indicators that citric acid cycle has slowed down)

Requires a second mole of ATP-Mg2+ complex

Prevent later products from diffusing out of cell

Rate determining stepCommit the cell to glycolysis

4. Cleavage of FBP

2-

2- 2-

2-

OH

H2C

H

CH2

OH H

H OHO

OOO3P PO3

CH2OPO3

C

CH2OH

O+CH

CHO

CH2OPO3

OH

1

2

3

6

4

5

Products: Dihydroxyacetone phosphate (DHAP) from C1-C3Glyceraldehyde-3-phosphate (GAP) from C4-C6

Enzyme = aldolaseCovalent catalysisAcid-base catalysisElectrostatic stabilization of intermediates

G’° = 23.8 kJ/mol (note standard)Reaction = aldol cleavage

Reverse of aldol condensationCommon C-C bond cleavage reactionTwo carbonyl products (aldehyde and ketone), each with 3 carbons

Would not be true of G6P

Mechanism of the Aldolase Reaction

5. Interconversion of GAP and DHAP

2-

2-CH2OPO3

C

CH2OH

O

CH

CHO

CH2OPO3

OH

GAP is the only substrate for the next reaction in glycolysis

DHAP is converted to isomeric GAP to prevent loss of 3 carbon unit

Enzyme = triose phosphate isomerase (TPI or TIM)

Rate of reaction is diffusion controlled

Product formation occurs as quickly as E and S collide

Catalytic perfection

G’° = 7.5 kJ/mol

Formation of GAP

End of Stage 1

1 mol glucose→ 2 mol GAP

Next…payoff

6. Oxidation/phosphorylation of GAP to 1,3-bisphosphoglycerate (1,3-BPG)

2-

2-

NAD + Pi

NADH + H

HC

CHO

CH2OPO3

OH

2-

HC

C

CH2OPO3

OH

O PO3O

Aldehyde is oxidized and phosphorylated

Oxidizing agent = NAD+

Enzyme = glyceraldehyde-3-phosphate

dehydrogenase

Binds GAP and NAD+

G’° = 6.3 kJ/mol

1,3-BPG contains high-energy bond

Used in next step to generate ATP

Mechanism of the Glyceraldehyde-3-phosphate Dehydrogenase Reaction

7. Phosphoryl group transfer to form 3-phosphoglycerate (3PG)

2-

2-

ADP

ATP

HC

COO

CH2OPO3

OH

2-

HC

C

CH2OPO3

OH

O PO3O

Phosphoryl group transfers from 1,3-BPG to ADP

Substrate-level phosphorylation

ATP is produced (2 mol in overall pathway)

Enzyme = phosphoglycerate kinase (PGK)

Named for reverse reaction

Mg2+ required

Two domains

ADP

1,3-BPG

Domains swing

together to create

water-free active site,

as with hexokinase

G’° = -18.5 kJ/mol

Equilibrium slightly shifted to products

8. Interconversion of 3PG and 2-phosphoglycerate (2PG)

2-

HC

COO

CH2OPO3

OH

2-HC

COO

CH2OH

OPO3

3PG has low phosphoryl-group-transfer potential

Isomerization of 3PG to 2PG is first step to forming a molecule with a high energy phosphate bond

Enzyme = phosphoglycerate mutase (PGM)

An isomerase

Mg2+? No, but does need BPG to seed the rxn

Phosphoryl group from active site on enzyme transferred to substrate

Bisphospho intermediate 2,3-BPG

Phosphoryl group from C3 transfer back to His

G’° = 4.4 kJ/mol

The Phosphoglycerate Mutase Reaction

9. Dehydration of 2PG to form phosphoenolpyruvate (PEP)

2-HC

COO

CH2OH

OPO3

H2O

2-C

COO

CH2

OPO3

Dehydration of primary alcohol

Elimination of H2O from C2 and C3

PEP has high phosphoryl-group-transfer

potential due to enol

Phosphoryl group restricts keto-enol

tautomerization

Enzyme = enolase

Requires Mg2+ for activity (F- inhibits)

G’° = 7.5 kJ/mol

Synthesis of pyruvate from PEP

ADP

ATP

2-C

COO

CH2

OPO3

C

COO

CH3

O

Enzyme = pyruvate kinase (PK)

Requires both Mg2+ and K+

G’° = -31.4 kJ/mol

Phosphoryl group transfer to ADP

Substrate-level phosphorylation

Overall, 2 mol ATP produced

Reaction is irreversible

Large decrease in free energy as keto tautomer formed from enolpyruvate

Activity, part 1

Activity, part 2

Fate of Pyruvate

Pyruvate does not accumulate

Undergoes one of 3 possible enzyme-catalyzed reactions

Reaction depends on type of cell or species or the availability of Oxygen

Fate of Pyruvate

Anaerobic organisms / conditions:

Need to oxidize NADH to regenerate NAD+ for glycolysis to continue

Pyruvate converted to waste products (ethanol, lactate, acetic acid, etc.) through fermentation

Mammals: homolactic or lactate fermentation (lactate dehydrogenase)

Yeast/microorganisms: alcoholic fermentation (pyruvate decarboxylase)

Aerobic organisms:

NADH oxidized by oxidative phosphorylation (using molecular oxygen)

Oxidative decarboxylation of pyruvate to form CO2 and an acetyl group (of Acetyl-CoA)

Enzyme = pyruvate dehydrogenase

Requires coenzyme A and NAD+

Acetyl-CoA becomes fuel for the citric acid cycle (formation of CO2 and H2O) and building block for fatty acid synthesis

Regulation of glycolysis

Glycolysis operates continuously under steady-state conditions

Flux varies to meet the needs of organism

Regulation primarily controlled by allosteric enzymes

Hexokinase (reaction 1)

Inhibited by uncomplexed ATP (no Mg2+) and G6P (product of rxn 1)

PFK (reaction 3)

Inhibited by ATP and citrate

Pyruvate kinase (reaction 10)

Inhibited by ATP

Activated by FBP (product of rxn 3)

Reactions of these enzymes are irreversible

Regulation of glycolysis

Regulatory enzymes● Hexokinase – inhibited by Glc-6-P● Glucokinase – activated by insulin

– inhibited by Fru-6-P

● 6-phosphofructokinase-1 (PFK-1)

– activated by insulin, ↑AMP / ATP

- inhibited by ↑ ATP /AMP, citrate

● Pyruvatekinase

– activated by insulin, Fru-1,6-bisP

- inhibited by glucagon, ↑ ATP /AMP, acetyl-CoA

Metabolism of other carbohydrates

Fructose, galactose, and

mannoseConvert to glycolytic

intermediatesMetabolized by glycolytic

pathway

Pentose phosphate pathwayAlternative pathway to glycolysis

Glucose degradation

Products = ribose-5-phosphate

and NADPHBiosynthetic precursors

Production of ATP in glycolysis

conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate

conversion of phosphoenolpyruvate (PEP) to pyruvate

These reactions are examples of substrate level phosphorylation!

Pentose phosphate pathway

• substrate: Glc-6-P• product: CO2, NADPH + H+

• function: gain of NADPH + H+, production of rib-5-P for nucleotide synthesis, mutual conversions of monosacharides

• subcellular location: cytosol• organ location: all tissues• regulatory enzyme: glucose 6-phosphate

dehydrogenase

Pentose phosphate pathway – oxidative stage produces rib-5-P

Figure is found on http://web.indstate.edu/thcme/mwking/pentose-phosphate-pathway.html

Pentose phosphate pathway – non-oxidative stage includes interconversions of

monosaccharides

Figure is found on http://web.indstate.edu/thcme/mwking/pentose-phosphate-pathway.html

Glycogen synthesis (glycogenesis)

• substrate: Glc-6-P• product: glycogen• function: glucose storage in the form of glycogen• cellular location: cytosol• organ location: especially in the liver and

skeletal muscles, other tissues have lower glycogen storage

• regulatory enzyme: glycogen synthase

Enzyme glycogen synthase is inhibited by phosphorylation (glucagon in liver and epinephrine in muscles)!

Glycogen synthesis• Glc-6-P → Glc-1-P• Glc-1-P + UTP → UDP-Glc + PPi

Glycogen synthase catalyzes the formation of (1→4) glycosidic bonds.

Branching (formation of (1→6) glycosidic bonds) is performed by enzyme amylo-(1,4 – 1,6)-transglycosylase („branching enzyme“).

Figure is found on http://en.wikipedia.org/wiki/Glycogen

Metabolic pathways serving to supplementation of Glc into the

bloodstream – glycogen degradation and gluconeogenesis

Glycogen degradation (glycogenolysis)● substrate: glycogen• product: Glc-6-P• function: releasing of Glc from glycogen• subcellular location: cytosol• organ location: liver, skeletal muscles, but also other tissues• regulatory enzyme: glycogen phosphorylase

Enzyme glycogen phosphorylase is activated by phosphorylation which is induced by hormones glucagon and epinephrine. Insulin

inhibits enzyme phosphorylation.

Glycogen degradation

Glycogen (n Glc) + Pi → Glc-1-P + glycogen (n - 1 Glc) Enzyme glycogen phosphorylase catalyzes the cleavage of 1→4 bonds.Enzyme amylo-1→6-glucosidase („debranching enzyme“) cleaves 1→6 bonds. Glc-1-P ↔ Glc-6-P phosphoglucomutase

Glc-6-P glucose-6-phophatase (liver, kidneys, enterocytes)

Glc

Gluconeogenesis

• substrates: lactate, pyruvate, glycerol, amino acids – Ala, Asp, Gln etc.

• product: glucose

• function: synthesis of Glc from non-sugar precursors

• subcellular location: mitochondrial matrix + cytosol

• organ location: liver + kidneys

• regulatory enzymes: pyruvate carboxylase and PEP carboxykinase

Regulatory enzymes are activated by hormones glucagon and cortisol. Insulin inhibits them.

Scheme of gluconeogenesis

Figure is found on http://web.indstate.edu/thcme/mwking/gluconeogenesis.html

Gluconeogenesis

Synthesis of PEP is divided into 2 steps:

• Pyr → matrix of mitochondria → Pyr is carboxylated to oxaloacetate (OA) by pyruvate carboxylase

CH3-CO-COO- + CO2 + ATP → -OOC-CH2-CO-COO- + ADP + Pi

• OA is transported to the cytosol and decarboxylated to PEP by PEP carboxykinase

-OOC-CH2-CO-COO- + GTP → PEP + CO2 + GDP

Synthesis of 1 mol Glc consumes 4 mol ATP and 2 mol GTP!

Figure was assumed from http://www.biochem.arizona.edu/classes/bioc462/462b/glycolysis.html

Regulation of gluconeogenesis

Hormones:• activation: cortisol, glucagon, epinephrine• inhibition: insulin

Enzyme pyruvate carboxylase• activation: acetyl-CoA from β-oxidation of FA → source of ATP

Enzyme fructose-1,6-bisphosphatase• activation: citrate, starvation• inhibition: AMP, Fru-2,6-bisP

Enzyme glucose-6-phosphatase (in ER of liver, kidneys and enterocytes !)

Cori cycle

Figure was assumed from http://web.indstate.edu/thcme/mwking/gluconeogenesis.html

Glucose-alanine cycle

Figure is found on http://web.indstate.edu/thcme/mwking/gluconeogenesis.html

Fructose metabolism

• Fru is a component of sucrose (Glc + Fru)• part of Fru in converted to Glc in enterocytes: Fru-6-P → Glc-6-P → Glc

• part of Fru is absorbed and it is transferred via blood into liver:Fru + ATP → Fru-1-P + ADP by enzyme fructokinase

• Fru-1-P is broken down to glyceraldehyde (GA) and dihydroxyacetonephosphate (DHAP) by aldolase

• DHAP enters glycolysis and GA → glyceraldehyde-3-P → glycolysis

Galactose metabolism• Gal is a component of lactose (Glc + Gal)• Gal is absorbed by the same mechanism in

enterocytes like Glc → liver• Gal is phosphorylated in liver to form Gal-1-P: Gal + ATP → Gal-1-P + ADP by enzyme

galactokinase• Gal-1-P is converted to UDP-Gal:

Gal-1-P + UTP → UDP-Gal + PPi by

uridyltransferase• UDP-Gal is used to lactose synthesis in mammary

gland during lactation • epimerization of UDP-Gal to UDP-Glc → glycogen

synthesis / synthesis of glucuronic acid / glycoprotein synthesis