Metabolism of carbohydrates. Sources of glucose (Glc) ● from food (4 hours after meal) ● from...
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Transcript of Metabolism of carbohydrates. Sources of glucose (Glc) ● from food (4 hours after meal) ● from...
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