Biochemie-6-2-metabolismus_sacharidů
Cellular metabolism
glucose metabolism
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cellular metabolism glucose metabolism
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Metabolism
Living organism requires a constant supply of energy
and the creation and restoration of building materials.
Metabolism - marches , in which a living organism
uses and produces energy .
A summary of all the reactions occurring in the
body .
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Functions of metabolism
•maintenance of energy (catabolic - degradation )
• synthesis of molecules (anabolic -storage )
• both processes are interdependent --- Energy
Affinity
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Types of organisms by metabolism
• by source of energy:
• phototrophs (use of solar energy, for example green
plants)
• chemotrophs (oxidation of nutrients )
• by source of building material:
• autotrophs (synthesized substances from anorg. sources)
• heterotrophic (use org. substanes)
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CO2 Small
molecules
Macromolecules
Proteins,
carbohydrates,
lipids
Energy
O2
Metabolism of human
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• consistently receive nutrients with high enthalpy ( = energy ) and low
entropy ( = complex and ordered structure )
• nutrients are converted to waste products with low enthalpies and
entropies high (= simple structure )
• Gibbs energy released during these processes is maintained in the
course of biochemical processes , ensuring highly organized cell
structure
• part of the energy is converted to a usable form of heat
Organisms as open systems
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Energy in chemical reactions
Gibbs free energy
( G  )
the maximum
amount of useful
energy that can be
obtained for the
reaction constant .
pressure and
temperature
A+B  C+D
ba
dc
BA
DC
RTGG
][][
][][
ln0

During the conversion of nutrients into the waste
substances are released Gibbs energy that keeps running
biochemical processes and ensures a highly organized
cellular structure. Unfortunately, you can not use all the
released energy - part of it is always converted to a form
unusable = heat.
Gibbs energy (G ) can be defined as the maximum
amount of useful energy that can be obtained in the
reaction at constant pressure and temperature. For the
reaction A + B → C + D , it can be
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 G0´ (pH = 7, 0 25 oC)
Biochemical processes
 Steady-state ( dynamic equilibrium ) .
 Reaction of the successive product of one reaction is the substrate for
subsequent reactions .
 Concentration do not meet the standard.
With regard to the Gibbs energy in the body, we can distinguish two different
types of processes :
a) Exergonic processes
b) endergonic processes
Endergonic processes (G > 0) can take place only in coupling reactions with
exergonic (G < 0) . The transfer of energy from one process to another takes
place by means of energy-rich molecules - most often used ATP ( energy
released in the particular process is transferred via phosphoryl groups -PO32to
other substances) .
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Processes
exergonic endergonic
Endergonic reaction can take place only in coupling
reactions with exergonic.
Energy transfer from one process to another takes
place by means of energy-rich molecules.
The most commonly used is ATP.
When coupling that transfers phosphoryl groups -
PO3
2- other substances
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-PO3
2- is by enzyme of kinase transported from ATP
to glucose.
Principles of coupling
Go´ = +13,8 kJ/mol
Go´ = -30,5 kJ/mol
Example 1:
Formation of glucosa-6-phosphate
glucose + Pi  glucose-6-P + H2O
Go´ = - 16,7 kJ/molglucose + ATP  glucose -6-P + ADP
ATP + H2O  ADP + Pi
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G 1 > 0pyruvate + HCO3
- oxalacetate
Example 2: Carboxylation of pyruvate
HCO3
- + ATP  ADP + -OCO-O-PO3
2-
phosphocarbonate
-OCO-O-PO3
2- + biotin  -OOC-biotine + Pi
-OOC-biotin + pyruvate  oxalacetate
biotin + ATP + HCO3
- → carboxybiotin + ADP + Pi
carboxybiotin + pyruvate → biotin + oxalacetate
G 2 < 0
G < 0
Partial reaction:
ATP ADP + Pi
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ATP + HCO3
-  ADP + -O -P-O-C-O-
OO
O phosphocarbonate
N NH
O
S CO enzym
C
O
OCarboxylate
anion is activated by binding Pi and by biotin is
transfer to pyruvate.
Carboxylation of biotin
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The term " high energy compound"
(also called " energy- rich compound "
" Macroergic Compound " )
The compound to hydrolytic cleavage of its bonds provide
approximately the same or greater energy than is
G0´for ATP hydrolysis
Most often, these functional derivatives of phosphoric acid
.
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The high-energy/macroergic phosphate compound
the rest contain phosphoric acid . linked most often :
 anhydride ,
 amide,
 enol esters bond.
(esters of phos. Acid are not macroergic)
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Univerzal phosphate high-energic compound is
ATP
Provides energy in reactions:
ATP + H2O  ADP + Pi  G0´ = -30,5 kJ/mol
ATP + H2O  AMP + PPi  G0´ = -32,0 kJ/mol
reaction must be enzyme-catalyzed
Similarly, provide energy: GTP, UTP a CTP
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Another high-energy phosphate compounds
These substances are formed during the metabolism.
Their reactions with ADP may give ATP = substrate phosphorylation
Compound  G0 (kJ/mol) type of compounds
phosphoenolpyruvate -62 enolester
Carbamoyl-P -52 mixed anhydride
1,3-bisphosphoglycerate -50 mixed anhydride
phosphokreatin -43 amide
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The energy- rich compounds can also be thioesters
( e.g. , an acyl group linked to coenzyme A)
 G0 = -31,0 kJ/mol
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How to get high- energy compound by metabolism?
„burning nutrients “
• nutrients in the diet (lipids and carbohydrates, proteins
partially ) contain carbon atoms with a low oxidation state
• are sequentially dehydrogenated to various intermediates,
in which decarboxylation reactions cleave CO2
• electrons and hydrogen atoms are transferred to the
redox cofactors (NADH, FADH2 ) and transported to the
respiratory chain
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Macroergic compounds
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 75.
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The above- mentioned energy compounds in metabolism
resulting from the combustion of nutrients.
The nutrients contained in food can be divided into lipids,
carbohydrates and proteins.
These compounds contain a carbon with a low degree of
oxidation . The main reactions in the combustion of nutrient
oxidation, which takes the form of the dehydrogenation .
Sequential dehydrogenation rise to various intermediates ,
releasing CO2 , electrons and hydrogen ( H). Hydrogen along
with electrons are transferred to the oxidation-reduction
cofactors and transported to the respiratory chain. 20
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 75.
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•during degradation of the nutrients may also be formed
directly high energy compound, or ATP providing
subsequent substrate phosphorylation
• reoxidation of the energy released is used to generate ATP
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Formation of ATP in cells
• Majority of formation of ATP
aerobic phosphorylation
= direct reaction between Pi and ADP
ADP + Pi  ATP catalyzed ATP-synthase
- the use of energy produced by oxidation of NADH
a FADH2
- Occurs in coupling in respiratory chain
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the use of energy produced by oxidation
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• Other possibilities of formation of ATP
transport -PO3
2- from high energetic rich compound to
ADP
SUBSTRATE PHOSPHORYLATION
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Examples of substrate phosphorylation
Reaction of glycolysis
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 84-85.
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Citrate cycle
thiokinase
Muscle:
ADP ATP
kreatinkinase
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 153, 234.
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ATP in cells
 Life time of ATP in cell cca 2 min
 They must be constantly replenished
 Instant ATP content in the body is about 100 g per day, daily is
produced 60-70 kg
 Adenylatekinase keep balance between ATP, ADP and AMP
ATP + ADP 2 ADP
 In health cell ratio [ATP]/[ADP] = 5-200
Energetic charge of cell:
when drops to zero , the cell die
   
     AMPADPATP
ADPATP


 2
1
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•Metabolism is regulated at several levels
• regulation of enzyme activity ( allosteric effects
product inhibition , substrate availability )
• covalent modification enzymes ( phosphorylation )
• regulation of the synthesis of enzymes
• compartmentalization and organ specialization
• hormonal regulation
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Different metabolic pathways are affected by the
condition of the body
• condition after eating x starvation
•Rest x severe physical strain
• rest x Stress
• physiological state xdisease
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Metabolisms glucose in cells
Metabolism of carbohydrates
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Sources of glucose in the diet
 Glucose can be taken from food :
 a) free
 b ) chemically bonded
 Free glucose gain eg . From grapes (glucose = dextrose ) and other fruits ( resp. Fruit
juices ) and honey.
 May be chemically bound glucose in polysaccharides and disaccharides .
 The main sources of glucose in food is starch. It is a polysaccharide composed glucose
subunits. We distinguish its two parts :
 a) a linear amylose =
 b ) branched amylopectin =
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 76.
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 In addition to its structure, these two also differ in their biological effect .
This difference describes variable called the glycemic index. The glycemic
index is related to the speed at which there is an increase in blood glucose the
lower the index , thus the food is "better" (glucose is released from the
more slowly , resulting in smaller demands on the pancreas and insulin
production ) .
 Starch sources are, for example . Potatoes ( the main source of our food ) ,
as well as bread, rice , pasta , corn and others. An important source of
starch are also the pulses , which have the advantage that they contain lots
of straight-chain amylose ( from the glucose is cleaved more slowly ), and
hence have a low glycemic index.
 Other sources of glucose, in addition to the polysaccharides are
disaccharides :
 a) sucrose
 b ) Lactose
 c ) maltose
 From the food can also receive free fructose .
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Entry of glucose into cells
glucose transporters
transmembrane proteins facilitating transport of
glucose into cells
- type GLUT (1-14)* or SGLT**
* glucose transporter
** sodium-coupled glucose transporter
Glucose molecules are highly polar , they can not diffuse
through hydrophobic lipid bilayer membrane (hydrogen
bonds between the OH groups and water )
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GLUT 1-GLUT 14, similarities:
 500 AK, 12 transmembrane helixes mechanism:
facilitated diffusion through a membrane ( extends
over the concentration gradient does not require
energy )
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Why are so many types of carriers ?
• different affinity for glucose
• They can be regulated in different ways
• They occur in various tissues
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Glucose Transport
• Na+-Independent Facilitated Diffusion
Glucose Transporters (GLUT 1-14)
With concentration gradient
Energy Independent
• Na+-Monosaccharide Cotransporter:
Against concentration gradient
Energy dependent
Carrier-mediated (SGLT)
Coupled to Na+ transport
Small intestine, renal tubules & choroid plexus
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Glucose Transporters
• Tissue-specific expression pattern
GLUT-1 RBCs and brain
GLUT-2 Liver, kidney & pancreas
GLUT-3 Neurons
GLUT-4 Adipose tissue & skeletal muscle
GLUT-5 Small intestine & testes
GLUT-7 Liver (ER-membrane)
• Functions:
GLUT-1, 3 & 4 Glucose uptake from blood
GLUT-2 Blood & cells (either direction)
GLUT-5 Fructose transport
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Glucose Transport:
Facilitated Diffusion
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Glucose transport via GLUT
Facilitated diffusion mechanism NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 78
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NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 78
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NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 78
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Receptor type GLUT 4 are regulated by insulin
Intracellular membrane
vesicles are " sleeping "
glukosovými transporters.
If insulin is not binding to the
receptor , glucose can not
enter into the cell .
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 78
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After binding to the
insulin receptor vesicles
with membrane
transporters move
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 78
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Binding of insulin to the receptor
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Glucose transporters penetrate
into the membrane , glucose
transport into the cell can begin.
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 78
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Glucose transport into cells
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Glucose transport into the cells of the intestinal
mucosa and ledviných tubules ( SGLT )
Mechanism: co-transport with sodium
Secondary active transport
•at two specific sites linked glucose transporter and Na +
• their transport runs in parallel ( without energy consumption)
• Na + is subsequently pumped from the cell ATPase ( ATP
consumption )
• Glucose is subsequently transported out of the cell via GLUT2
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Mucous membrane of small intestine
cells ( enterocytes )
The mechanism of glucose cotransport of
Na +
Lumen of the
small intestine
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 79
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After binding of Na + and
glucose transporter changes
the conformation and glucose
and Na + enter the cell
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 79
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Na + and glucose are transported to the cell
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 79
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At the opposite pole of the cell ( serosal side ) is Na + over Na + / K
+ -ATPase is transported out of the cell ( active transport )
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 69
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cell
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Glucose is the serosal side of the enterocyte transported out of the
cell via GLUT -2 ( passive transport )
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 79
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glucose + ATP glucose-6-P + ADP
Glucose metabolism in cells
Enzymes hexokinase or glucokinase
Formation of glucose-6- phosphate after the entry of glucose
into the cell :
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Significance of the formation of glucose-6 -P for further glucose
metabolism
phosphorylation
glucose
glucose
ATP
ADP
glucose-6-P
Hexokinase,
(glucokinase)
Catalyzes the reverse
reaction of glucose-6
- P phosphatase
It is only in the liver
( and kidneys )
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•The conversion of glucose to Glc -6-P in the cell allows additional
supply of glucose along a concentration gradient
• Once phosphorylated glucose can not have a cell out ( trap glucose )
• Only the liver ( and kidneys ) can be converted Glc -6-P back to
glucose and send that back to the blood
Consequences:
SODERBERG, Tim. ChemWiki: The Dynamic Chemistry E-textbook. Dostupné z:
http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Bi
ological_Emphasis/Chapter__3%3A_Conformations_and_Stereochemistry/Sectio
n_3.2%3A_Conformations_of_cyclic_organic_molecules
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Glucose concentration and the rate of phosphorylation
of glucokinase and hexokinase
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 81
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glucokinase X HEXOKINASe
Characteristic Hexokinase glucokinase
location
specify
inhibition
Affinity to Glc
inductive
KM (mmol/l)
Most tissues
broad (hexoses)
Glc-6-P
high
no
0,1
Hepatocytes
Islet cells (pancreas)
narrow (glucose)
no inhibition
low
insulin
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• phosphorylation in cells other than hepatic ( hexokinase )
takes place only if the glucose to be metabolized
• phosphorylation of glucokinase in the liver occurs at a
sufficient supply of glucose in the liver ( after meal )
• at lower concentrations it hexokinase
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Role glukokinasy v pankreatu
 glucokinase in the pancreatic cells - sensor in blood
glucose
 At elevated blood glucose levels , glucose enters into cells
of the pancreas ( GLUT2 ) and is phosphorylated by
glucokinase
 Another glucose metabolism mediated insulin release from
cells 
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Transformation of Glc-6P in cell and
significance
pathway significance
glycolysis
Synthesis of glycogen
Pentose cycle
Synthesis of Glc
derivates
Energy, transformation of acetylCoA to FA
Storage of Glc
Source of pentose, source of NADPH
Synthesis of glycoprotein's and proteoglycans
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GLYCOLYSIS
 Glycolysis occurs in almost every living cell.
 It occurs in cytosol.
 It was the first metabolic sequence to be studied.
 Most of the work done in 1930s by the German biochemist G.
Embden Meyerhof Warburg.
 That is why it is also called Embden-Meyerhof pathway.
 It is a Greek word.
 Glykos  sweet
 Lysis  loosing
 Glycolysis  loosing or splitting of glucose
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Glycolysis
Glykos (sugar) lysis (cleavage)
• Meaning : energy yield , production of other substances ,
including metabolism, galactose and fructose
• In virtually all cells
• Location: cytoplasm
• Reversible enzyme- catalyzed reactions
• Three reactions are irreversible
aerobic glycolysis
oxygen , pyruvate is converted
to acetyl-CoA
anaerobic glycolysis
in the absence of oxygen ,
pyruvate is converted into
lactate
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Stages of Glycolysis
Three stages
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Glycolysis: 1
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Glycolysis: 2
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Glycolysis: 3
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Glucose phosphorylation: step 1
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Induced fit in hexokinase
Conformation changes
on binding glucose,
the two lobes of the
enzyme come
together and surround
the substrate
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Hexokinase
Glucokinase
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Hexokinase Glucokinase
Site Most tissues Hepatocytes
Islet cells (pancreas)
Kinetics Low Km
(0.1mmol/l)
Low Vmax
High Km (1mmol/l)
High Vmax
Regulation G-6-phosphate F-6-phosphate
Insulin: Induction
Function Low glucose conc. High glucose conc.
Glucose sensor
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glucokinase X HEXOKINASe
Characteristic Hexokinase glucokinase
location
specify
inhibition
Affinity to Glc
inductive
KM (mmol/l)
Most tissues
broad (hexoses)
Glc-6-P
high
no
0,1
Hepatocytes
Islet cells (pancreas)
narrow (glucose)
no inhibition
low
insulin
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Difference between hexokinase and glucokinase
 Hexokinase:
 Its function is to make sure there is enough glc for the tissues,
even in the presence of low blood glc concentrations, by
phosphorylating all the glc concentration gradient between the
blood and the intracellular environment.
 Glucokinase:
 Its function is to remove glc from the blood following a meal.
 Hexokinase, phosphofructokinase and pyruvate
kinase are 3 regulatory enzymes of glycolysis.
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Hexokinase vs glucokinase
 Liver has an additional enzyme, glucokinase, that phosphorylate only
glc
1. Glucokinase has a high Km, because it phosphorylates glc only when
its concentration is high. This occurs during the brief period after
consumption of a carbohydrate rich meal, when high glc are delivered
by portal vein.
2. Glucokinase has a high Vmax, allowing the liver to remove effectively
this flood of glc from the portal blood. So this prevents extreme
hyperglycemia after meals.
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SUMMARY
Hexokinase Glucokinase
Tissue dist all liver
Km low high
Vmax low high
Substrate D-glc and other D-glc only
Inhibition by G-6-P Yes no
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More about HK
 Hexokinase, like adenylate kinase and all other
kinases, requires Mg (or Mn) for activity.
 Hexokinase is also one of the induced-fit model
enzymes.
 It has two lobes that move towards each other when Glc is
bound!
 Substrate-induced cleft closing is a general feature of
kinases.
 Other kinases (Pyruvate kinase, phosphoglycerate
kinase and PFK) also contain clefts between lobes that
close when substrate is bound.
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Formation of Glc-6-P: cleavage of glycogen (without ATP)
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 82.
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Formation of fructose-6-phosphate: step 2
by phosphoglucose isomerase
Conversion of an aldose
to a ketose
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2. ISOMERIZATION OF G-6-P
 The isomerization of G-6-P (an aldose sugar) to F-6-P (a
ketose sugar) is catalyzed by phosphoglucose
isomerase. The reaction is readily reversible, is NOT a
rate limiting or regulated step.
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3. PHOSPHORYLATION OF F-6-P
 Irreversible phosphorylation reaction catalyzed by
PFK (phosphofructokinase) is the most important
control point of glycolysis.
 Within the cell, the PFK reaction is the rate-limiting
step in the glycolytic breakdown of glc. It is
controlled by the concentrations of the substrates
ATP and F-6-P
80
by phosphofructokinase (PFK): an allosteric enzyme
that regulates the phace of glycolysis.
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3. Formation of fructose 1,6-bisphosphate
The rate of reaction is determinative of the rate of
glycolysis whole
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 82.
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Principles of regulation phosphofructokinase
allosteric inhibition of ATP and citrate
allosteric activation of AMP , ADP and hepatic
fructose 2,6 - bisphosphate - *
( * Fru - 2,6- Bispo arises under hormonal control )
What are allosteric enzymes ?
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Properties of enzymes which limit the rate of reaction
• slowest enzyme pathway
• operating at Vmax ( the only way to increase the speed
of response is to add more enzyme - no more substrate).
The rate of reaction is independent of [S].
• the reaction is irreversible ( the reaction takes place in
the opposite direction , it is necessary the action of a
different enzyme). Other enzymes tracks may be
reversible.
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The regulatory effect of fructose -2,6- biP in
glycolysis and gluconeogenesis in the liver
2,6-biP is allosteric efector
Phosphofructokinase fructosa 1,6-bisphosphatase
(glycolysis ) ( gluconeogenesis )
Activation Inhibition
Formation of fructose-2,6-biP
 Stimulation of fructosa-6P  inhibition glucagon
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Cleavage of six-carbon sugar: step 4
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5. ISOMERIZATION OF DIHYDROXYACETONE-P
 Triose phosphate isomerase (TIM) catalyzes the
reversible interconversion of dihydroxyacetone
phosphate and glyceraldehyde 3-phosphate.
 X-ray crystallographic and other studies showed
that Glu 165 plays the role of a general acid-base
catalyst.
 TIM has 8 parallel beta and 8 alpha helices (a
barrel). This structure is also found in
 Aldolase
 Enolase
 Pyruvate kinase
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Salvage of three-carbon fragment: step 5
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5. ISOMERIZATION OF DIHYDROXYACETONE-P
 Triose phosphate isomerase (TIM) catalyzes the
reversible interconversion of dihydroxyacetone
phosphate and glyceraldehyde 3-phosphate.
 X-ray crystallographic and other studies showed
that Glu 165 plays the role of a general acid-base
catalyst.
 TIM has 8 parallel beta and 8 alpha helices (a
barrel). This structure is also found in
 Aldolase
 Enolase
 Pyruvate kinase
90
6. OXIDATION OF GLYCERALDEHYDE 3-P.
 Because there is only a limited amount of NAD+ in the
cell, the NADH formed in this reaction must be
reoxidized back to NAD+ for glycolysis to continue. Two
major mechanisms for oxidizing NADH are
 The NADH-linked conversion of pyruvate to lactate
 Oxidation by the respiratory chain.
 The high energy P group at carbon of 1,3-bisPG
conserves much of the free energy produced by the
oxidation of Glycerate-3-P
91
Formation of 1,3-Bisphosphoglycerate: step 6
Done in two steps
92
Two-process reaction
Aldehyde Acid
93
Glyceraldehyde 3-phosphate dehydrogenase
Active site configuration
94
7: FORMATION OF ATP FROM 1,3-BisPGLYCERATE AND ADP
 This step is a substrate-level phosphorylation in which the
production of a high-energy P is coupled to the conversion of
substrate to product, instead of resulting from oxidative
phosphorylation. The energy trapped in this new high-energy P
will be used to make ATP in the next reaction of glycolysis. The
formation of ATP by P group transfer from a substrate such as
1,3-bisphosphoglycerate is referred to as a substrate-level
phosphorylation. Unlike most other kinases, this reaction is
reversible.
 2 mols 1,3biPGlycerate  2ATP replaces the 2ATP consumed
earlier with the formation of G-6-P and fructose 1,6bisP.
95
Formation of ATP from 1,3-Bisphosphoglycerate: step 7
High phosphoryltransfer
potential
Biochemie-6-2-metabolismus_sacharidů
7. Formation of 3-phosphoglycerate and ATP
Formation of ATP on the principle of substrate phosphorylation :
1.3 BPG is a high energy compound ( mixed anhydride ) ,
Energy released during the transmission PO32- is utilized to
synthesize ATP NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 84.
96
Biochemie-6-2-metabolismus_sacharidů
The formation of 2,3- bisfosfoglycerátu - side road in
erythrocytes
Binding to
Hb
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 84.
97
98
Rearrangement: step 8
99
An enol phosphate is formed: step 9
Dehydration elevates the transfer
potential of the phosphoryl group,
which traps the molecule in an
unstable enol form
Enol: molecule with hydroxyl group next to double bond
Biochemie-6-2-metabolismus_sacharidů
enolase
(inhibition F- - arrest of glycolysis by
taking a blood sample )
9. Formation of fosfoenolpyruvate
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 84.
100
101
Formation of Pyruvate & ATP: step 10
Biochemie-6-2-metabolismus_sacharidů
9. Formation of pyruvate
pyruvate kinase
activation of fructose -1,6- BISP
hormonally regulated glucagon (
inactivation )
ATP
(substrate
phosphorylation )
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 85.
102
103
Maintaining Redox Balance
NAD+ must be regenerated
for glycolysis to proceed
Glycolysis is similar in all cells,
the fate of pyruvate is variable
Biochemie-6-2-metabolismus_sacharidů
Conversion of pyruvate
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 85.
104
TEST
PYRUVATE
pyruvate oxalacetate
only
microorganism
Biochemie-6-2-metabolismus_sacharidů
Conversion of pyruvate
1) During anaerobic glycolysis, pyruvate is reduced to lactate
2 ) During aerobic glycolysis ( under air ) followed by oxidative
decarboxylation of pyruvate , which is then converted to acetyl-CoA .
3 ) carboxylation of pyruvate (one of anaplerotic reaction of the citric
acid cycle ) for energy arises oxaloacetate , which can be used in various
orbits (conversion to aspartate , CC ).
4 ) transamination of pyruvate obtained Alanine - reaction involved
glutamate ( Glu) and produces the 2-oxoglutarate (2- OG) .
5 ) Another possibility is the degradation of pyruvate alcohol
microorganisms. In this reaction, ethanol is formed .
105
Fermentation
 An anaerobic process beyond glycolysis.
 In our body it is used to make NAD+ when
there is not enough oxygen.
 NAD+ must be regenerated from NADH or
glycolysis will stop.
 We’ll look at two types of fermentation:
 Lactate and Ethanol.
Lactate fermentation
 Lactate
 Produced by muscles when the body
can’t supply enough oxygen.
 pyruvate lactate
 Anaerobic conversion of pyruvate to lactate
permits regeneration of NAD+.
 Body can then make more ATP - at a cost.
 Creates an oxygen debt.
 Body must take in extra O2 to oxidize lactate.
NADH + H+ NAD+
Biochemie-6-2-metabolismus_sacharidů
The formation of lactate - anaerobic
glycolysis
meaning :
Regeneration of NAD + consumed in the formation of 2,3- BisP -
glycerate
if not enough NAD + , not more glucose
molecules to enter glycolysis
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 86.
108
Pyruvate lactate
Biochemie-6-2-metabolismus_sacharidů
Lactate dehydrogenase ( LD )
Katalyzuje reakci v obou směrech
Isoenzymes LD1 - LD5
Subunit H
(heart)
Subunit M
(muscle)
LD1 LD2 LD3 LD4 LD5
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 86.
109
Pyruvate lactate
Biochemie-6-2-metabolismus_sacharidů
• in the short term intensively working muscle 14 %
• in erythrocytes ( have mitochondria) 25%
• Skin 25 %
• Brain 14 %
• in intestinal cells 8 %
The formation of lactate
average of 1.3 mol / day ( 70 kg man )
The concentration of lactate in the blood : 1 mmol / l
changes during intensive muscle work (up to 30 mmol / l)
110
Biochemie-6-2-metabolismus_sacharidů
Cori cycle - removing lactate from tissues to the
liver and utilization for gluconeogenesis
LIVER
glucose
MUSCLE
glucose
pyruvate
lactate
BLOOD
lactate
pyruvate
Coriovi – Nobel price 1947
LD
LD
111
Biochemie-6-2-metabolismus_sacharidů
Energy gain of glycolysis
1. direct profit ATP - substrate phosphorylation
2 ATP for on glucoseconsumption
ATP
gain ATP
- +
This gain is true for aerobic and anaerobic process.
For anaerobic glycolysis is only profit of ATP .
112
Biochemie-6-2-metabolismus_sacharidů
- NADH reoxidation reaction of 5 (glyceraldehyd-P1,3-bisPglycerate)
:
 transmission with " shuttles " to the respiratory chain - profit
reoxidace NADH - profit 2x 2-3 ATP
2. ATP further gains in aerobic glycolysis:
- transmission of pyruvate to acetylCoA (2 NADH) 2x3 ATP
- transmission of acetylCoA in CAC 2x12 ATP
113
Biochemie-6-2-metabolismus_sacharidů
The overall balance of aerobic glycolysis
Reaction profit ATP
glucose 2 pyruvate (substrate phos.)
2 NADH  2NAD+
2
4-6*
After aerobic glycolysis pyruvate
:
* It depends on the type of the shuttle
Further conversion of pyruvate :
Reaction profit ATP
2 pyruvate  2 acetylCoA + 2 NADH
2 acetyl CoA  2 CO2 + 6 NADH + 2 FADH2
6*
2x 12
The total maximum gain of glycolysis 36-38 ATP
* (2x NADH to RCh)
114
Biochemie-6-2-metabolismus_sacharidů
Balance anaerobic glycolysis
After anaerobic glycolysis pyruvate :
Reaction profit ATP
glukosa 2 x pyruvát (substrátová f.)
2 NADH  2NAD+
2
0
Creation and consumption of NADH in anaerobic glycolysis
Reaction profit/ loss NADH
2 glyceraldehyd-P  2 1,3-bisP-glycerát
2 pyruvát  2 laktát
total
+2
- 2
0
115
Biochemie-6-2-metabolismus_sacharidů
• During anaerobic glycolysis , the net energy yield 2 ATP
phosphorylation of substrate
• it's just a small fraction of the energy stored in the molecule of
glucose
• but has a role in process when supply of oxygen is limited
• tissue does not have mitochondria ( ERCS , leukocytes , ..)
• it is necessary that lactate is saved for gluconeogenesis
116
Biochemie-6-2-metabolismus_sacharidů
Oxidative decarboxylation of pyruvate
• Pyruvate dehydrogenase complex
CH3COCOOH + CoA-SH + NAD+
CH3COSCoA + CO2 + NADH + H+
Summary equation:
acetyl-CoA
matrix of
mitochondria
• Conversion of pyruvate to acetyl-CoA
cofactors: thiamine pyrophosphate, lipoic acid, CoA, FAD, NAD+
117
Biochemie-6-2-metabolismus_sacharidů
1. binding of pyruvate to thiamine pyrophospate
Reactions
118
Biochemie-6-2-metabolismus_sacharidů
2. decarboxylation and acetyl transfer to lipoate
lipoate
119
Biochemie-6-2-metabolismus_sacharidů
3. transfer of acetyl to Coenzyme A
120
Biochemie-6-2-metabolismus_sacharidů
4. re-oxidation of lipoate, transfer of hydrogen via FAD
to NAD+
121
Biochemie-6-2-metabolismus_sacharidů
Gluconeogenesis
• Glucose is not an essential nutrient. Human body can produce
it through a series of reactions which are called
gluconeogenesis
(GNG) .
• GNG is one of the factors that postresorption phase
(starvation) is ensured the maintenance of blood glucose levels
within the physiological range from 3.1 to 5.0 mmol / l .
• Other factors are glycogenolysis (a major factor in
postresorption phase) and consumption of carbohydrates (the
main factor in the resorptive phase - phase after a meal) .
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 91 .
122
 Glucose metabolism – and its amount in the blood –
the influence of hormones.
 Insulin, glucagon, adrenaline (instantaneous stress hormone)
and cortisol (primary stress hormone). Basic information on
individual hormones are given in table.
 Gluconeogenesis - in the liver (to a small extent in the
kidney), specifically in the cytosol of cells.
 Glucose is synthesized from simpler non-sucrose substances :
 lactate
 pyruvate
 glucogenic AA
 glycerol
Biochemie-6-2-metabolismus_sacharidů
123
Biochemie-6-2-metabolismus_sacharidů
Resorptive phase Postresorption
phase, starvation
Carbohydrates from
food
Glycogenolysis (liver)
Gluconeogenesis (liver,
kidney)
3,1-5,0 mmol/l
Blood glucose levels
124
Biochemie-6-2-metabolismus_sacharidů
Hormone Source
Effect on Glc level
in blood
Insulin -cells of pancreas 
Glucagon a-cells of pancreas 
Adrenaline
Cortisol
Adrenal medulla
Adrenal cortex


The main hormones in the metabolism of glucose
125
Biochemie-6-2-metabolismus_sacharidů
The different reactions of glycolysis and GNG
• For the synthesis of glucose are used enzymes and reactions of glycolysis
- but not all , since three reactions of glycolysis are irreversible and need
to be replaced .
• These are the reactions:
• 1 ) Glucose + ATP → glucose-6- phosphate + ADP
• 2 ) fructose - 6-phosphate + ATP → fructose 1,6-bisphosphate + ADP
• 3 ) PEP + ADP → pyruvate + ATP
• These reactions cannot be carried out in reverse for a simple reason – the
reverse reaction in neither of the cases releases enough energy to produce
the products – e.g. substrate phosphorylation (originated by ATP) - reverse
reaction is not possible because the cleavage of glucose-6 -phosphate does
not provide enough energy needed for the synthesis of ATP.
126
Biochemie-6-2-metabolismus_sacharidů
Gluconeogenesis - synthesis of glucose de novo
• Organ : liver (kidney)
• Location: cytoplasm of cells
• Source for synthesis : a non-sugar substances
• (lactate, pyruvate , glucogenic amino acids, glycerol)
• enzymes of glycolysis , only 3 irreversible reactions are
replaced by other enzymes
127
Biochemie-6-2-metabolismus_sacharidů
1. Glc + ATP  Glc-6-P + ADP
(replaced by another enzyme)
2. Fru-6-P + ATP  Fru-1,6-bisP
(replaced by another enzyme)
3. PEP + ADP  pyruvate + ATP
(replaced by " bypass " )
Irreversible reactions of glycolysis (kinase
reaction)
128
Biochemie-6-2-metabolismus_sacharidů
glucose
Glc-6-P
Fru-6-P
Fru-1,6-bisP
Glyceraldehyde-3-
P
Dihydroxyacetone-2-P
1,3-bis-P-glycerate
3-P-glycerate
2-P-glycerate
phosphoenolpyru-
vate
pyruvate
Irreversible
reaction of
glycolysis
Glycolysis x gluconeogenesis
129
Biochemie-6-2-metabolismus_sacharidů
Specific reactions of gluconeogenesis
1. Synthesis of phosphoenolpyruvate
Why does this reaction not occur in reverse?
 Go = -61,9 kJ/mol
ATP conversion to ADP does not provide enough energy for a reverse reaction
ATPADP
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 242 .
130
Biochemie-6-2-metabolismus_sacharidů
The emergence of phosphoenolpyruvate is
divided into two stages :
1. the establishment of oxaloacetate decarboxylation to pyruvate *
localization : liver and kidney mitochondria
enzyme: pyruvate carboxylase
Energy : consumption of 1 ATP
2. The conversion of oxalacetate to phosphoenolpyruvate
localization : cytoplasm (mitochondria)
enzyme: phosphoenolpyruvatecarboxykinase
Energy : consumption of 1 GTP
* Note .: carboxylation of pyruvate is also anaplerotical reaction of the citric
acid cycle
131
Biochemie-6-2-metabolismus_sacharidů
•dehydrogenation of malate (CAC)
Other ways the formation of oxaloacetate in
mitochondria
•aspartate transamination
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 236.
WikiSkripta: Aminotransferázy [online]. [cit. 2014-07-17]. Dostupné z:
http://www.wikiskripta.eu/index.php/Aminotransfer%C3%A1zy
132
Biochemie-6-2-metabolismus_sacharidů
The mitochondrial membrane is not
permeable to oxaloacetate .
How to get oxalacetate from mitochondria
to the cytoplasm ?
It is transported in the form of malate or aspartate.
Oxaloacetate formed in the mitochondrial matrix . The
decarboxylation takes place partly in the cytoplasm :
133
Biochemie-6-2-metabolismus_sacharidů
pyruvát
pyruvate
laktát
oxaloacetate
glucogenic AA
malate
acetyl-CoA
citrate
fosfoenolpyruvát
oxaloacetát
Vznik
fosfoenolpyruvátu
schematicky
a
b
c
mitochondria
cytoplasmpyruvate
lactate
alanine
aspartate
CC
Synthesis and transport of oxaloacetate
134
Biochemie-6-2-metabolismus_sacharidů
pyruvate
pyruvate
oxaloacetate
glucogenic AA
malate
acetyl-
CoA
citrate
mitochondria
cytoplasm
phosphoenolpyruvate
oxaloacetate
a
b
c
malate
alanine
lactate
aspartate
aspartate
Synthesis of phosphoenolpyruvate
CC
135
Biochemie-6-2-metabolismus_sacharidů
1. Conversion of pyruvate to phosphoenolpyruvate
• Carboxylation of pyruvate (matrix of mitochondria)
a
CarboxybiotinCH3
C=O
COOH
pyruvate carboxylase
biotin
Pyruvate
Oxaloacetate NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 37.
136
Biochemie-6-2-metabolismus_sacharidů
• Transport of oxaloacetate into cytoplasm in form of malate
oxaloacetate
malate
NADH + H+
NAD+
oxalacetate
malate
NADH + H+
NAD+
cytoplasm
mitochondria
b
137
Biochemie-6-2-metabolismus_sacharidů
(*lecture Respiration – aspartate/malate shuttle).
• Transport of oxaloacetate into cytoplasm in form of aspartate
138
Biochemie-6-2-metabolismus_sacharidů
• decarboxylation of oxaloacetate
(PEP)
participates in reversible reactions of glycolysis
c
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 94.
139
Biochemie-6-2-metabolismus_sacharidů
Synthesis of phosphoenolpyruvate from pyruvate
or lactate requires 2 ATP
Coupled carboxylation and subsequent
decarboxylation enables the occurence of otherwise
energetically unfavorable reaction.
(also in the synthesis of fatty acids)
140
Biochemie-6-2-metabolismus_sacharidů
Why is pyruvate carboxylation preferred instead of
decarboxylation during GNG?
Organisms is under the influence of glucagon
Fatty acids are released from adipose tissue
-oxidation of fatty acids takes place in liver
Acetyl-CoA is present in liver
Acetyl-CoA: inhibits pyruvatedehydrogenase
activates pyruvatecarboxylase
141
Biochemie-6-2-metabolismus_sacharidů
2. Dephosphorylation of fructose-1,6-bisphosphate
Fructose-1,6-bisphosphatase
Allosteric inhibition by AMP,
activation by ATP
Inhibition by fructose-2,6bisphosphate
(glucagon
downregulates its level)
hydrolytic cleavage
Second special reaction in GNG
H2O
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 92.
142
Biochemie-6-2-metabolismus_sacharidů
3. Dephosphorylation of glucose-6-P
The enzyme is located in liver and
kidneys, not in muscles.
Third special reaction in GNG
Enzyme is
localized in
ER
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 92.
143
Biochemie-6-2-metabolismus_sacharidů
Energetic requirements of GNG
reaction ATP/glucose
2 pyruvate → 2 oxaloacetate -2
2 oxaloacetate → 2 phosphoenolpyruvate -2 (GTP)
2 3-phosphoglycerate → 2 1,3-bisphosphoglycerate -2
-6 ATP/glucose
The source of energy is mainly -oxidation of FA
144
Biochemie-6-2-metabolismus_sacharidů
2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 2H+
glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi
Summary equation of GNG
-6 ATPConsumption:
145
Biochemie-6-2-metabolismus_sacharidů
Lactate
created in tissues, transported via blood into liver
lactate + NAD+  pyruvate + NADH + H+
(cytoplasm)
(Cori cycle)
Substrates for GNG - origins
146
Biochemie-6-2-metabolismus_sacharidů
Glycerol
• created in adipocytes during the cleavage of triacylglycerols
• transported via blood to liver
• liver (cytoplasm):
glycerol + ATP  glycerol-3-P + ADP
glycerol-3-P + NAD+  dihydroxyacetone-P + NADH + H+
What is the energy consumption for synthesis of 1 mol
glucose from glycerol?
147
Biochemie-6-2-metabolismus_sacharidů
Glucogenic amino acids
their metabolism yields pyruvate or other
byproducts of citric acid cycle, which can
be transformed to oxaloacetate
Acetyl-CoA – it is not a direct source in
gluconeogenesis !!!
it is metabolised in CC to CO2
in animals, fatty acids are not transformed into
glucose
148
Biochemie-6-2-metabolismus_sacharidů
The most important glucogenic amino acid is alanine
It is released form muscle where it is created by transamination
from pyruvate, then it is transported into liver where it is
trasformed back into pyruvate
liver muscle
glucose
pyruvate
lactate
alanine
lactate
alanine
pyruvate
glucose
amino acid
2-oxo acid
glutamate
2-oxoglutarate
149
Biochemie-6-2-metabolismus_sacharidů
Glukoneogenesis from lactate and glycerol
requires NAD+
Sometimes during metabolism the NADH/NAD+ ratio can be
high – glukoneogenesis does not take place
NADH/NAD+ ratio rises e.g. during ethanol metabolism
(alcohol dehydrogenase), therefore the intake of alcohol can
inhibit gluconeogenesis  hypoglycemia
150
Biochemie-6-2-metabolismus_sacharidů
The main factors in gluconeogenesis regulation
Availability of substrates.
Regulation of irreversible reactions allosterically or by hormones.
Allosteric effects are fast (immediate response)
Hormones can influence GNG by:
• direct effect by second messenger – activation or inhibition
(fast)
• regulation of synthesis by induction or repression (slow –
hours to days)
151
Biochemie-6-2-metabolismus_sacharidů
Enzyme Activator Inhibitor
Hexokinase Glucose-6-phosphate
Phosphofructokinase 5´AMP, fructose-6phosphate,
fructose-
2,6-bisphosphate
Citrate, ATP,
glucagon
Pyruvatekinase fructose-1,6-
bisphosphate,
insulin
ATP, alanine,
glucagon,
noradrenalinw
Pyruvatedehydrogena
-se
CoA, NAD+,
insulin, ADP,
pyruvate
Acetyl-CoA, NADH,
ATP
Pyruvatecarboxylase Acetyl-CoA ADP
Phosphoenolpyruvate
carboxykinase
Glucagon ?
Effects of activators and inhibitors on enzymes in glycolysis and gluconeogenesis
152
Biochemie-6-2-metabolismus_sacharidů
Enzyme Inductor Repressor
Glucokinase Insulin Glucagon
Fosfofruktokinasa Insulin Glucagon
Pyruvatekinase Insulin Glucagon
Pyruvatecarboxylase Glucokorticoids
Glucagon
Adrenaline
Insulin
Phophoenolpyruvate
carboxykinase
Glucocorticoids
Glucagon
Adrenaline
Insulin
Glucose-6-phosphatase Glucocorticoids
Glucagon
Adrenaline
Insulin
Effects of hormones on enzymes in glycolysis and gluconeogenesis
153
Biochemie-6-2-metabolismus_sacharidů
Gluconeogenesis in kidneys
Kidneys can produce glucose by GNG
They can release it into bloodstream – during
postresorption phase, fasting or acidosis
Substrates - lactate, glycerol a glutamine
154