Download Pyruvate Dehydrogenase

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
starch
lactate
glucose
Lactate,
amino
acids,
glycerol
H2O+CO2
Pentose phosphate
pathway
Ribose, NADPH
§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
glycolytic pathway
pyruvate
lactic acid
1) Glycolytic pathway :
G → pyruvate
including 10 reactions.
(1) G phosphorylated into glucose 6-phosphate
HO CH2
H
H
OH
O
H
OH
H
H
OH
ATP
ADP
2+
Mg
Hexokinase
OH
G
P O CH2
H
H
OH
O
H
OH
H
OH
H
OH
G-6-P
• Phosphorylated G cannot get out of cell
• Hexokinase , HK (4 isoenzymes) ,
glucokinase, GK in liver ;
• Irreversible .
Comparison of hexokinase and
glucokinase
hexokinase
glucokinase
occurrence in all tissues
only in liver
Km value
0.1mmol/L
10mmol/L
Substrate
G, fructose,
mannose
glucose
Regulation
G-6-P
Insulin
(2) G-6-P → fructose 6-phosphate
P O CH2
H
H
OH
P O CH2
H
H
OH
OH
H
O
O H
OH
G-6-P
isomerase
CH2OH
OH
OH
H
OH
H
F-6-P
(3) F-6-P → fructose 1,6-bisphosphate
P O CH2
O
PFK-1
OH
H
OH
H
OH
P O CH2
CH2OH
H
F-6-P
H
2+
Mg
ATP
CH2 O P
O
OH
OH
H
ADP
OH
H
F-1,6-BP
• The second phosphorylation
• phosphofructokinase-1, PFK-1
(4) F-1,6-BP → 2 Triose phosphates
CH2 O P
C O
HO C H
H C OH
CH2 O P
C O
aldolase
H C OH
CH2 O P
F-1,6-BP
• Reversible
CH2OH
CHO
+
CHOH
CH2 O P
dihydroxyacetone glyceraldehyde
3-phosphate,
phosphate,
GAP
DHAP
(5) Triose phosphate isomerization
CH2 O P
C O
CH2OH
DHAP
CHO
phosphotriose
isomerase
CHOH
CH2 O P
GAP
G→2 molecule glyceraldehyde-3-phosphate,
consume 2 ATP .
(6) Glyceraldehyde 3-phosphate →
glycerate 1,3-bisphosphate
CHO
CHOH
CH2 O P
glyceraldehyde
3-phosphate
Pi NAD+
NADH+H +
glyceraldehyde
3-phosphate
dehydrogenase,
GAPDH
O C O~ P
CHOH
CH2 O P
glycerate
1,3-bisphosphate,
1,3-BPG
(7) 1,3-BPG → glycerate 3-phosphate
O C O~ P
CHOH
CH2 O P
glycerate
1,3-bisphosphate
ADP
ATP
Phosphoglycerate
kinase
COOCHOH
CH2 O P
glycerate
3-phosphate
• Substrate level phosphorylation
(8) Glycerate 3-phosphate → glycerate 2phosphate
COO-
COO-
CHOH
CH O P
CH2OH
CH2 O P
glycerate
3-phosphate
Mutase
glycerate
2-phosphate
(9) Glycerate 2-phosphate →
phosphoenol pyruvate
COOCH O P
enolase
CH2OH
glycerate
2-phosphate
COOC O ~ P + H2O
CH2
PEP
(10) PEP →pyruvate
COOC O~ P
CH2
PEP
ADP
ATP
pyruvate kinase
COOC O
CH3
Pyruvate
• Second substrate level phosphorylation
• irreversible
2) Pyruvate → lactate
NADH + H+
COO
C O
CH3
Pyr
NAD+
COO
CHOH
Lactate dehydrogenase,
CH3
LDH
Lactic acid
Summary of Glycolysis
ADP
ADP
ATP 2+
ATP 2+
Mg
Mg
F- 6-P
F- 1,6-BP
G
G-6-P
HK
Isomerase
PFK-1
Aldolase
lactate
NAD+
DHAP
GAP
LDH
H3PO4
NAD+
glyceraldehyde
NADH+H+
pyruvate
3-phosphate
+
NADH+H
dehydrogenase
ATP
pyruvate kinase
ADP
PEP
glycerate
1,3-bisphosphate
ADP
Enolase
ATP
Mutase
glycerate
glycerate
H2O
2-phosphate
3-phosphate
Phosphoglycerate
kinase
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
Pyruvate Kinase
(active)
ADP
PKA
cAMP
Glucagon
Pyruvate Kinase- P
(inactive)
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 energyyielding 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
cytosol
first
stage
G
Pyr
glycolytic
pathway
Mitochodria
third
second
stage
stage
Pyr
CH3CO~SCoA
CO2 + H2O+ATP
TAC
1) Oxidative decarboxylation of
Pyruvate to Acetyl CoA
COO-
NAD+
C O + HSCoA
CH3
NADH + H + O
Pyruvate
dehydrogenase
complex
pyruvate
• irreversible;
• in mitochodria.
H3C C ~SCoA + CO2
Acetyl CoA
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)
Pyruvate dehydrogenase complex:
HSCoA
NAD+
The structure of
pyruvate dehydrogenase complex
N
H3C C
N
NH2
C
HC
C
C
H
S
N
C
H2
O-
+
O-
C
CH2CH2 O
P
O P
C
CH3
O
O
O-
TPP
H2
C
+2H
CH (CH2)4 COOH
H2C
S
H2
C
S
lipoic acid
-2H
H2C
SH
CH (CH2)4 COOH
SH
dihydrolipoic acid
HSCoA
4'-phosphopantotheine
OH CH3
HS CH2 CH2 NH C CH2 CH2 NH C C
O
¦Â-mercaptoethylamine
O H
¦Â-alanine
OH
OH
C CH2 O P O P O 3'AMP
CH3
O
O
pantoic acid pyrophosphate
pantothenic acid
CO2
NADH
+H+
NAD+
CoASH
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
CH3CO~SCoA
acetyl CoA HSCoA
H2O
CH2 COO
CH2 COO
CO COO
CH2 COO
oxaloacetate
NADH+H+
H2O
HO C
citrate
synthase
COO
CH2 COO
malate dehydrogenase
CH
COO
aconitase
NAD
HO CH
COO
malate
CH2 COO
fumarase
HC
COO
Citrate cycle
isocitrate CH
HO CH
CH2 COO
COO
COO
+
NAD
fumarate
isocitrate dehydrogenase
succinyl CoA
syntetase
CH2 COO
NADH+H+
NAD+
succinate
ADP
CO~ SCoA
CH2 COO
CH2
CH2
CoASH GTP GDP+Pi
CO2
NADH+H+
succinate dehydrogenase
FAD
CH2 COO
H2O
CH2 COO
OOC CH
FADH2
COO
cis-aconitate
citrate
+
H2O
C
aconitase
CO2
HSCoA
COCOO
succinyl-CoA ¦Á-ketoglutarate ¦Á-ketodehydrogenase glutarate
ATP
complex
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
ADP + Pi
ATP
CH3
Biotin
C H2
C O + CO2
pyruvate carboxylase
COOH
CH3
COOH
C
O
COOH
NADPH+H+
C O + CO2
COOH
NADP+
COOH
NAD+
NADH+H+
C H2
C H2
malic enzyme
CHOH
COOH
COOH
malic acid DH
C
O
COOH
(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
allosteric activators:
AMP, CoA,
NAD+,Ca2+
allosteric inhibitors:
ATP, acetyl CoA,
NADH, FA
Pyruvate dehydrogenase
(active form)
Pi
ATP
pyruvate dehydrogenase
phosphatase
H2O
Ca2+,insulin
pyruvate dehydrogenase
kinase
ADP
pyruvate dehydrogenase P
(inactive form)
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
6-phosphogluco6-Phosphogluconate
G-6-P
G-6-P
nolactone
6-Phospho
dehydrogenase
NADP+
gluconolactonase
6-phosphogluconate
dehydrogenase
Ribose 5-P
Isomerase
Xylulose 5-P
NADPH+H+
CO2
Ribulose 5-P
Epimerase
2) Non-Oxidative Phase
Ribose 5-p
Fructose 6-p
Glycolysis
Fructose 6-p
Xylulose 5-p
Xylulose 5-p
Glyceraldehyde 3-p
• Transketolase: requires TPP
• Transaldolase
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 biosynthesis 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;
H2O2
2GSH
NADP+
glutathione reductase
2H2O
G-S-S-G
NADPH + H+
So, NADPH, glutathione and glutathione
reductase together will preserved the integrity
of RBC membrane.
Deficiency of glucose 6-phosphate
dehydrogenase results in hemolytic
anemia.
favism
 NADPH serves as the coenzyme of
mixed function oxidases (monooxygenases). 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.
1. Glycogen synthesis (Glycogenesis)
• The process of glycogenesis
occurs in cytosol of liver and
skeletal muscle mainly.
ATP ADP
G
G-6-P
HK or GK
UDP
Gn+1
G-1-P
UDPG
glycogen
UDPG
synthase
pyrophosphorylase
UTP
PPi
Gn
• UDPG: G active pattern, G active donor.
• In glycogen anabolism, 1 G consumes
2~P.
• Glycogen synthase: key E.
O
CH2OH
HN
O
H
H
OH
H
O
H
O
OH
H
OH
P
O
O
O
O
P
O
CH2
O
O
H
H
OH
H
OH
H
UDPG
N
Branching enzyme
2. Glycogen catabolism (glycogenolysis)
Pi
Gn-1
Gn
H2O
G-1-P
Phosphorylase
G-6-P
Pi
G-6-Pase
G
Phosphorylase: key E;
The end products: 85% of G-1-P and 15%
of free G;
There is no the activity of glucose 6phosphatase (G-6-Pase) in skeletal muscle.
Debranching enzyme:
glucan transferase
-1,6-glucosidase
(1→6) linkage
Nonreducing ends
Glycogen
phosphorylase
Transferase activity of
debranching enzyme
(1→6) glucosidase activity of
debranching enzyme
Glucose
3. Regulation of glycogenesis and
glycogenolysis
1) Allosteric regulation
In liver:
G
phosphorylase
glycogenolysis
In muscle:
AMP
Ca2+
phosphorylase-b
ATP
G-6-P
phosphorylase-a
glycogenolysis
2) Covalent modification
Glucagon
epinephrine
cAMP
receptor
PKA
G protein
Adenylyl
cyclase
Phosphorylase
Glycogen synthase
glycogenolysis
glycogenesis
Blood sugar
glucagon, epinephrine
active
adenylate cyclase
inactive
adenylate cyclase
cAMP
phosphorylase b
kinase
ATP
ATP
inactive
PKA
ATP
active
PKA
glycogen
synthase
(active)
glycogen
synthase
(inactive)
Pi
H2O
ATP
H2O
phosphorylase b
kinase
ATP
P
inhibitor-1
(inactive)
P
ADP
ADP
Pi
ADP
P
phosphorylase b phosphorylase a
Pi
protein
phosphatase-1
inhibitor-1
(active)
P
H2O
§6 Gluconeogenesis
• Concept:
The process of transformation of noncarbohydrates 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
GDP
CO 2
COO
-
CH O ~ P
PEP carboxykinase
GTP
COO
£¨ 1/3Mt.
. 2/3cytosal£©
-
CH2
PEP
ADP
Pyr kinase
ATP
ADP+Pi
ATP
CO
COO
2
C O
Biotin
C O
CH2
Pyr carboxylase
CH3
COOH
£¨ Mt.£©
pyruvate
oxaloacetic acid
2) F-1, 6-BP →F-6-P
ATP
ADP
PFK-1
F-6-P
Fructosebisphosphatase
Pi
H2O
F-1,6-BP
3) G-6-P →G
ATP
ADP
HK
G
Glucose-6phosphatase
H2 O
Pi
• 2 lactic acid
G-6-P
G
consume
ATP?
glucose
glycogen
G-6-P
G-1-P
gluconeogenesis
CYTOSOL
MITOCHONDRIA
F-6-P
NAD+
glyceraldehyde 3-P
glutamate
glutamate
¦Á-ketoglutarate
NADH+H+
DHAP
malic acid
malic acid
F-1,6BP
OAA
Asp
¦Á-ketoglutarate
NADH+H+
Asp
1.3-bisphospho2/3
glycerate
CO2
ADP
GDP
ATP
glycerate 3-P
phosphoenol
pyruvate
ADP
glycerate 2-P
PK
ATP
NAD+ NADH+H+
lactate
pyruvate
OAA
GTP
GTP
glycerol
NAD+
ADP
CO2
GDP
1/3
phosphoenol
pyruvate
ATP
CO2
pyruvate
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
gluconeogenesis:
F-6-P
Pi
F-2,6-BP
PFK-1
FBPase-1
AMP
H2O
ATP
F-1,6-BP
glycolysis
ADP
F-2,6-BP
glucagon
PEP
ADP
F-1,6-BP
glucagon
Ala in liver
insulin
OAA
ATP
Pyr
acetyl CoA
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
Lactic acid cycle
glucose
glucose
gluconeogenesis
glucose
glycolytic
pathway
pyruvate
pyruvate
NADH+H+
NAD+
NAD+
NADH+H+
lactate
liver
lactate
lactate
blood
muscle
§6 Blood Sugar and Its
Regulation
1. The source and fate of blood sugar
origin (income)
fate (outcome)
CO2 + H2O + energy
dietary supply
liver glycogen
non-carbohydrate
(gluconeogenesis)
other saccharides
blood sugar
glycogen
3.89¡« 6.11mmol/L
other saccharides
non-carbohydrates
(lipids and some
amino acids)
>8.89¡« 10.00mmol/L
(threshold of kidney)
urine glucose
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