Transcript 1. dia - Semmelweis

Reaction mechanism of pyruvate carboxylase
acidic anhydride bond
biotin carboxylase
domain
- enzyme
biotin
transcarboxylase dom.
-enzyme
carboxybiotin
oxaloacetate
Pyruvate carboxylase:
found in mitochondrial matrix
homotetramer
prosthetic group: biotin
carboxylates pyruvate to oxaloacetate
ligase:
requires ATP breakdown to ADP + Pi
has 3 domains: biotin carboxylase (BC),
biotin carrier part (BCCP),
transcarboxylase = carboxytransferase (CT)
needs Mg2+ as cofactor to bind ATP
K+, VO2+ as activators to bind HCO3Mn2+ to maintain enzyme structure
acetyl-CoA is an obligate allosteric activator
(derived from glucose in well-fed and from fatty acids or ketone bodies
in fasting state)
Localization of PC
high amount in gluconeogenetic: liver and kidney cortex
and fat producing tissues: adipose, lactating mammary gland, liver
and pancreas beta-cells
moderate amount in astrocytes in brain, heart, adrenal gland,
small amount: white blood cells, fibroblasts
missing from neurons, red blood cells...
Wellfed liver cell
•glycogenesis (glycogen synthesis)
•glycolysis
•amino acid degradation
•protein synthesis
•ornithine cycle
•FA and TAG synthesis
•PL, SL, VLDL production
•cholesterol, its ester synthesis
•bile acid and bile production
insulin secretion
?
NADPH
M
carbohydrates
↓
↓
↓
gal
glu
fru
↓
↓
glycogen
pyr
↓
‫ ۝‬PDHC
mt.bm.
pyr → AcCoA
pyruv.carbox. ↓
OA → citr
↑
↓
NAD
↓
NADH+H+ █
M ←← αKG
ADP+P
ATP
In pancreatic beta cells the high blood and cytoplasmic glucose cc. is the main signal
of insulin secretion.
Aerobic glucose degradation, high ATP, NADPH etc. production are required
cytopl.
carbohydrates
VLDL
lipids
bile
↓
↓
↓
↑
↓
↓
↑
gal
glu
fru
TAG,PL,SL←FA cholest.
↓
↓
glycogen
pyr
malate
↓
NADPH
OA
matrix
pyr → AcCoA
PC ↓
→AcCoA
OA → citr
O citr
↑
↓
NAD
↓
NADH+H+ █
M ←← αKG
ADP+P
slows down
ATP
1.) In triacylglycerol synthesizing hepatocytes, white adipocytes, lactating
mammarian cells fat is produced mainly from carbohydrates,
Pyruvate carboxylase is necessary to increase citrate level, which will be the
precursor and allosteric activator of FA synthesis,
so PC has an anaplerotic role for citric acid cycle.
2.) Phospholipid (PL) and sphingolipid (SL) synthesis also requires sufficient
carbohydrate level normally everywhere.
3.) In brain the FA transport across the blood-brain barrier is not significient,
therefore CNS must synthesize its onw fats and can not take it up.
In case of PC deficiency the fatty acid, phospholipid, myelin sheat
synthesis is impossible causing action potential transmission disturbancies and
neurological signs in utero and early in life.
4.) In other tissues both TAG and PL synthesis can be normal, because FA uptake
can replenish lipid synthesis from carbohydrates.
proteins
carbohydrates
VLDL
lipids
bile
↓
↓
↓
↑
↓
↓
↑
amino acids
gal
glu
fru
TAG,PL,SL←FA cholest.
NH3
↓
↓
glycogen
pyr
malate
citr ac c.
↓
AcCoA
OA
pyr → AcCoA
Glu
↓
→AcCoA
AS
Asp
O
Asp ← OA → citr
O citr
↑
NH3 ↑
↓ NAD
citr
citr
↓
NADH+H+ █
‫۝‬
M ←← αKG
Arg
orn
orn
NH3←↑
ADP+P
Glu
urea
ATP
fum
mal
NH3 ←
Gln
Anabolic reactions that whithraw intermediets from the citric acid cycle and
speeding up the cycle require anaplerotic, filling up reactions, e.g. pyruvate
carboxylase, glutaminase, glutamate dehydrogenase.
a.) In liver NH3 is biult in and detoxified to urea = carbamide in ornithine = urea cycle.
It needs Asp that is derived from oxaloacatete which comes from pyruvate.
In pyruvate carboxylase deficiency ammonia is not eliminated good enough,
ornithine cycle intermediets are increased: e.g. citrulline.
Hyperammonemia is harmful for brain.
NH3 is derived from amino acid degradation (from tissues, gut lumen, liver).
Amino acid concentration is high after protein rich meal (from gut) and
in fasting, when muscle and liver proteins are degraded.
b.) malate-aspartate shuttle can export cytoplasmic glycolytic NADH hydrogen to
mitochondrial matrix to electron transport chain
alpha-ketoglutarate-malate and aspartate-glutamate(+H+) antiporters take part
In PC deficiency the NAD/NADH ratio is abnormal, mitochondrial
membrane potential is disrupted, mitochondrial structural abnormalities
occur.
c.) in brain the astrocytes produce glutamine from glutamate, that is derived from
a-ketoglutarate. It withraws CAC intermediate. Glutamine is taken up and
transformed to glutamate in neurons. Glutamate is the main stimulatory
neurotransmitter. Aspartate comes from oxaloacetate, GABA from Glu.
Asp and GABA are also neurotransmitters.
In PC deficiency these amino acid neurotransmitters can not be produced properly,
signal transduction is disturbed.
Treatment:
- Supplementation of biotin, the prosthetic group.
- Aspartate that can take part in urea cycle and fills up citric acid cycle.
- Triheptanoin = triglyceride containing 7 carbon fatty acids, that degraded to
propionyl-CoA, converted to succinyl-CoA filling up CAC in most cells.
In liver 5 C containing ketone bodies are produced, transported to CNS and
fill up the citric acid cycle there, improving all the metabolic and neurological
disturbances.
Fasting in liver
•
•
•
•
•
•
•
glycogenolysis = glycogen degradation
gluconeogenesis
amino acid degradation
urea cycle
FA beta-oxidation
ketone body production
(VLDL production and glyceroneogenesis
from lactate or amino acids)
adip. TAG
↓
glycerol
↓
glyc3P
brain, rbc, heart
↑
glucose
↑
glycogen
DHAP
PEP
↑
OA
fum → mal
Arg
urea
rbc, white muscle
↓
lactate
↓
amino acids
pyruvate
pyr
PC↓
Asp → OA →
P
‫۝‬
citr ←
citr
orn →
orn ←
AcCoA
citr
↓
↓
M ← ← αKG
↑
↑
amino acids
NH3
liver and muscle proteins
C atoms of lactate, glycerol, amino acids are built into glucose
N atoms of amino acids are built into urea
1.) Pyruvate carboxylase is one of the regulated enzymes of gluconeogenesis,
if the precursors are lactate, and amino acids degraded to pyruvate.
In liver and kidney cortex gluconeogenetic regulated enzymes are
- induced by adrenalin, noradrenalin leading to cAMP elevation,
- by cortisol
- repressed by insulin.
In PC deficiency gluconeogenesis is not efficient causing hypoglycemia, which is
wrong for the obligate glucose consuming brain, red blood cell, white
skeletal muscle, kidney medulla etc.
Pyruvate is converted to lactate and alanine in the cytoplasm, causing lactic acidosis,
Metabolic acidosis deteorates brain.
2.) Low blood glucose level causes glucagon secretion, causing FFA increase,
leading to ketone body production i.e. ketoacidosis.
adip. TAG
↓
glycerol
↓
glyc3P
brain, rbc, heart
↑
glucose
↑
glycogen
DHAP
PEP
↑
OA
Arg
urea
rbc, white muscle
↓
lactate
↓
amino acids
pyruvate
ketone bodies
pyr
↓
Asp → OA →
P
fum → mal
brain, muscles
‫۝‬
citr ← citrul.
‫۝‬
orn → orn ←
AcCoA ←← FA-CoA
citrate
↓
↓
M ← ← αKG
↑
↑
amino acids
NH3
liver and muscle proteins
adip. TAG
FFA
FA
cytolasm
mitoch
matrix
Pyruvate carboxylase deficiency – group I.
point mutations: Val145 → Ala, or Arg451→Cys in biotin carboxylase domain
→ enzyme acitivity↓↓ in North Amariacan native people
signs: lactic acidemia (metabolic acidosis) mild to moderate (pyr and Ala ↑, too)
delayed development
psychomotor retardation
Pyruvate carboxylase deficiency group II.
point mutation: Ala610 →Thr or Met743 →Ile in transcarboxylase domain →
protein instabilty and degradation → missing PC enzyme in UK and French
patients
signs: serious lactic acidemia, Pyr and Ala are high
improper gluconeogenesis causes hypoglycemia
oxaloacetate ↓→ Asp ↓→ urea cycle in liver ↓→ NH3 is not detoxified ↓→
brain damage and citrullin ↑ in blood
anaplerotic reac. of citrate cycle ↓→ Citric ac. cycle ↓→ ATP ↓→ brain damage
Regulation of pyruvate carboxylase
glucose → ? → P2= distal promoter activated
pyr. carb. transcription → ? → insulin secretion
PPAR → P1= proximal promoter activated
→ pyr. carb. gene transcription →
lipogenesis
glucagon, adrenalin... → CREB → P1
promoter activated → PC expression →
gluconeogenesis