gluconeogenesis for medical school
TRANSCRIPT
GLUCONEOGENESIS
V.S.RAVIKIRAN, MSc.
V.S.RAVIKIRAN, MSc., Department of Biochemistry,
ASRAM Medical college, Eluru-534005.AP, [email protected]
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GLUCONEOGENESIS
The de novo synthesis of glucose and its
role in preventing hypoglycemia
Gluconeogenesis occurs mainly in liver.
Gluconeogenesis occurs to a more limited extent in kidney & small intestine under some conditions.
Synthesis of glucose from pyruvate utilizes many of the same enzymes as Glycolysis.
Three Glycolysis reactions have such a large negative G that they are essentially irreversible. Hexokinase (or Glucokinase) Phosphofructokinase Pyruvate Kinase.
These steps must be bypassed in Gluconeogenesis.Two of the bypass reactions involve simple hydrolysis reactions.
Gluconeogenesis: Overview
General Features
Tissues: liver (80%)kidneys (20%)
Subcellular location of enzymespyruvate
carboxylase: mitochondrial
glucose-6-phosphatase: ER
all other enzymes cytoplasmic
Pathway of Gluconeogenesis
The distinctive reactions and enzymes of this pathway are shown in red.The other reactions are common to glycolysis. The enzymes for gluconeogenesis are located in the cytosol, except for pyruvate carboxylase (in the mitochondria) and glucose 6-phosphatase (membrane bound in the endoplasmic reticulum).The entry points for lactate, glycerol, and amino acids are shown.
Compartmental Cooperation
Oxaloacetate utilized in the cytosol for gluconeogenesis is formed in the mitochondrial matrix by carboxylation of pyruvate. Oxaloacetate leaves the mitochondrion by a specific transport system (not shown) in the form of malate, which is reoxidized to oxaloacetate in the cytosol.
Generation of Glucose from Glucose 6-Phosphate
Several endoplasmic reticulum (ER) proteins play a role in the generation of glucose from glucose 6-phosphate. T1 transports glucose 6-phosphate into the lumen of the ER, whereas T2 and T3 transport Pi and glucose, respectively, back into the cytosol. Glucose 6-phosphatase is stabilized by a Ca2+-binding protein (SP).
Glyceraldehyde-3-phosphate Dehydrogenase
Phosphoglycerate Kinase
Enolase
PEP Carboxykinase
glyceraldehyde-3-phosphate
NAD+ + Pi
NADH + H+
1,3-bisphosphoglycerate
ADP
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate H2O
phosphoenolpyruvate
CO2 + GDP
GTP oxaloacetate
Pi + ADP
HCO3 + ATP
pyruvate
Pyruvate Carboxylase
Gluconeogenesis
Summary of Gluconeogenesis Pathway:
Gluconeogenesis enzyme names in red.
Glycolysis enzyme names in blue.
Glucose-6-phosphatase
Fructose-1,6-bisphosphatase
glucose Gluconeogenesis
Pi
H2O glucose-6-phosphate
Phosphoglucose Isomerase
fructose-6-phosphate
Pi
H2O fructose-1,6-bisphosphate
Aldolase
glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate Isomerase (continued)
Malate Shuttle
OAA produced in mitochondria
mitochondrial membrane impermeable to OAA
malate transporter in mito. Membrane
malate dehydrogenase in both mito and cyto
NADH produced in cyto also used in gluconeogenesis.
Energetics of Gluconeogenesis
Pyruvate Carboxylase 2 ATPs
PEP Carboxykinase 2 GTPs
3-P-glycerate kinase 2 ATPs
Glyceraldehyde-3-P dehydrogenase 2NADH
Glycolysis & Gluconeogenesis are both spontaneous. If both pathways were simultaneously active in a cell, it would constitute a "futile cycle" that would waste energy.
Glycolysis: glucose + 2 NAD+ + 2 ADP + 2 Pi
2 pyruvate + 2 NADH + 2 ATPGluconeogenesis: 2 pyruvate + 2 NADH + 4 ATP + 2 GTP glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi
Questions:1. Glycolysis yields how many ~P ? 2. Gluconeogenesis expends how many ~P ? 3. A futile cycle of both pathways would waste how many ~P per cycle ?
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Precursors for gluconeogenesis
Glycerolderived from adipocyte lipolysishepatic glycerol kinase
Precursers for gluconeogenesis
LactateRBCmusclethe Cori Cycle
The lactic acid (Cori) and glucose-alanine cycles.
Cori Cycle
Liver Blood Muscle Glucose Glucose 2 NAD+ 2 NAD+
2 NADH 2 NADH 6 ~P 2 ~P 2 Pyruvate 2 Pyruvate 2 NADH 2 NADH 2 NAD+ 2 NAD+ 2 Lactate 2 Lactate
Precursers for gluconeogenesis
Alanine and other amino acids transamination of pyruvate pyruvate derived from glycolysis or from amino acid
degradation alanine cycle
Hexokinase or Glucokinase (Glycolysis) catalyzes:glucose + ATP glucose-6-phosphate + ADP
Glucose-6-Phosphatase (Gluconeogenesis) catalyzes: glucose-6-phosphate + H2O glucose + Pi
H O
OH
H
OHH
OH
CH2OH
H
OH
HH O
OH
H
OHH
OH
CH2OPO32
H
OH
HH2O
1
6
5
4
3 2
+ Pi
glucose-6-phosphate glucose
Glucose-6-phosphatase
H O
OH
H
OHH
OH
CH2OH
H
OH
HH O
OH
H
OHH
OH
CH2OPO32
H
OH
HH2O
1
6
5
4
3 2
+ Pi
glucose-6-phosphate glucose
Glucose-6-phosphatase
Glucose-6-phosphatase enzyme is embedded in the endoplasmic reticulum (ER) membrane in liver cells.
The catalytic site is found to be exposed to the ER lumen. Another subunit may function as a translocase, providing access of substrate to the active site.
Phosphofructokinase (Glycolysis) catalyzes: fructose-6-P + ATP fructose-1,6-bisP + ADP
Fructose-1,6-bisphosphatase (Gluconeogenesis) catalyzes:
fructose-1,6-bisP + H2O fructose-6-P + Pi
fructose-6-phosphate fructose-1,6-bisphosphate
Phosphofructokinase CH2OPO3
2
OH
CH2OH
H
OH H
H HO
O6
5
4 3
2
1 CH2OPO32
OH
CH2OPO32
H
OH H
H HO
O6
5
4 3
2
1ATP ADP
Pi H2O
Fructose-1,6-biosphosphatase
Bypass of Pyruvate Kinase:
Pyruvate Kinase (last step of Glycolysis) catalyzes:
phosphoenolpyruvate + ADP pyruvate + ATP
For bypass of the Pyruvate Kinase reaction, cleavage of 2 ~P bonds is required.
G for cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP).
PEP has a higher negative G of phosphate hydrolysis than ATP.
Bypass of Pyruvate Kinase (2 enzymes):
Pyruvate Carboxylase (Gluconeogenesis) catalyzes:pyruvate + HCO3
+ ATP oxaloacetate + ADP + Pi
PEP Carboxykinase (Gluconeogenesis) catalyzes:oxaloacetate + GTP PEP + GDP + CO2
C
C
CH 2
O O
O PO 32
C
C
CH 3
O O
O
A T P A D P + P i C
CH 2
C
C
O
O O
O O
HC O 3
G T P G D P
CO 2
p y r u v a te o x a lo a c e ta te P E P
P y ru v a te C a rb o x y la s e P E P C a rb o x y k in a s e
Biotin has a 5-C side chain whose terminal carboxyl is in amide linkage to the -amino group of an enzyme lysine.
The biotin & lysine side chains form a long swinging arm that allows the biotin ring to swing back & forth between 2 active sites.
Pyruvate Carboxylase uses biotin as prosthetic group.
CHCH
H2CS
CH
NHC
HN
O
(CH2)4 C NH (CH2)4 CH
CO
NH
O
biotin
N subject to carboxylation
lysine residue
H3N+ C COO
CH2
CH2
CH2
CH2
NH3
H
lysine
Biotin carboxylation is catalyzed at one active site of Pyruvate Carboxylase.
ATP reacts with HCO3 to yield carboxyphosphate.
The carboxyl is transferred from this ~P intermediate to N of a ureido group of the biotin ring. Overall:
biotin + ATP + HCO3 carboxybiotin + ADP + Pi
O P O
O
OH
C O
O
carboxyphosphate
CHCH
H2CS
CH
NHC
N
O
(CH2)4 C NH (CH2)4 CH
CO
NH
O
CO
-O
carboxybiotin
lysine residue
At the other active site of Pyruvate Carboxylase the activated CO2 is transferred from biotin to pyruvate:
carboxybiotin+ pyruvate
biotin + oxaloacetate
CHCH
H2CS
CH
NHC
N
O
(CH2)4 C NH R
O
CO
-OC
C
CH3
O O
O
C
CH2
C
C
O
O O
OO
CHCH
H2CS
CH
NHC
HN
O
(CH2)4 C NH R
O
carboxybiotin
pyruvate
oxaloacetate
biotin
PEP Carboxykinase catalyzes GTP-dependent oxaloacetate PEP. It is thought to proceed in 2 steps: Oxaloacetate is first decarboxylated to yield a
pyruvate enolate anion intermediate. Phosphate transfer from GTP then yields
phosphoenolpyruvate (PEP).
C
C
C H 2
O O
O P O 32
C
C H 2
C
C
O
O O
O O
C O 2
C
C
C H 2
O O
O
G T P G D P
o x a lo a c e ta te P E P
P E P C a rb o x y k in a se R e a c tio n
Coordinated Regulation of Gluconeogenesis and Glycolysis
Gluconeogenesis and Glycolysis are regulated by similar effector molecues but in the opposite directionavoid futile cycles
PK vs PC & PEPCK PFK-1 vs FDP’taseGK vs G6P’tase
Coordinated Regulation of Gluconeogenesis and Glycolysis
Regulation of enzyme quantity
Fasting: glucagon, cortisol induces gluconeogenic
enzymes represses glycolytic enzymes liver making glucose
Feeding: insulin induces glycolytic enzymes represses gluconeogenic
enzymes liver using glucose
Coordinated Regulation of Gluconeogenesis and Glycolysis
Short-term Hormonal Effects Glucagon, Insulin
cAMP & F2,6P2
PFK-2 & FBPase-2 A Bifunctional enzyme cAMP
Inactivates PFK-2 Activates FBPase-2 Decreases F2,6P2
Reduces activation of PFK-1 Reduces inhibition of FBPase-1
Low blood sugar results in Hi gluconeogenesis Lo glycolysis
Coordinated Regulation of Gluconeogenesis and Glycolysis
Allosteric EffectsPyruvate kinase vs Pyruvate carboxylase
PK - Inhibited by ATP and alaninePC - Activated by acetyl CoAFasting results in gluconeogenesis
PFK-1 vs FBPase-1FBPase-1 inhibited by AMP & F2,6P2
PFK-1 activated by AMP and & F2,6P2
Feeding results in glycolysis
Reciprocal Regulation of Gluconeogenesis and Glycolysis in the Liver
The level of fructose 2,6-bisphosphate is high in the fed state and low in starvation. Another important control is the inhibition of pyruvate kinaseby phosphorylation during starvation.
Pathway of Gluconeogenesis
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