1. BIOC 460 - DR. TISCHLER LECTURE 26 GLYCOLYSIS AND GLUCONEOGENESIS-2

2. OBJECTIVES Gluconeogenesis Pathway
Key liver gluconeogenic precursors; 4 unique rxns
relationship between nutritional status (i.e., fed or starved) and the amount of phosphoenolpyruvate carboxykinase in liver cells as well as the significance of these responses .
Significance of the glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions in gluconeogenesis

3. Regulation of Glycolysis and Gluconeogenesis
1. Kinetics and allosteric regulation of glucokinase and hexokinase; logic for differences between the two kinases.
2. Regulation of PFK-1 and F-1,6-BPase:
a) compare allosteric regulation.
b) regulation coordinates glycolysis and gluconeogenesis
c) metabolism of F-2,6-BP; role of bifunctional protein
d) insulin via F-2,6-BP ensures during the fed state that liver glycolysis is active and gluconeogenesis is inactive
3. Regulation of pyruvate kinase via allosteric control and by covalent modification (phosphorylation/ dephosphorylation)

4. G6Pase
unique
Glucose 6-P
Glucose
precursor
Fructose 6-P
glycolytic enzyme also
F1,6BPase
F-1,6-BP
G-3-P + DHAP
Glycerol
Lactate
NADH
LDH
NAD+
NAD+
G-3-P DH
Figure 1.
Gluconeogenic pathway for lactate, ala, asp, glycerol
NADH
Alanine
ALT
ADP + Pi
PGK
ATP
Pyruvate
CYTOPLASM
PEP PC
PEPCK
Pyruvate
OAA
PEP
MITOCHONDRION
Aspartate
AST

5. pyruvate + ATP + CO2  oxaloacetate + ADP + Pi
Unique Reaction 1:
Pyruvate carboxylase catalyzes the reaction:
pyruvate + ATP + CO2  oxaloacetate + ADP + Pi
CO2
CO2
O=C-OH
C=O
CH3
CH2
O=C-OH
C=O
CO2
requires biotin
CO2
CO2
O=C-OH

6. oxaloacetate + GTP  phosphoenolpyruvate + GDP + CO2
Unique Reaction 2:
Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the reaction:
oxaloacetate + GTP  phosphoenolpyruvate + GDP + CO2
PO3
CO2
PO3
CO2
O=C-OH
C=O
CH2
PO3
CO2
O=C-OH
C-OPO32-
CH2
PO3
PO3
CO2
CO2

7. Fructose-1,6-bisphosphate  Fructose-6-phosphate + Pi
Unique Reaction 3:
Fructose-1,6-bisphosphatase catalyzes the reaction:
Fructose-1,6-bisphosphate  Fructose-6-phosphate + Pi
PO3
PO3
PO3
PO3 O
CH2OPO32- HO OH
CH2OH O
CH2OPO32- HO OH

8. Glucose-6-phosphate  Glucose + Pi
Unique Reaction 4:
Glucose-6-phosphatase catalyzes the reaction:
Glucose-6-phosphate  Glucose + Pi
PO3
PO3
PO3 O
CH2OH OH O
CH2OPO32- OH

9. COMPARATIVE KINETICS OF HEXOKINASE AND GLUCOKINASE
has low Km (high affinity) for glucose
ensures brain, red blood cells, etc can use
glucose even when blood low in starvation
Glucokinase:
has high Km (low affinity) for glucose
ensures that liver or pancreas only uses glucose
in response to high blood levels after a meal

10. Figure 2. Regulation of glucokinase and hexokinase
ADP
ATP
Hexose
(e.g.,glucose,
fructose, galactose)
Glucose
(liver; pancreas)
Glucokinase
Feedback inhibition
by glucose-6-P
No allosteric control
Glucose-6-P
Hexose-6-P
(G6P most
Tissues)
Figure 2. Regulation of glucokinase and hexokinase

11. - = allosteric inhibition; + = allosteric activation
Fructose-6-phosphate
ATP Pi
ATP -
Citrate
fructose-2,6-bisP
AMP -
PFK-1 H+
F-1,6-BPase
(glycolysis) +
AMP
(gluconeogenesis) +
citrate
fructose-2,6-bisP
H2O
ADP
Fructose-1,6-bisphosphate
Figure 3 – Reciprocal regulation of glycolysis and gluconeogenesis
at reactions catalyzed by phosphofructokinase-1
and fructose-1,6-bisphosphatase.
- = allosteric inhibition; + = allosteric activation

12. Bifunctional protein – dephosphorylated
(by protein phosphatase):
PFK-2 - active
FBPase-2 - inactive
fructose-6-phosphate
fructose-2,6-bisphosphate
ATP
ADP
fructose-6-phosphate
Bifunctional protein – phosphorylated (by protein kinase A):
FBPase-2 - active
PFK-2 - inactive
fructose-2,6-bisphosphate
H2O Pi
Figure 4. Reactions in the metabolism of fructose-2,6-bisphosphate.

13. X fructose-6-P ATP fructose-6-P PFK-2 + ATP active inactivation PFK-1
fructose-2,6-bisP
ATP
ADP
fructose-6-P + Pi
fructose-1,6-bisP
inactivation
FBPase-1
gluconeogenesis X
fructose-6-P
+ ATP
fructose-1,6-bisP
+ ADP
PFK-1
glycolysis
activation
Figure 5. Formation of F-2,6-bisP in the fed state leads to activation of PFK-1 in glycolysis and inactivation of gluconeogenesis.

14. - Figure 6. Regulation of liver pyruvate kinase. ATP ADP
protein kinase A +
Glucagon OH
Pyruvate
Kinase
(active)
+ ATP
Phosphoenolpyruvate
+ ADP
OPO32-
Pyruvate
kinase
(inactive) -
citrate
ATP
alanine Pi
protein
phosphatase +
Insulin
Figure 6. Regulation of liver pyruvate kinase.