Gluconeogenesis;

Regulation of Glycolysis &

Gluconeogenesis
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.
 Glucose-6-phosphatase
6 CH OPO 2 CH2OH
2 3
5 O O
H H H H
H H2O H
4
OH H 1
OH H + Pi
OH OH OH OH
3 2
H OH H OH
glucose-6-phosphate glucose

Hexokinase or Glucokinase (Glycolysis) catalyzes:

glucose + ATP glucose-6-phosphate + ADP
Glucose-6-Phosphatase (Gluconeogenesis) catalyzes:

glucose-6-phosphate + H2O glucose + Pi

 Glucose-6-phosphatase
6 CH OPO 2 CH2OH
2 3
5 O O
H H H H
H H2O H
4
OH H 1
OH H + Pi
OH OH OH OH
3 2
H OH H OH
glucose-6-phosphate glucose

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
6 CH OPO 2 1CH2OH 6 CH OPO 2 1CH2OPO32
2 3 2 3
O ATP ADP O
5 H HO 2 5 H HO 2

H 4 3 OH H 4 3 OH
Pi H2O
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate
Fructose-1,6-biosphosphatase

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
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.
 Pyruvate Carboxylase PEP Carboxykinase
O O
O C O
O O
C ATP ADP + Pi C O GTP GDP C
C O CH2 C OPO32
HCO3 C CO2
CH3 CH2
O O
pyruvate oxaloacetate PEP

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
 Pyruvate Carboxylase PEP Carboxykinase
O O
C
O O O O
C ATP ADP + Pi C O GTP GDP C
C O CH2 C OPO32
HCO3 C CO2
CH3 CH2
O O
pyruvate oxaloacetate PEP

Contributing to spontaneity of the 2-step

process:
Free energy of one ~P bond of ATP is conserved
in the carboxylation reaction.
Spontaneous decarboxylation contributes to
spontaneity of the 2nd reaction.
Cleavage of a second ~P bond of GTP also
contributes to driving synthesis of PEP.
Pyruvate N subject to O
Carboxylase carboxylation
uses biotin as C
prosthetic HN NH
lysine
group. CH CH residue
O O C
H2C CH
S (CH2)4 C NH (CH2)4 CH

biotin NH

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.
 O
-O C
C N NH lysine
O
CH CH residue
O O C
H2C CH
S (CH2)4 C NH (CH2)4 CH

carboxybiotin NH

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
At the other O O
active site of C -O C
Pyruvate C N NH
Carboxylase C O O carboxybiotin
the activated CH CH
CH3
CO2 is H2C CH O
transferred pyruvate
S (CH2)4 C
from biotin to NH R
pyruvate:
O O O
carboxybiotin
C
+ pyruvate C
C O HN NH
biotin
biotin + CH2 CH CH
oxaloacetate C H2C CH O
View an animat O S (CH2)4 C
O NH R
ion oxaloacetate
.
Pyruvate Glucose-6-phosphatase
Carboxylase glucose-6-P glucose
(pyruvate Gluconeogenesis Glycolysis
oxaloactate)
is allosterically pyruvate
activated by fatty acids
acetyl CoA. acetyl CoA ketone bodies
[Oxaloacetate]
tends to be oxaloacetate citrate
limiting for
Krebs cycle.
Krebs Cycle

When gluconeogenesis is active in liver,

oxaloacetate is diverted to form glucose.
Oxaloacetate depletion hinders acetyl CoA entry into
Krebs Cycle. The increase in [acetyl CoA] activates
Pyruvate Carboxylase to make oxaloacetate.
Avidin, a protein in egg whites with a
barrel structure, tightly binds biotin.
Excess consumption of raw eggs can
cause nutritional deficiency of biotin.
The strong avidin-to-biotin affinity is avidin
used by biochemists as a specific "glue." with bound biotin

If it is desired to bind 2 proteins together for

an experiment, biotin may be covalently
linked to one protein and avidin to the other.
Explore with Chime the biotinyl domain of a
carboxylase and the avidin-biotin complex.
 O O
PEP Carboxykinase Reaction
C O
O GTP GDP O O
C O C C
CH2 C O C OPO32
CO2
C CH2 CH2
O O
oxaloacetate PEP

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).
In the bacterial enzyme, ATP
is Pi donor instead of GTP.
In this crystal structure of an
E. Coli PEP Carboxykinase,
pyruvate is at the active site
as an analog of PEP/
oxaloacetate.

A metal ion such as Mn++ is required for the PEP

Carboxykinase reaction, in addition to a Mg++ ion that
binds with the nucleotide substrate at the active site.
Mn++ is thought to promote Pi transfer by interacting
simultaneously with the enolate oxygen atom and an
oxygen atom of the terminal phosphate of GTP or ATP.
The source of pyruvate and oxaloacetate for
gluconeogenesis during fasting or carbohydrate
starvation is mainly amino acid catabolism.
Some amino acids are catabolized to pyruvate,
oxaloacetate, or precursors of these.
Muscle proteins may break down to supply
amino acids. These are transported to liver
where they are deaminated and converted to
gluconeogenesis inputs.
Glycerol, derived from hydrolysis of
triacylglycerols in fat cells, is also a significant
input to gluconeogenesis.
 glyceraldehyde-3-phosphate
NAD+ + Pi Glyceraldehyde-3-phosphate
NADH + H+ Dehydrogenase
1,3-bisphosphoglycerate
ADP
Summary of Phosphoglycerate Kinase
ATP
Gluconeogenesis 3-phosphoglycerate
Pathway: Phosphoglycerate Mutase
2-phosphoglycerate
Gluconeogenesis
enzyme names in H2O Enolase

red. phosphoenolpyruvate
CO2 + GDP
PEP Carboxykinase
Glycolysis enzyme GTP
names in blue. oxaloacetate
Pi + ADP
Pyruvate Carboxylase
HCO3 + ATP
pyruvate Gluconeogenesis
 glucose Gluconeogenesis
Pi
Glucose-6-phosphatase
H2O
glucose-6-phosphate
Phosphoglucose Isomerase
fructose-6-phosphate
Pi
Fructose-1,6-bisphosphatase
H2O
fructose-1,6-bisphosphate
Aldolase

glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
(continued)
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
ATP
Gluconeogenesis:
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
2. Gluconeogenesis expends how many ~P ?
3. A futile cycle of both pathways would waste6 how
many
~P per cycle ?4
 Phosphofructokinase
6 CH OPO 2 1CH2OH 6 CH OPO 2 1CH2OPO32
2 3 2 3
O ATP ADP O
5 H HO 2 5 H HO 2

H 4 3 OH H 4 3 OH
Pi H2O
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate
Fructose-1,6-biosphosphatase
To prevent the waste of a futile cycle, Glycolysis
& Gluconeogenesis are reciprocally regulated.
Local Control includes reciprocal allosteric
regulation by adenine nucleotides.
Phosphofructokinase (Glycolysis) is inhibited
by ATP and stimulated by AMP.
Fructose-1,6-bisphosphatase
(Gluconeogenesis) is inhibited by AMP.
The opposite effects of adenine nucleotides
on
Phosphofructokinase (Glycolysis)
Fructose-1,6-bisphosphatase (Gluconeogenesis)

insures that when cellular ATP is high (AMP would

then be low), glucose is not degraded to make ATP.
When ATP is high it is more useful to the cell to
store glucose as glycogen.
When ATP is low (AMP would then be high), the cell
does not expend energy in synthesizing glucose.
Global Control in liver cells includes reciprocal
effects of a cyclic AMP cascade, triggered by
the hormone glucagon when blood glucose is
low.
Phosphorylation of enzymes & regulatory
proteins in liver by Protein Kinase A (cAMP
Dependent Protein Kinase) results in
inhibition of glycolysis
stimulation of gluconeogenesis,

making glucose available for release to the

blood.
Enzymes relevant to these pathways that
are phosphorylated by Protein Kinase A
include:
Pyruvate Kinase, a glycolysis enzyme that is
inhibited when phosphorylated.
CREB (cAMP response element binding protein)
which activates, through other factors,
transcription of the gene for PEP
Carboxykinase, leading to increased
gluconeogenesis.
A bi-functional enzyme that makes and
degrades an allosteric regulator, fructose-2,6-
bisphosphate.
Reciprocal regulation by fructose-2,6-
bisphosphate:
Fructose-2,6-bisphosphate stimulates
Glycolysis.
Fructose-2,6-bisphosphate allosterically activates
the Glycolysis enzyme Phosphofructokinase.
Fructose-2,6-bisphosphate also activates
transcription of the gene for Glucokinase, the
liver variant of Hexokinase that phosphorylates
glucose to glucose-6-phosphate, the input to
Glycolysis.
Fructose-2,6-bisphosphate allosterically
inhibits the gluconeogenesis enzyme
Fructose-1,6-bisphosphatase.
 Recall that Phosphofructokinase, the rate-limiting
step of Glycolysis, is allosterically inhibited by ATP.
At high concentration, ATP binds at a low-affinity
regulatory site, promoting the tense conformation.

60

50 low [ATP]

40
PFK Activity

Sigmoidal
dependence of 30
reaction rate on high [ATP]
20
[fructose-6-
phosphate] is 10
observed at high
[ATP]. 0
0 0.5 1 1.5 2
[Fructose-6-phosphate] mM
 60

PFK activity in 50 low [ATP]

the presence of the
40
globally controlled

PFK Activity
allosteric regulator 30
fructose-2,6- high [ATP]
20
bisphosphate is
similar to that at low 10
ATP.
0
0 0.5 1 1.5 2
[Fructose-6-phosphate] mM

Fructose-2,6-bisphosphate promotes the relaxed

state, activating Phosphofructokinase even at high [ATP].
Thus activation by fructose-2,6-bisphosphate, whose
concentration fluctuates in response to external hormonal
signals, supersedes local control by [ATP].
The allosteric regulator fructose-2,6-bisphosphate is synthesized &
degraded by a bi-functional enzyme that includes 2 catalytic
domains:

Phosphofructokinase-2
(PFK2) domain catalyzes:
Fructose-6-phosphate + PFK2/FBPase2 homodimer PDB
2BIF
ATP fructose-2,6-
bisphosphate + ADP
PFK-2
Fructose-Biophosphatase- domain
2 (FBPase2) domain
catalyzes:
Fructose-2,6-
bisphosphate + H2O
fructose-6-phosphate + Pi
Bifunctional
FBPase-2
PFK2/FBPase2 assembles domain
into a homodimer. with bound
fructose-6-P
in active site
 PFK2/FBPase2 homodimer PDB
2BIF

PFK-2
domain

FBPase-2
domain
with bound
fructose-6-P
in active site
Adjacent to the PFK-2 domain in each copy of the liver
enzyme is a regulatory domain subject to
phosphorylation by cAMP-dependent Protein Kinase.
Which catalytic domains of the enzyme are active
depends on whether the regulatory domains are
phosphorylated.
 (active as Phosphofructokinase-2)
Enz-OH
ATP ADP

fructose-6-P fructose-2,6-bisP

Pi View an
Enz-O-PO32 animation.
(active as Fructose-Bisphosphatase-2)

cAMP-dependent phosphorylation of the bi-

functional enzyme activates FBPase2 and
inhibits PFK2.
[Fructose-2,6-bisphosphate] thus decreases in
liver cells in response to a cAMP signal cascade,
activated by glucagon when blood glucose is low.
 (active as Phosphofructokinase-2)
Downstream Enz-OH
effects of ATP ADP
the cAMP
cascade: fructose-6-P fructose-2,6-bisP

Pi
Enz-O-PO32
(active as Fructose-Bisphosphatase-2)

Glycolysis slows because fructose-2,6-bisphosphate

is not available to activate Phosphofructokinase.
Gluconeogenesis increases because of the
decreased concentration of fructose-2,6-bisphosphate,
which would otherwise inhibit the gluconeogenesis
enzyme Fructose-1,6-bisphosphatase.
 Glycogen Pyruvate

X Gluconeogenesis

Glucose-1-P Glucose-6-P Glucose + Pi

Glucose-6-Pase
X

Glycolysis
Pathway

Summary of effects of glucagon-cAMP cascade in liver:

Gluconeogenesis is stimulated.
Glycolysis is inhibited.
Glycogen breakdown is stimulated.
Glycogen synthesis is inhibited.
Free glucose is formed for release to the blood.
The Cori Cycle operates during exercise.
For a brief burst of ATP utilization, muscle
cells utilize ~P stored as phosphocreatine.
Once phosphocreatine is exhausted, ATP is
provided mainly by Glycolysis, with the input
coming from glycogen breakdown and from
glucose uptake from the blood.
(Aerobic fat metabolism, discussed elsewhere,
is more significant during a lengthy period of
exertion such as a marathon run.)
 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

Lactate produced from pyruvate passes via the blood to

the liver, where it may be converted to glucose.
The glucose may travel back to the muscle to fuel
Glycolysis.
 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

The Cori cycle costs 6 ~P in liver for every 2 ~P made

available in muscle. The net cost is 4 ~P.
Although costly in ~P bonds, the Cori Cycle allows the
organism to accommodate to large fluctuations in energy
needs of skeletal muscle between rest and exercise.
The equivalent of the Cori Cycle also
operates during cancer.
If blood vessel development does not keep
pace with growth of a solid tumor,
decreased O2 concentration within the
tumor leads to activation of signal processes
that result in a shift to anaerobic
metabolism.
 Liver Blood Cancer Cell

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

Energy dissipation by the Cori Cycle, which expends

6 ~P in liver for every 2 ~P produced via Glycolysis for
utilization within the tumor, is thought to contribute to
the weight loss that typically occurs in late-stage
cancer even when food intake remains normal.