Chem*3560 Lecture 15: Gluconeogenesis

Gluconeogenesis is the synthesis of "new" glucose in

the cytoplasm, and is undertaken when there is little
demand for energy, and unused substrate is available.
The liver is the major organ responsible responsible
for gluconeogenesis in animals, and may take up
lactate or amino acids from blood as a source of
substrate (Lehninger p.723-729).

The pathway may start in the cytoplasm from lactate,

or in the mitochondrion use amino acids by removing
the amino group.

The gluconeogenesis pathway broadly follows

glycolysis sequence in reverse. Eight reactions out of
eleven are close to equilibrium (∆∆ G close to zero),
and are therefore easily made to run in the opposite
direction (‡ symbols in the diagram).

Three reactions, hexokinase, phosphofructokinase,

and pyruvate kinase have large negative ∆ G at
cellular concentrations, and would be difficult or
impossible to reverse. A different reaction or
sequence is needed for each of these steps to
proceed in the direction needed for gluconeogenesis.

Conversion of pyruvate to phosphoenolpyruvate

In animals, this is a multistep sequence. It is too difficult to add phosphate to pyruvate in a single
reaction, because phosphoenolpyruvate (PEP) has an exceptionally high ∆ Go ' of hydrolysis (–61.9
kJ/mol). The strategy used is to add a carboxylate group to pyruvate first, which yields oxaloacetate.
Since decarboxylation always releases considerable energy, an ATP must be used as an energy
source when carboxylate is added. Then in a second reaction, the added carboxylate is lost again; with
the help of GTP as a phosphate donor, the energy made available by this decarboxylation is used to
drive the reaction in the direction of PEP formation.
pyruvate carboxylase
pyruvate + CO2 + ATP → oxaloacetate + ADP + Pi
PEP carboxykinase
oxaloacetate + GTP → PEP + CO2 + GDP
Overall cost is 2 ATP equivalents to make PEP from pyruvate (Lehninger p. 726-727).
Pyruvate carboxylase

The enzyme, pyruvate carboxylase uses

the coenzyme biotin to activate and localize
the -CO2 – group. The enzyme has four
identical subunits, with each polypeptide
made up of three domains:

Biotin carrier domain contains biotin

covalently bonded to a lysine side chain.
This forms a long "arm", and gives the biotin
freedom of movement between the two
catalytic sites.

The biotin carboxylase domain uses ATP

hydrolysis to drive addition of a bicarbonate
ion HCO3 – to biotin (step 1), making
carboxybiotin.

The carboxyltransferase domain transfers

the carboxylate group to the ultimate
substrate, pyruvate, to yield oxaloacetate
(step 2).

This pattern of three components arranged

around a bound biotin coenzyme is used by
several other carboxylases, including acetyl
CoA carboxylase (Lehninger p. 585-6).

PEP carboxykinase

PEP carboxykinase uses the decarboxylation of oxaloacetate to create the unstable enolpyruvate isomer
of pyruvate, which can then accept phosphate from GTP.
Location of pyruvate carboxylase and PEP carboxykinase

Pyruvate carboxylase is located in mitochondria,

whereas PEP carboxykinase is located in both
cytoplasm and in mitochondria. Which is used
depends on the starting substrate used.

When lactate is the starting substrate, lactate

dehydrogenase produces pyruvate and NADH in the
cytoplasm. This cytoplasmic NADH is needed later
to reduce 1,3-bisphosphophoglycerate. The
cytoplasmic pyruvate must be imported through the
mitochondrial membrane via a specific transporter,
and is then converted to PEP in the mitochondria.
PEP is exported by another transporter (Lehninger p.
728).

When amino acids are used as starting

substrate, the amino group is first removed and
the carbon structures are converted to pyruvate,
oxaloacetate or malate in mitochondria. Of
these, only malate has an export
transporter. The malate is then oxidized in the
cytoplasm to provide the NADH that will be
needed to reduce 1,3-bisphosphoglycerate.
There is no transporter for NADH, so it
must be generated in the cytoplasm when
needed for gluconeogenesis.

Once PEP is made and conditions favour

gluconeogenesis, the enzymes of glycolysis can
run in the opposite direction as far as
fructose-1,6-bisphosphate.

Fructose-1,6-bisphosphatase bypasses the phosphofructokinase step

fructose-1,6-bisphosphatase
fructose-1,6-bisphosphate + H2 O → fructose-6-phosphate + Pi ∆ Go ' = –15.9 kJ/mol

This reaction is not the reverse of phosphofructokinase, because no ATP is produced. Both
phosphofructokinase and fructose-bisphosphatase can have –ve ∆Go ', because they are different
reactions (Lehninger p. 728).
Glucose-6-phosphatase bypasses the hexokinase step

Glucose-6-phosphate isomerase is freely reversible, but hexokinase represents the one-way reaction,
which is bypassed by glucose-6-phosphatase

glucose-6-phosphatase
glucose-6-phosphate + H2 O → glucose + Pi ∆ Go ' = –13.8 kJ/mol

Again, this is not the reverse of hexokinase because no ATP is produced (Lehninger p.729).

Hydrolysis of Glucose-6-phosphate is coupled to export of glucose

Most other enzymes of glycolysis and gluconeogenesis are located in the cytoplasm.
Glucose-6-phosphatase is located in the cell membrane, and substrate is bound on the cytoplasmic
side, but glucose product is released on the outside of the cell. This means that glucose is exported
from the cell that makes it.

Glucose-6-phosphatase is primarily an enzyme of the liver and kidneys, which routinely export
glucose to maintain the blood glucose level. Muscles lack glucose-6-phosphatase, and direct
glucose-6-phosphate to glycogen synthesis, keeping the glucose as a reserve within the cell than made
it.

Overall energy cost of gluconeogenesis is 4-6 ATP per glucose made

Taking lactate as starting point, 2 lactates are needed per glucose

2 NADH made by lactate dehydrogenase
2 ATP needed by pyruvate carboxylase
2 GTP needed by PEP carboxykinase
2 ATP needed by phosphoglycerate kinase
2 NADH used glyceraldehyde-3-phosphate dehydrogenase
No ATP made by fructose-1,6-bisphosphatase or glucose-6-phospahtase

Net 6 ATP equivalents are needed to make one glucose (Lehninger p. 729).

If the starting point is malate derived from amino acids, the pyruvate carboxylase step can be skipped,
so that only 4 ATP are consumed.