GLUCONEOGENESIS

Introduction:

Gluconeogenesis converts other foodstuffs into glucose when it is not available at sufficient
levels in diet

GLUCOSE:

it is an indispensable metabolite
brain requires approx. 50% of its calories in form of glucose
RBCs solely exist on them
Its a precursor of other sugars needed in biosynthesis of nucleotides, glycoproteins, and
glycolipids
Is needed to replenish NADPH supplies reducing power for biosynthesis and detoxification
Thus, cannot leave blood glucose level up to vagaries of dietary supply

Overview of Gluconeogenesis:

*draw gluconeogenesis path here along with the notes written on diagram; p.102*
Both dietary and endogenous protein is the major substrate supply for this pathway
Protein first broken down into amino acids, which can then be converted to pyruvate
OR any of TCA cycle intermediates can serve as substrates for gluconeogenesis these
intermediated would be called glucogenic
Leucine, lysine, and aromatic amino acids are degraded to acetyl-CoA (can be converted to
ketone bodies) / Acetoacetate (is a ketone body) thus, these amino acids are called ketogenic
Was believed ketogenic amino acids cannot be converted to glucose in metabolism not true
ketone body acetone can be converted to pyruvate; but its contribution to gluconeogenesis is
likely minor
Gluconeogenesis proceeds ONLY in liver and the kidneys; liver synthesizes most of glucose as it
is 5x larger than both kidneys combined
along with glycogen degradation, gluconeogenesis ensures stable blood glucose levels b/w
meals
gluconeogenesis also enables us to maintain necessary glucose levels when on a diet thats rich
in protein but low in carbs

Reactions in gluconeogenesis:

most shared with glycolysis

final reaction in glycolysis (PEP to pyruvate which produces ATP) is irreversible due to
strongly exergonic nature of accompanying rearrangement of pyruvate from enol to keto form
In gluconeogenesis: takes 2 enzymatic steps to convert pyruvate back into PEP
o 1. Pyruvate carboxylase Carboxylates pyruvate to oxaloacetate
o 2. PEP carboxykinase converts oxaloacetate to PEP
 The pyruvate carboxylase reaction
o *draw rxn*
o Just like plants we are able to metabolically fix CO2 with this reaction
o The very same CO2 molecule gets released again in the next step
o Whole purpose of transient CO2 fixation is to enable this subsequent reaction shown in
carboxylation of pyruvate
The active site of E.coli biotin carboxylase
o In human metabolism: pyruvate carboxylase reaction occurs in two separate steps
carried out in two distinct active sires of a single enzyme molecule
o In E.coli: two activities found on separate enzyme molecules; first enzyme activity is
biotin carboxylase attaches CO2 to the coenzyme biotin
Reaction involves bicarbonate and ATP; terminal phosphate of ATP fits into
space b/w ADP, arginine 292, & bicarbonate
Roles of arginine 338 and glutamate 296 illustrated next
Activation of bicarbonate and carboxylation of biotin
o *draw rxn here*
o Glutamate 296 deprotonates bicarbonate, this in turn attacks the terminal phosphate
of ATP; this yields carboxyphosphate, which in turn deprotonates biotin
o Arginine 338 stabilizes anionic biotin intermediate (forms transiently at this stage);
biotin anion finally attacks carboxyphosphate, producing phosphate and carboxybiotin
o Note: carboxyphosphate (carbonic-phosphoric anhydride) occurs as an intermediate in
carbamoylphosphate Synthetase rxn which is first step in Urea cycle
The carboxylation of pyruvate
o * draw rxn here*
o Second active site (human) / second enzyme (e.coli) transfers carboxyl group from
biotin to pyruvate; the rxn begins with pyruvate adopting enoyl config.
Electrons of C=C double bond perform nucleophilic attack on carboxyl group
which produces biotin readily product is oxaloacetate
The PEP carboxykinase rxn
o *draw rxn here*
o In this rxn CO2 tht was just attached to substrate, leaves again; this gives rise to an
enolpyruvate anion intermediate which attacks and acquires the terminal phosphate of
GTP
o Product of this rxn is PEP
o All rxns b/w PEP and fructose-1,6-bisphosphate are reversible; thus skip ahead to latter
metabolite
Fructose-1,6-bisphosphate and glucose-6-phosphate
o *write out rxns here*
o These rxns revert substrate phosphorylations tht occur in first & 3rd step of glycolysis
(hexokinase and phosphofructokinase rxns)
o In gluconeogenesis phosphate groups are simply hydrolyzed off (not a difficult rxn)
Energy Balance of Gluconeogenesis

Gluconeo. Needs input of 6 equivalents of ATP/ GTP for each glucose molecule
o Expenditure of these 6 molecules makes it exergonic to convert pyruvate back to
glucose
Glycolysis net gain of only 2 ATP per glucose makes it exergonic to turn glucose into pyruvate

Interactions of Gluconeogenesis with other Pathways

Substrate carbon for gluconeo. Acrrues mostly from amino acid degradation at level of
pyruvate or TCA cycle intermediates
Pyruvate Carboxylase important in gluconeo. And replenishment of TCA cycle intermediates
which may become depleted through diversion to biosynthesis of amino acids or of heme
Therefore, this enzyme expressed ubiquitously

Mitochondrial substrate transport in gluconeogenesis
o *draw rxn path here*
o Pyruvate carboxylase resides inside the mitochondria
o PEP carboxykinase catalyzes next step in gluconeogenesis & occurs in cytosol; thus
oxaloacetate must be exported from mitochondria again
o Already know [oxaloacetate] is low in mitochondria that malate dehydrogenase
equilibrium favours malate
Thus substrate export occurs at level of malate which is exchanged for
phosphate by dicarboxylate carrier (a mitochondrial transport protein)
o Malate dehydrogenase rxn then reversed in cytosol; NADH can be used in reversal of
glyceraldehyde 3-dehydrogenase rxn (occurs later in gluconeo.)
o phosphate (entered mito. In exchange for malate) can be used by ATP synthase &
ATP be exchanged for cytosolic ADP this balances entire transport cycle and supplies
1 ATP to cytosol where it can for ex. Be used by phosphoglycerate kinase in gluconeo.
o Methylmalonate inhibits mito. Export of malate; thioester of methylmalonate occurs
in metabolic utilization of fatty acids with uneven numbers of carbon atoms
o This pathway requires vitamin B12 lack of vitamin will cause free methylmalonate to
accumulate which may inhibit gluconeo and thus account for clinical hypoglycemia tht
sometimes accompanies vitamin B12 deficiency
o
Ethanol degradation inhibits gluconeogenesis
o *draw path here*
o Ethanol degradation occurs in liver
o Utilization of 1 ethanol molecule by alcohol dehydrogenase & then aldehyde
dehydrogenase yields acetate tht is cconverted to acetyl-CoA by acetate thiokinase
o For each ethanol molecule degraded 2 equivalents of NAD+ reduced to NADH this
raises cytosolic [NADH]/[NAD+] ratio this in turn reduces both pyruvate and
oxaloacetate, thus depriving gluconeo of its substrates
o In alcoholic patients this problem often compounded by low intake of carbohydrates
Regulation of Gluconeogenesis

Simultaneous activity of glycolysis and gluconeogenesis creates futile cycles

o Draw the cycles
o each of these cycles combines 1 enzyme from glycolysis with one/two enzymes from
gluconeo
o net result of each cycle is the consumption of ATP/ GTP and the release of heat
o these cycles run in living cells possibly for sake of heat production such substrate
cycles also sharpen up regulatory responses
o their activity must be kept in check so excessive wastage of ATP can be avoided
Glucose phosphorylation cycling involves two separate compartments
o *draw cycle here*
o Hexokinase/ glucokinase operates in cytosol here
o Glucose-6-phosphatase located in ER
o Flow through cycle limited by capacity of glucose-6-phophate transport to ER
o Transporter and phosphatase are expressed in many tissues
o Not clear what function this might serve other than futile cycling
Allosteric regulation limits fructose-6-phosphate phosphorylation cycling
o Phosphofructokinase inhibited by ATP
o Depletion of ATP results in a buildup of ADP and AMP; thus also makes sense that ADP
and AMP stimulate phosphofructokinase (sxn 2.5)
o Fructose-1,6-bisphosphatase stimulated by ATP and inhibited by AMP; this behaviour
opposite to phosphofructokinase & it ensures that only one of 2 enzymes will be fully
active at any given time this helps limit rate of futile cycling
Hormonal control of phosphofructokinase and fructose-1,6-bisphosphatase
o *draw cycle*
o Phosphofructokinase & fructose-1,6-bisphosphatase also show opposite responses to a
third allosteric effector fructose-2,6-bisphosphate synthesized solely for sake of its
regulation & it occurs at much lower levels than fructose-1,6-bisphosphate
o [fructose-2,6-bisphosphate] under control of hormones via secondary messenger 3, 5-
cyclo-AMP (cAMP)
1. Hormones bind to their receptors on cell surface this promotes formation
of cAMP by adenylate cyclase (glucagon and epinephrine) or its degradation by
phosphodiesterase (insulin)
2. cAMP binds to & activates protein kinase A this phosphorylates enzyme
phosphofructokinase 2 (PFK 2); a protein phosphatase can reverse this
phosphorylation
3. PFK 2 has 2 opposite activities;
1) in dephosphorylated state it acts as kinase and thus raises level of
fructose-2,6-bisphosphate; in phosphorylated state it acts as
corresponding phosphatase and thus decreases level of fructose-2,6-
bisphosphate
 o fructose-2,6-bisphosphate activates phosphofructokinase & inhibits fructose-1,6-
bisphosphate:
the upshot of glucagon and epinephrine action is to promote gluconeo & inhibit
glycolysis
insulin has opposite effect
The secondary messengers cAMP and fructose-2,6-bisphosphate
o Among the 3 allosteric effectors of phosphofructokinase and fructose-1,6-
bisphosphatase ATP and AMP reflect energetic situation of cell itself whereas level of
fructose-2,6-bisphophate is regulated by hormonal stimulation and thus controls same
enzymes on behalf of needs of body as a whole
Regulation of pyruvate kinase
o Allosteric inhibition by ATP
o Allosteric activation by alanine and fructose-1,6-bisphosphate
o Inhibition by PKA mediated phosphorylation
o In third cycle shown under Simultaneous activity of glycolysis. throughput limited
under PEP and pyruvate
o Liver form of pyruvate kinase allosterically activated by fructose-1,6-bisphosphate;
inhibited by alanine, ATP, and through phosphorylation downstream of cAMP
o Alanine key substrate for hepatic gluconeo, since it carries substrate carbon and
surplus nitrogen from muscle to liver in catabolic conditions; thus makes sense that
alanine should inhibit glycolysis