GLYCOLYSIS, GLUCONEOGENESIS, PENTOSE PHOSPHATE PATHWAY

Fates of Glucose:
1) Stored as glycogen, starch, and sucrose
3) Ribose 5-phosphate (oxidation via pentose phosphate pathway)

2) Pyruvate (oxidation via glycolysis)

4) Extracellular matrix and cell wall polysaccharides

GLYCOLYSIS: Anaerobic process (fermentation), converts 1 molecule of glucose to 2 molecules of pyruvate.

2 Glucose + 2NAD+ + 2ADP + 2Pi 2 Pyruvate + 2NADH + 2 ATP + 2H2O + 2H+
1. Glucose Glucose 6-phosphate
Enzyme: Hexokinase [Class: Transferase]
-First priming action
-Hexokinase requires Mg2+
-Hexokinase undergoes induced fit
-ATP is required (ATPADP)
-Irreversible (Large, negative G)
2. Glucose 6-phosphate Fructose 6-phosphate
Enzyme: Phosphohexose isomerase [Class: Isomerase]
-Rearrangement of the carbonyl and hydroxyl groups occur at C1 & C2
-Reversible, small change in free energy
3. Fructose 6-phosphate Fructose 1,6-bisphophaste
Enzyme: Phosphofructokinase-1 (PFK-1) [Class: Transferase]
-First committed step
-ATP is required (ATP ADP)
-PFK-1 activity increased when ATP supply is depleted
-PFK-1 activity is also increased when ADP/AMP accumulate
-Fructose 2,6-bisphosphate is a potent allosteric activator of PFK-1
4. Fructose 1,6-bisphosphate Glyceraldehyde 3-phosphate + Dihydroxyacetone phosphate
Enzyme: Aldolase [Class: Lyase]
-Cleaves to Ketose (Dihydroxyacetone) & Aldose (Glyceraldehyde 3-phosphate)
-Lysine (+ Charge) attacks substrate carbonyl (First step)
5. Glyceraldehyde 3-phosphate + Dihydroxyacetone phosphate Glyceraldehyde 3-phosphate
Enzyme: Triose phosphate isomerase [Class: Isomerase]
-Ketose converted to aldose rapidly
-Only Glyceraldehyde 3-phosphate can go on
6. Glyceraldehyde 3-phosphate (2) 1,3-Bisphosphoglycerate (2)
Enzyme: Glyceraldehyde 3-phosphate dehydrogenase [Class: Oxidoreductase]
-Oxidation & phosphorylation
-2NADH produced + H+
-2Pi and 2NAD+ required
-Cystine residue important
7. 1,3-Bisphosphoglycerate (2) 3-Phosphoglycerate (2)
Enzyme: Phosphoglycerate kinase [Class: Transferase]
-2ATP produced (Substrate-level phosphorylation)
8. 3-Phosphoglycerate (2) 2-Phosphoglycerate (2)
Enzyme: Phosphoglycerate mutase [Class: Isomerase]
-Reversible shift of the phosphoryl group between C-2 and C-3
-Mg2+ essential
9. 2-Phosphoglycerate (2) Phosphoenolpyruvate (2)
Enzyme: Enolase [Class: Lyase]
-Removes H2O
10. Phosphoenolpyruvate (2) Pyruvate (2)
Enzyme: Pyruvate kinase [Class: Transferase]
-2ATP produced

Fates of Pyruvate:
1) Lactate

2) Acetyl-CoA Citric Acid Cycle

1) Pyruvate Lactate
-NAD+ produced is cycled back for use in glycolosis

3) Ethanol + CO2
2) Pyruvate Acetyl-CoA

3) Pyruvate Ethanol
-TPP is important coenzyme, important in citric acid cycle
-TPP cleaves bonds adjacent to a carbonyl group
-Only occurs in yeast and other microorganisms

GLYCOLYSIS SUMMARY
-Glycolysis is a near-universal pathway by which a glucose molecule is oxidized to two molecules of pyruvate, with energy conserved as ATP
and NADH
-All 10 glycolytic enzymes are in the cytosol, and all 10 intermediates are phosphorylated compounds of three of six carbons
-Glycolysis is tightly regulated in coordination with other energy-yielding pathways to assure a steady supply of ATP
Feeder Pathways for Glycolysis:

GLYCOGEN BREAKDOWN
-Glycogen and Starch are degraded by
phosphorolysis
-Glycogen phosphorylase attacks the
non-reducing end of glycogen,
breaking the -1-4 glycosidic bond
using inorganic phosphate to generate
glucose 1-phosphate
-Glucose 1-phosphate is converted to
Glucose 6-phosphate via
phosphoglucomutase
GLYCOGEN SYNTHESIS:
-Glucose 6-phosphate is isomerized to
glucose 1-phosphate by
phosphoglucomutase

-Glycogen synthase is inactivated by phosphorylation, catalyzed by cAMP dependent protein

kinase; it is activated by dephosphorylation, catalyzed by phosphoprotein phosphatase.
-Glycogen phosphorylase is activated by phosphorylation catalyzed by phosphorylase kinase,
and it is inactivated by dephosphorylation, catalyzed by phosphorylase a phosphatase.

-UDP-glucose pyrophosphorylase
converts glucose 1-phosphate to UDP
glucose using UTP and producing
pyrophosphate
-Glycogen synthase attaches the UDPglucose to the nonreducing end of a
branched glycogen molecule

GLUCONEOGENESIS: Converts pyruvate and related three- and four-carbon compounds to glucose, takes place mainly in the liver
-Cori Cycle: Lactate produced by anaerobic glycolysis in skeletal muscle returns to the liver and is converted to glucose, which moves back to
muscle and is converted to glycogen.
-7/10 enzymatic reactions are the reverse of glycolytic reactions
-Hexokinase, PFK-1, and pyruvate kinase are irreversible, therefore bypassed
Pyruvate Phosphoenolpyruvate
1. Pyruvate is first transported from the cytosol into mitochondria or is generated from alanine within mitochondria
2. Pyruvate carboxylase converts pyruvate to oxaloacetate (requires coenzyme biotin) [Biotin acts as carrier of activated bicarbonate]
*Pyruvate + Bicarbonate (HCO3-) + ATP Oxaloacetate + ADP + Pi
3. Pyruvate carboxylase is the first regulatory enzyme in the gluconeogenic pathway requiring acetyl-CoA as a positive effector
*Pyruvate carboxylase reaction can replenish intermediates in the citric acid cycle
4. Mitochondrial membrane has no transporter for oxaloacetate; the oxaloacetate must be reduced to malate via malate dehydrogenase
*Oxaloacetate + NADH + H+ Malate + NAD+
5. Malate leaves mitochondrion thru transporter, and in the cytosol is reoxidized to oxaloacetate, producing NADH
*Malate + NAD+ Oxaloacetate + NADH + H+
6. Oxaloacetate is converted to Phosphoenolpyruvate by phosphoenolpyruvate carboxykinase, requires GTP
*Oxaloacetate + GTP Phosphoenolpyruvate + CO2 + GDP
OR if lactate is the glucogenic precursor:
Lactate is converted to Pyruvate by lactate dehydrogenase This creates NADH in the cytosol and the export of reducing equivalents (as
malate) is no longer necessary.
Fructose 1,6-bisphosphate Fructose 6-phosphate
-Catalyzed by fructose 1,6-bisphosphatase (FBPase-1)
-Irreversible hydrolysis:
Fructose 1,6-bisphosphate + H2O Fructose 6-phosphate + Pi
Glucose 6-phosphate Glucose
-Catalyzed by glucose 6-phosphatase
-Irreversible hydrolysis:
Glucose 6-phosphate + H2O Glucose + Pi
*Most amino acids derived from proteins are ultimately catabolized to pyruvate or to intermediates of the citric acid cycle. Such amino acids can
therefore undergo net conversion to glucose and said to be glucogenic. Alanine and Glutamine are important glucogenic amino acids in
mammals. After removal of their amino groups in liver mitochondria, the carbon skeletons remaining (pyruvate and -ketoglutarate) are readily
funneled into gluconeogenesis.

PENTOSE PHOSPHATE PATHWAY

Used in rapidly dividing cells, such as those of bone marrow, skin, and intestinal mucosa, and those of tumors to make RNA, DNA, and such
coenzymes as ATP, NADH, FADH2, and CoA. Some tissues need NADPH for biosynthesis (Reducing power).

In tissues that require primarily NADPH, the pentose phosphates produced in the oxidative phase of the pathway are recycled into glucose 6phosphate.
Nonoxidative phase:
1. Ribulose 5-phosphate is converted to xylulose 5-phosphate (catalyzed by ribosome 5-phosphate epimerase)
2. Transketolase is a TPP dependent enzyme; catalyzes the transfer of a two carbon fragment from xylulose 5-phosphate to ribose 5phosphate forming a seven carbon product and glyceraldehyde 3-phosphate

3. Transaldolase then converts fructose 6-phosphate

4. Fructose 6-phosphate can be isomerized to glucose 6-phosphate and re-initiate PPP

5. Meanwhile, transketolase uses erythrose 4-phosphate and xylulose 5-phosphate to produce fructose 6-phosphate (for isomerization)
6. Transketolase also converts glyceraldehyde 3-phosphate to fructose 6-phosphate

PRINCIPLES OF METABOLIC REGULATION

Coordinated Regulation of Glycolysis & Gluconeogenesis
Simultaneous operation of both glycolysis and gluconeogenesis would consume ATP without accomplishing any chemical or biological work.
For example, PFK-1 and FBPase-1 catalyze opposite reactions:
ATP + fructose 6-phosphate ADP + fructose 1,6-bisphosphate (PFK-1)
Fructose 1,6-bisphosphate + H2O fructose 6-phosphate + Pi (FBPase-1)
TOGETHER: ATP + H2O ADP + Pi + heat
= hydrolysis of ATP with no useful work being done
FUTILE CYCLE!
Hexokinase Regulation
-Hexokinase catalyzes the entry of free glucose into glycolysis
-When PFK-1 is inhibited both fructose 6-phosphate & glucose 6-phosphate build up
-Glucose 6-phosphate inhibits hexokinase
-Many distinct forms of hexokinase exist, which all convert glucose to glucose 6-phosphate (ISOZYMES)
-Isozymes resulting from gene duplication events allow evolution to tune the metabolic potential of cells (Different metabolic pattern in different
tissues, different locations and metabolic roles for isozymes in the same cell, different stages of development, different responses of isozymes
to allosteric modulators)
Example: Hexokinase expressed in the liver has distinct properties from the enzyme expressed in muscle.
PFK-1 Regulation
-The rate of fructose 6-phosphate to fructose 1,6-bisphosphate is limited by PFK-1 activity
-You can produce as much fructose 6-phosphate you want, but it wont automatically push glycolysis
-PFK-1 = valve & rate-limiting step
-PFK-1 is under complex metabolic regulation
-Fructose 2,6-bisphosphate activates the enzyme
-Fructose 2,6-bisphosphate is synthesized from fructose 6-phosphate by PFK-2
-PFK-2 is a unique enzyme because this polypeptide also acts as FBPase-2

Pyruvate Kinase Regulation

-Multiple isoforms or isozymes, which respond to distinct metabolic cues
-Pyruvate kinase found in muscle is activated by fructose 1,6-bisphosphate
-Inhibited by ATP and alanine (alanine serves as a monitor for biosynthetic precursors)
-Also under hormonal control (glucagon)

CITRIC ACID CYCLE

-Carbon skeletons of sugars (and fatty acids) are degraded to the acetyl group of acetyl-CoA to enter the citric acid cycle
-Pyruvate is converted to Acetyl-CoA via pyruvate dehydrogenase complex
Pyruvate Dehydrogenase Complex:
-Enzyme is subject to covalent modification and allosteric regulation
-Five cofactors participate in the reaction mechanism: CoA (reactive thiol), TPP, FAD, NAD, and Lipoate (electron carrier & acyl carrier)
-Complex is comprised of THREE enzymes:
1) Pyruvate dehydrogenase (E1): 24 copies attached to E2 core, each contains bound TPP
2) Dihydrolipoyl transacetylase (E2): Forms the core of the complex, 24 polypeptides, each containing 3 covalently bound lipoate
molecules. Lipoate is attached to the end of lysine chains providing long flexible arms of acyl group transfer
3) Dihydrolipoyl dehydrogenase (E3): 12 copies attached to E2 core, contains bound FAD
-2 Regulatory proteins:
1) Specific potein kinase phosphorylates a serine residue on one of the two subunits of E1
2) Phosphatase removes this phosphate to active the enzyme
*Allosteric Regulation.
-The enzyme complex is regulated by ATP, acetyl-CoA levels, NADH, and fatty acids
-Five steps in the decarboxylation and dehydrogenation of pyruvate
1) Pyruvate is decarboxylated; the C1 of pyruvate is released as CO2, while C2 and C3 remain fixed to TPP of E1
2) Group attached to TPP is oxidized to a carboxylic acid (acetate); the removed electrons reduce the disulfide bond of a lipoyl
group on E2; the acetate is transferred to one of the resulting sulfhydrl groups on the lipoyl molecule.
3) The acetate is then transferred to CoA to form Acetyl-CoA; subsequent reactions in this cycle regenerate the oxidized lipoyl group
on E2
4) Dihydrolipoyl dehydrogenase (E3) promotes the transfer of two H atoms from the reduced lipoyl groups on E2 to the FAD of E3
5) The reduced FADH2 of E3 transfers a hydride ion to NAD+ forming NADH
**The long lipollysl arms of E2 are central to the catalytic mechanism of this complex. They accept two electrons and the acetyl group from
pyruvate and pass them to E3.
**Also noteIntermediates along this pathway are never release: Channeling!
Citric Acid Cycle
-8 step cycle, 4 oxidation reactions (forming NADH, FADH2)
1) Citrate formation: Acetyl-CoA binds with Oxaloacetate forming Citrate
Enzyme: Citrate Synthase
2) Citrate Isocitrate
Enzyme: Aconitase
-Aconitase is a moonlighting protein
-Also acts as a mRNA regulatory factor
-Iron-Sulfur containing protein
3) Isocitrate -Ketoglutarate
Enzyme: Isocitrate dehydrogenase
-NAD(P)H produced, CO2 produced
-NAD serves citric acid cycle
-NADP form generates reducing power for anabolic reactions
3) -Ketoglutarate Succinyl-CoA
Enzyme: -Ketoglutarate dehydrogenase complex
-Enzyme comples similar to pyruvate dehydrogenase complex
-Requires TPP, bound lipoate, FAD, NAD and CoA.
4) Succinyl-CoA Succinate
Enzyme: Succinyl-CoA synthetase
-Reaction involves phosphorylated intermediate
-Two isozymes of succinyl-CoA synthetase:
-One generates ATP (plants) the other GTP (animals)

5) Succinate Fumarate
Enzyme: Succinate dehydrogenase
-Oxidation; Produces FADH2
-Succinate dehydrogenase is a potential branch point in oxidative phosphorylation (but get less ATP)
6) Fumarate Malate
Enzyme: Fumerase
7) Malate Oxaloacetate
Enzyme: Malate dehydrogenase
-NADH produced
CITRIC ACID CYCLE INTERMEDIATES:
-Used to synthesize other biomolecules
--Ketoglutarate and oxaloacetate serve
as precursors for aspartate and glutamate,
which can be subsequently be used for other
molecules.
-Oxaloacetate is converted to glucose via
gluconeogenesis
-Succinyl CoA Porphryin Rings
Removed intermediates are replenished via anaplerotic reactions:
Among other reactions, oxaloacetate can be generated from CO2 and pyruvate catalyzed by pyruvate carboxylase

Regulation of Citric Acid Cycle:

-Pyruvate dehydrogenase complex is tightly regulated
-High levels of acetyl CoA or high ratios of ATP/ADP and NADH/NAD inhibit
-Three factors govern flux thru TCA:
1. Substrate availability
2. Product inhibition
3. Allosteric feedback inhibition
-Regulated at its three exergonic steps:
1. Citrate Synthase
2. Isocitrate dehydrogenase
3. -Ketoglutarate dehydrogenase

Urea Cycle
1. Carbamoyl phosphate synthetase I (CPS-I)

(X) Congenital hyperammonemia, Type I

Bicarb + 2ATP +NH3 (1st N destined for urea) carbamoyl phosphate

Low pH inhibits, CO2- acid, mitochondria, rate

limiting step

+ N-acetyl glutamate
2. ornithine transcarbamoylase (OTC)

(X) Congential hyperammonemia, type II

Ornithine + Carbamoyl phosphate citrulline

(Transported into cytosol)

mitochondria
3. argininosuccinate synthetase (ASS)

(X) citrullinemia

Citrulline + Aspartate (2nd N destined for urea)

argininosuccinate

1 ATP
4. argininosuccinate lyase (ASL)

(X) Argininosuccinate aciduria

Argininosuccinate Arginine + Fumarate (enters

TCA)
5. arginase

(X) Arginemia

Arginine Ornithine + Urea

Fates of Urea
1. liver blood kidneys urine
2. liver blood intestine urease in bacteria, NH3 + CO2
3. feces or reabsorbed into blood
Regulation of Urea Cycle
LOW pH

Inhibits urea cycle in liver and shunts ammonia to hepatic circulation to become Gln that enters the kidney and increases the
pH of the urine.
N-acetylglutamate

+ activator of carbamoyl phosphate synthase I

Arginine

+ activator of NAGS (N-acetyl glutamate synthase)

Amino Acid Metabolism

Ketogenic vs. Glucogenic

Ketones in urine: acetyl-CoA and acetoacetyl-CoA derivatives

Which AA are solely ketogenic?

Lysine and leucine

Which AA are ketogenic and glucogenic?

Isoleucine, tryptophan, tyrosine

Methionine and Cysteine
What is the use of SAM in the degradation reaction?

Methyl donor
At the level of L-Homocysteine what are the two pathways it can take?

Methionine and Cysteine

Which one can lead to a folate trap?

Methionine because it uses b12 as a cofactor

What is the cofactor for cysteine formation?

B6
Tryptophan
Glucogenic Alanine pyruvate

Ketogenic acetoacetyl-coA

Vitaminogenic niacin
Precursor to serotonin
Glutamate
Gamma- aminobutyric acid (GABA)
o Neurotransmitter
o Cofactors: PLP
o Enzyme: glutamate decarboxylase
Glutamine
o Glutamine synthase
Alpha-ketoglutarate
o Glutamate dehydrogenase
Arginine
Citrulline and nitric oxide (vasodilator)
Creatine phosphate
o Needs to form w/ glycine
o Uses SAM
o Energy reserve
Histamine
Histidine histamine

Uses decarboxylase & PLP

Biosynthesis of AA
Transamination of metabolic intermediates:

Pyruvate Alanine

Oxaloacetate Aspartate

Alpha keto glutarate Glutamate

Asparagine

Asp Asn via asparagine synthetase with NH3 and ATP

Asp Asn via asparagine synthetase with glutamine and ATP

Glutamine

Glu Gln via glutamine synthetase

Serine

Glycine Serine via serine hydroxymethyltransferase and THF