CHEM 153A-XL
Summer 2022
MODULE 8 08/22/2023
DR. KAREN LOHNES
Metabolism
Involves the coupling of the exergonic reactions of nutrient
oxidation to the endergonic processes required to maintain
the living state.
1. Performance of mechanical work
2. Active transport of molecules against concentration
gradients.
3. Biosynthesis of complex molecules.
Metabolism
The energy required to drive unfavorable reactions is in two
main forms;
Reducing Power (NADH, NADPH and FADH2)
Non-Reducing Chemical Energy (phosphoryl group transfer potential)
(ATP)
Reduction of the nicotinamide ring
In the cell, the redox potential of these redox pairs:
[NAD+]/[NADH] = 1000
In contrast; [NADP+]/[NADPH] ≈ 0.01
Thus you can see NAD+ is a better oxidizing agent because the
concentration is high while NADPH is a better reducing agent.
NADH + ATP à NADPH + ADP
Can utilize the energy of ATP to shift the relative concentration of
nucleotides
The Big Picture of Metabolism
Anabolic Pathways;
- Biomolecular synthesis
converts small molecules
into large molecules
- These are energy requiring
processes. Require NADPH
as reducing power and ATP
as non-redox chemical
energy
Catabolic Pathways;
- Degrades large
molecules into
small ones
- Recover reducing power
and ATP
Overview of Catabolism; Activated Carriers
ATP
Phosphoryl Group Transfer Potentials
ATP can transfer its phosphoryl
Group more efficiently than other
Molecules such as Glycerol-3-Phosphate because of 3 factors;
1) Less resonance stabilization in
ATP than ADP and Pi
2) Negative charge on ATP
causes electrostatic repulsion
3) Water binds more effectively
to ADP and Pi than to
phosphoanhydride part of ATP
Acetyl-Coenzyme A
Activated Carriers
• All of these activated carriers are stable without the presence of a
catalyst; essential to their function since need control over these
with enzymes
• The fact that these activated groups are exchanged through a small
set of carriers also allows a small set of molecules to carry out a
wide range of tasks
As we discussed with enzymatic catalysis, there are 6 types of
reactions that are seen in metabolism based on the 6 classes of
enzyme;
1) Oxidation-Reduction
4) Group transfer
2) Ligation
5) Hydrolytic
3) Isomerization
6) Addition of functional groups (lyases)
Metabolic Regulation
Metabolic processes are regulated in 3 principal ways;
1) Amount of enzymes
2) Catalytic activities
3) Accessibility of substrates/energy
(1) Adjusted primarily by adjusting/changing the rate of transcription for the
genes encoding them
(2) Controlled in ways we’ve discussed previously;
- Allosteric control
- Covalent modification;
- Reversible
- Irreversible
- Compartmentalization (we haven’t discussed this yet);
- keeps biosynthetic and degradative pathways separate to
prevent a “futile cycle”
Energy status of the cell also plays a role;
Phosphorylation potential = [ATP]/([ADP][Pi])
Glycolysis; overview
Glucose; fuels the brain and only
Fuel that RBC’s use at all
Why glucose?
-Glucose is the one monosaccharide
that could have been formed primordially
-Has a low tendency (relative to other
monosaccharides) to nonenzymatically glycosylate proteins
-Has a strong tendency to exist in the
ring formation and thus little tendency
to modify proteins (b/c the ring
conformation of beta-glucose has
high relative stability)
Glycolysis; overview
The end product of glycolysis, pyruvate, is toxic and needs to be converted to another
molecule type.
Metabolic Functions of Eukaryotic Organelles
Glycolysis; overview
Strategy of glycolysis;
1) Oxidize carbon atoms to extract energy
2) Move phosphate groups to positions that have enough ΔG°’Hydrolysis to drive the
formation of ATP
Glycolysis; Overview
There are two “phases”
Of glycolysis;
1) Preparatory Phase/
Energy Investment
Phase;
during this phase,
phosphate groups are
placed at strategic
locations for the
second phase
2) Payoff Phase/
Energy Recovery Phase;
during this phase, you
implement energy
extraction strategies
and recover ATP and
reducing power
Second Phase
First Phase
Glycolysis; Phase I
Please refer to the following animated figure for an overview of glycolysis;
https://higheredbcs.wiley.com/legacy/college/voet/0470570954/animated_figs/4e_ch17/17-3_4e.html
Glycolysis; Hexokinase
Stage I:
Step 1
Glycolysis; Hexokinase
Stage I:
Step 1
Glycolysis; Phosphoglucose Isomerase
Fructose:
Stage I:
Step 2
C1-OH is a better nucleophile
Cleaved into two 3-Carbon Molecules
Glycolysis; Phosphoglucose Isomerase
Stage I:
Step 2
Glycolysis; Phosphofructokinase
(Second Use of ATP)
Stage I:
Step 3
NOTE: bisphosphate
versus diphosphate
Please refer to the following for comparison of PFK-1 activity vs F6P concentration;
https://higheredbcs.wiley.com/legacy/college/voet/0470570954/animated_figs/4e_ch17/17-33_4e.html
Glycolysis; Phosphofructokinase
Stage I:
Step 3
Glycogen
Glucose-6-P
Fructose-6-P
Glycolysis
Pentose-P Pathway (NADPH)
• Phosphofructokinase (PFK) is the first committed step which sets the pace of glycolysis
• It’s allosterically regulated; dependent of availability of glucose, need for glycolysis, etc
• Rate determining step
Glycolysis; Aldolase
Stage I:
Step 4
Please refer to the following for information on the mechanism used by aldolase;
https://higheredbcs.wiley.com/legacy/college/voet/0470570954/animated_figs/4e_ch17/17-9_4e.html
Glycolysis; Triose Phosphate Isomerase (TIM or TPI)
Stage I:
Step 5
Glycolysis; Triose Phosphate Isomerase (TIM or TPI)
Glycolysis; Triose Phosphate Isomerase (TIM or TPI)
Flexible loop closes over TIM active site
Loop prevents methylglyoxal formation
Glycolysis; Phase II
Stage I: Glucose + 2 ATP à 2 GAP + 2 ADP
Stage II: 2 GAP + 2 NAD+ + 4 ADP + 2 Pi à
2 Pyruvate + 2 NADH + 2H+ + 4 ATP + 2 H2O
Net:
Glucose + 2 NAD+ + 2 ADP + 2 Pi à
2 Pyruvate + 2 NADH + 2H+ + 2 ATP + 2 H2O
*NAD+ must be regenerated for additional passes of glycolysis
Glycolysis; Glyceraldehyde 3 phosphate
dehydrogenase (GAPDH)
Stage II:
Step 6
Please refer to the following for an animated figure of the reaction mechanism of this enzyme;
https://higheredbcs.wiley.com/legacy/college/voet/0470570954/animated_figs/4e_ch17/17-14_4e.html
Glycolysis; Glyceraldehyde 3 phosphate
dehydrogenase (GAPDH)
Stage II:
Step 6
Glycolysis; Glyceraldehyde 3 phosphate
dehydrogenase (GAPDH)
Glycolysis; Phosphoglycerate kinase
Stage II:
Step 7
Glycolysis; Phosphoglycerate mutase
Stage II:
Step 8
Mechanism of Phosphoglycerate Mutase:
Base Catalyzed Phosphoryl Transfer
Mechanism of Phosphoglycerate Mutase:
Acid Catalyzed Phosphoryl Transfer
2,3-BPG Affects Oxygen Carrying Ability
2,3-BPG Affects Oxygen Carrying Ability
Glycolysis; Enolase
Stage II:
Step 9
Glycolysis; Pyruvate Kinase
Stage II:
Step 10
Pyruvate Tautomerization
Drives ATP Production
Glucose + 2 NAD+ + 2 ADP + 2 Pi à 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP
§Used:
§ 1 glucose; 2 ATP; 2 NAD+
§Made:
§ 2 pyruvate
§ various different fates
§ 4 ATP
§ used for energy-requiring processes within the cell
§ 2 NADH
§ must be reoxidized to NAD+ in order for glycolysis to continue
§Glycolysis is heavily regulated.
§ ensure proper use of nutrients
§ ensure production of ATP only when needed
Feeder Pathways for Glycolysis
§Glucose molecules are cleaved from glycogen
and starch by glycogen phosphorylase.
§yielding glucose-1-phosphate
§uses inorganic phosphate as phosphate source
§Disaccharides are hydrolyzed.
§lactose: glucose and galactose
§sucrose: glucose and fructose
§Monosaccharides fructose, galactose, and mannose
enter glycolysis at different points.
Oxidation of Multiple Carbohydrates
Involves Glycolysis
Fates of Pyruvate
Anaerobic Glycolysis:
Fermentation
• Generation of energy (ATP) without consuming
oxygen or NAD+
• No net change in oxidation state of the sugars
• Reduction of pyruvate to another product
• Regenerates NAD+ for further glycolysis under
anaerobic conditions
• The process is used in the production of food from
beer to yogurt to soy sauce.
Animals Undergo
Lactic Acid Fermentation
• Reduction of pyruvate to lactate, reversible
• During strenuous exercise, lactate builds up in the
muscle.
– generally less than 1 minute
• The acidification of muscle prevents its continuous
strenuous work.
• The lactate can be transported to the liver and
converted to glucose there.
• Requires a recovery time
– high amount of oxygen consumption to fuel gluconeogenesis
– restores muscle glycogen stores
Lactic Acid Fermentation
Glycolysis Occurs at
Elevated Rates
in Tumor Cells
• Tumor cells often grow faster
than angiogenesis allows
growth of capillaries to
aerate them, resulting in
anaerobic metabolism.
• Tumor cells increased
expression of LDH.
• Compounds that inhibit key
steps in glycolysis can kill
cancer cells by limiting
energy production.
Yeast Undergo
Ethanol Fermentation
• Two-step reduction of pyruvate to ethanol
• Humans do not have pyruvate decarboxylase.
• We do express alcohol dehydrogenase for ethanol
metabolism, but is largely used in the reverse reaction.
• CO2 produced in the first step is responsible for:
– carbonation in beer
– dough rising when baking bread
• Both steps require cofactors.
– pyruvate decarboxylase: Mg++ and thiamine pyrophosphate
– alcohol dehydrogenase: Zn++ and NAD+
Ethanol Fermentation
TPP Is a Common
Acetaldehyde
Carrier
• TPP forms a covalent
bond with carbonyl
carbon, forming an
alcohol, and resulting in
release of CO2.
• TPP then allows
rearrangement of and
protonation of carbonyl
carbon to release from
complex.
TPP Is a Common Acetaldehyde Carrier
Gluconeogenesis:
Making “New” Glucose
Notice that mammals cannot
convert fatty acids to sugars.
Glycolysis versus Gluconeogenesis
Glycolysis occurs mainly
in the muscle and brain.
Gluconeogenesis occurs
mainly in the liver.
Glycolysis versus Gluconeogenesis
• Opposing pathways that are both thermodynamically
favorable
– operate in opposite direction
• end product of one is the starting compound of the other
• Reversible reactions are used by both pathways.
• Irreversible reaction of glycolysis must be bypassed
in gluconeogenesis.
– no ATP generated during gluconeogenesis
– different enzymes in the different pathways
– differentially regulated to prevent a futile cycle
Steps 1 and 2 : Pyruvate to
Phosphoenolpyruvate
• Requires two energy-consuming steps
• The first step, pyruvate carboxylase converts
pyruvate to oxaloacetate.
– carboxylation using a biotin cofactor
– requires transport into the mitochondria
• The second step, phosphoenolpyruvate
carboxykinase converts oxaloacetate to PEP.
– phosphorylation from GTP and decarboxylation
– occurs in mitochondria or cytosol depending on the
organism
During this conversion, the same carbon is added and
immediately removed from the structure.
Synthesis of Oxaloacetate
Biotin Is a CO2 Carrier
Oxaloacetate to Phosphoenolpyruvate
First Gluconeogenic
Steps Travel Through
Mitochondria
• The inner mitochondrial
membrane is selectively
permeable: Malate, PEP, and
pyruvate are permeable, while
oxaloacetate cannot escape.
• Oxaloacetate can be utilized in
the citric acid cycle (Kreb’s cycle)
if needed.
• Oxaloacetate can be converted
to PEP or malate to allow
transport to cytosol for
gluconeogenesis.
Additional Bypasses
• Catalyze reverse reaction of opposing step in glycolysis
• Are irreversible themselves
• Fructose 1,6-bisphosphate à fructose 6-phosphate
–
–
–
–
by fructose bisphosphatase-1
coordinately/oppositely regulated with PFK
cleaves phosphate with water
DOES NOT generate ATP
• Glucose 6-phosphate à glucose
–
–
–
–
by glucose 6-phosphatase
segregated in the endoplasmic reticulum
vleaves phosphate with water
DOES NOT generate ATP
Gluconeogenesis Is Expensive
2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4 H2O à
Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+
• Costs 4 ATP, 2 GTP, and 2 NADH
• Physiologically necessary: Brain, nervous system, and
red blood cells generate ATP ONLY from glucose.
• Allows generation of glucose when glycogen stores
are depleted:
– during starvation
– during vigorous exercise
– can generate glucose from amino acids, but not fatty acids
Precursors for Gluconeogenesis
• Animals can produce glucose from sugars or
proteins.
– sugars: pyruvate, lactate, or oxaloacetate
– protein: from amino acids that can be converted to citric
acid cycle intermediates (or glucogenic amino acids)
• Animals cannot produce glucose from fatty acids.
– product of fatty acid degradation is acetyl-CoA
– cannot have a net converstion of acetyl-CoA to
oxaloacetate
• Plants, yeast, and many bacteria can do this, thus
producing glucose from fatty acids.
TABLE 14-4
Glucogenic Amino Acids, Grouped by Site of Entry
Pyruvate
Alanine
Cysteine
Glycine
Serine
Threonine
Tryptophana
α-Ketoglutarate
Arginine
Glutamate
Glutamine
Histidine
Proline
Succinyl-CoA
Isoleucinea
Methionine
Threonine
Valine
Fumarate
Phenylalaninea
Tyrosinea
Oxaloacetate
Asparagine
Aspartate
Note: All these amino acids are precursors of blood glucose or liver glycogen, because
they can be converted to pyruvate or citric acid cycle intermediates. Of the 20 common
amino acids, only leucine and lysine are unable to furnish carbon for net glucose synthesis.
aThese amino acids are also ketogenic (see Fig. 18-15).
Two Phases of the Pentose Phosphate
Pathway Simplified
Pentose Phosphate Pathway
• The main products are NADPH and ribose 5-phosphate.
• NADPH is an electron donor.
– reductive biosynthesis of fatty acids and steroids
– repair of oxidative damage
• Ribose-5-phosphate is a biosynthetic precursor of
nucleotides.
– used in DNA and RNA synthesis
– or synthesis of some coenzymes
NADPH Regulates Partitioning into Glycolysis
versus Pentose Phosphate Pathway