control sites in metabolic pathways

Enzymes catalyzing irreversible reaction in metabolic pathways are potential control

sites.
If there is a reaction in a metabolic pathway only going in one direction, that is a clue
that there is going to be a relatively large negative change in free energy and be a
control enzyme.

Control Sites of Glycolysis [Regulatory Enzymes]

There are 3 steps that have irreversible reactions

Step 1: hexokinase
Step 3: phosphofructokinase (PFK) [most important]
Step 10: pyruvate kinase
The control mechanisms for these step are different in different cells
--- can be controlled either allosterically or through covalent modification.

Why is phosphofructokinase (PFK) the most important regulatory enzyme in glycolysis?

Because it is the first regulatory enzyme after there is no more ability of metabolites to
to spin off the metabolic pathway (WTF??? THIS IS WORD FOR WORD WHAT SHE
SAID)

Although Hexokinase (also a regulatory enzyme] is involved earlier in the pathway,

there are multiple uses for G6P (the product of hexokinase) other than glycolysis. G6P
can also be shuttled off to form glycogen.
The next regulatory enzyme is PFK, and everything past it can only be used in
glycolysis.
Regulation of PFK in Muscle [Energy Charge]
Phosphofructokinase is the key regulator of glycolysis in mammals.
---the entire pathway is regulated by feedback at different enzymes.
The PFK is allosterically inhibited by ATP and allosterically stimulated by AMP.
Determined by "energy charge" = [ATP]/[AMP]
--- higher energy charge implies a higher concentration of ATP which results in PFK
being inhibited (negative effector)
--- lower energy charge implies a higher concentration of AMP which results in PFK
being stimulated (positive effector)
Regulation of Glycolysis in Muscle
The major role of glycolysis in muscle is to provide ATP for muscle contraction.
Therefore, the enzymes are going to be controlled by the energy needs of the cell.
at rest --> glycolysis inhibited (no ATP needs to be produced); negative effectors
present
during exercise --> glycolysis stimulated (ATP needs to be produced); positive
effectors present
Regulation for this pathway is controlled by PFK and Pyruvate Kinase (see above for
why it's not hexokinase) and is determined by the energy need (energy charge) of
the cell
AT REST
ATP is not being used, so the energy charge is high (more ATP than AMP)
At a high energy charge, PFK is allosterically inhibited by ATP
At a high energy charge, Pyruvate Kinase is also inhibited
DURING EXERCISE
ATP is being used so the concentration goes down; thus the energy charge is low (more
AMP than ATP)
At low energy charge, PFK is allosterically stimulated by AMP
As the PFK speeds up, the concentration if its product, FBP, is increased. F-1,6-BP
(fructose 1,6-bisphosphate) can allosterically bind to Pyruvate Kinase to speed
up that enzyme.

A high energy charge is a (positive/negative) effector for PFK.

A low energy charge is a (positive/negative) effector for PFK.
A high energy charge is a negative effector for PFK.
--- implies that more ATP is present than AMP, and so ATP will allosterically bind and
inhibit the enzyme
A low energy charge is a positive effector for PFK.
--- implies that more AMP is present than ATP, and so AMP will allosterically bind and
stimulate the enzyme
Why is PFK stimulated by AMP and not ADP?

ADP does seem more likely since its the product of hydrolyzed ATP
BUT when the ADP concentration gets high and ATP needs are great (REALLY low on
energy), the enzyme adenylate kinase will covert 2 ADP into ATP +AMP
Therefore, the presence of AMP is a signal that energy levels are getting really low

Allosteric Regulation of Phosphofructokinase in Muscle

High ATP slows PFK (plenty of ATP present so no need to run glycolysis faster)
Ribbon Diagram of PFK
a homo-tetramer (4 [quaternary] subunits)
catalytic sites (where F6P and ATP come in)
allosteric sites (where extra ATP will change the conformation and slow the enzyme)
[negative effector]

Regulation of Pyruvate Kinase in Muscle

AT REST
PK is inhibited by the high energy charge (idk if ATP allosterically binds like with
PFK???)
DURING EXERCISE
PFK is no longer inhibited, so it is producing FBP. FBP (Fructose 1-6 Bisphosphate)
allosterically binds to Pyruvate Kinase, stimulating the enzyme.
[RECHECK this part] ATP and Alanine are allosteric binders that are negative effectors
of pyruvate kinase
Regulation of Glycolysis in the Liver
more complicated than muscle glycolysis because the liver has more functions than just
moving -- it's the place where a lot of biosynthesis is occuring
Regulators of PFK in liver (most important)
Citrate: reports on the status of the citrus acid cycle --> inhibits PFK
fructose 2,6-bisphosphate --> powerful activator of PFK
--- NOT the same as FBP
(see terms below)
ATP/AMP --> still present but energy charge is not as relevant in the liver
Regulators of Glucokinase aka Hexokinase IV (the specific hexokinase in the
liver)
Glucokinase is only active when blood glucose levels are high (aka after a meal)
--- allosterically regulated
Why?
You don't want the liver pulling glucose out of the blood stream when other organs need
the sugar
Regulation of Pyruvate Kinase
Similarily, Pyruvate Kinase is only active when blood glucose levels are high
--- regulated both allosterically AND through covalent modification
Allosteric: PFK is no longer inhibited at high glucose levels, so it is producing F-1,6-
BP. FBP allosterically binds to Pyruvate Kinase, stimulating the enzyme.
Covalent modification: Low blood glucose leads to the phosphorylation and
inhibition of liver pyruvate kinase.
Alanine --> also a negative effector
energy charge in liver
It is present in the liver but it is not as influential because the energy charge does not
change drastically in the liver (Since it doesn't move)

Instead, the key regulators of phosphofructokinase in the liver are citrate, which reports
on the status of the citric acid cycle, and fructose 2,6-bisphosphate.
Citrate (Citric acid)
Regulator of PFK in liver
Inhibits PKA
An intermediate in the citric acid cycle that is also used a storing material for
biosynthesis
If the citrate concentration is high, it implies that the biosynthetic needs of the cell are
being met ---> therefore glycolysis is slowed
fructose 2,6-bisphosphate (F-2,6-BP) as a regulator
Regulator of PFK in liver
Activates PKA
When fructose 2,6-bisphosphate concertation is high, it indicates that there is a high
concertation of blood glucose.
This will positively affect glycolysis because high blood sugar levels is dangerous,
so more glucose will be pulled though glycolysis
--- if you don't need the energy, then it will be stored as fat
F-1,6-BP vs. F-2,6-BP
F-1,6-BP = product of PFK and regulator of Pyruvate Kinase
F-2,6-BP = regulator of PFK in liver

Regulation of Pyruvate Kinase in Liver

Pyruvate Kinase is only active when blood glucose levels are high
--- regulated both allosterically AND through covalent modification
Allosteric
PFK is no longer inhibited at high glucose levels, so it is producing FBP. FBP
allosterically binds to Pyruvate Kinase, stimulating the enzyme.
At high blood concentration
Covalent Modification
At low blood glucose levels, pyruvate kinase is phosphorylated by ATP and is in
its LESS active state
At high blood glucose levels, pyruvate kinase is DEphosphorylated and is in
its MORE active state
Alanine is also a negative effector
binds allosterically
pyruvate can be converted to alanine
if there's a high concetration of alanine, it indicates that there is enoug pyruvate which
will slow pyruvate kinase

For Pyruvate Kinase in Liver, F-1,6-BP is a (negative/positive) effector, and ATP is

a (negative/positive) effector.
For Pyruvate Kinase in Liver, the allosteric binding of FBP is a positive effector, and
ATP (which adds phosphate to the enzyme [covalent modification]) is
a negative effector.
FBP = allosterically binds to ATP at high glucose levels and makes it more active
ATP = adds phosphate to the enzyme [covalent modification] at low blood glucose
levels that makes it less active
insulin and glucagon
Insulin -- signals that blood glucose level is high
Glucagon -- signals that blood glucose level is low
When insulin is triggered (blood glucose is high), it ultimately triggers
the dephosphorylation of pyruvate kinase
When glucagon is triggered (blood glucose is low), it ultimately triggers
the phosphorylation of pyruvate kinase
Effectors of Liver Glycolysis [get the pic]
Phosphofructokinase
Positive
F-2,6-BP
AMP
Negative
ATP
Citrate
H+
Pyruvate Kinase
Positive
F-1,6-BP
Negative
ATP
Alanine
Distinguish between the role of glycolysis in muscle and liver.
Muscle
More about meeting the energy needs of the cell
Liver
more about controlling blood glucose level and the amount of biosynthetic precursurs
that are need
Why can't cells continuously use the breakdown of glucose to pyruvate to generate
energy without further conversion of pyruvate via fermentation or respiration?
a. The rate of energy production by glycolysis alone is too slow for cells' biochemical
reactions.
b. The amount of energy produced by glycolysis alone is insufficient to fuel cell growth.
c. The amount of inorganic phosphate available becomes limiting.
d. The electron acceptor reduced during glycolysis must be regenerated.
e. The complete breakdown of pyruvate is necessary to produce the carbon dioxide
needed by cells.
d. The electron acceptor reduced during glycolysis must be regenerated.
which of the following is not an intermediate in the glycolytic pathway by which glucose
is oxidized to pyruvate?
a. 3-phosphoglycerate
b. fructose 1,6-bisphosphate
c. glucose 1-phosphate
d. glyceraldehyde 3-phosphate
e. phosphoenolpyruvate
c. glucose 1-phosphate
Gluconeogenesis
the metabolic pathway responsible for synthesizing glucose from pyruvate
--- essentially the reverse of glycolysis
occurs in the liver, although gluconeogenesis can occur in the kidney
There are many routes to getting to glucose (sugar, starch, glycogen), but in
a fasting/starvation state these sources are all used up.
--- glucose is essential because it is the primary fuel for the brain and the only fuel for
red blood cells.

T/F
All cells can undergo glycolysis and gluconeogenesis.
F
All cells can undergo glycolysis
ONLY liver and kidney cells can undergo gluconeogenesis
Glycolysis vs. Gluconeogenesis
Gluconeogenesis is basically reverse glycolysis except for the 3 irreversible steps
All the substrates are the same, except for one new one --> oxaloacetate
--- as a result, gluconeogenesis has 11 steps
pyruvate kinase --> pyruvate carboxylase + phosphoenolpyruvate carboxykinase
phosphofructokinase --> Fructose 1,6-biphosphatase
Hexokinase --> Glucose 6-phopshatase

Glucose Can Be Synthesized from Noncarbohydrate Precursors

lactate (lactic acid) (from anaerobic fermentation)
--- Pyruvate can be formed from muscle-derived lactate in the liver by lactate
dehydrogenase. (called Cori cycle)
glycerol
--- derived from the hydrolysis of triacylglycerols, can be converted into
dihydroxyacetone phosphate, which can be processed by gluconeogenesis or
glycolysis.
Some amino acids
--- some amnio acids can easily be converted to pyruvate
--- some amino acids can easily be converted to oxaloacetate
Conversion of Glycerol to Pyruvate
Glycerol, derived from the hydrolysis of triacylglycerols, can be converted into
dihydroxyacetone phosphate, which can be processed by gluconeogenesis or
glycolysis.

Cori Cycle
the cycle of lactate (lactic acid) to pyruvate between the muscle and liver
When oxygen is low in muscle, pyruvate will be converted to lactate in lactic acid
fermentation to regenerate NAD+.
Lactate will enter the blood stream.
If more energy is needed, the lactate can be converted to pyruvate by lactate
dehydrogenase.
If no more energy is needed, lactate will be taken up by the liver, converted to pyruvate
by lactate dehydrogenase, and then shuttled into the Gluconeogenesis
pathway to make more glucose.
Gluconeogenesis Steps
1) Pyruvate (3C) is converted to Oxaloacetate (4C) by Pyruvate
Carboxylase (requires 2 ATP)
--- occurs in the mitocondria
--- several important steps involved see terms below
2) Oxaloacetate is phosphorylated to phosphoenolpyruvate (PEP)
by Phosphoenolpyruvate Carboxykinase (requires 2 GTP)
--- oxaloacetate is shuttled into cytoplasm and a phosphate from GTP is added
3) phosphoenolpyruvate (PEP) gets converted to 2-Phoshoglycerate by Enolase
4) 2-Phoshoglycerate gets converted to 3-Phopshoglycerate by Phosphoglycerate
Mutase
5) 3-Phosphoglycerate gets phosphorylated into 1,3-Bisphosphoglycerate
by Phosphoglycerate Kinase (requires 2 ATP)
6) 1,3-Bisphosphoglycerate gets dephosphorylated to Glyceraldehyde 3-
phosphate by Glyceraldehyde 3-phosphate Dehydrogenase (involves oxidation of
2 NADH)
7) Glyceraldehyde 3-phosphate gets converted to DHAP by Triose Phosphate
Isomerase
8) Glyceraldehyde 3-phosphate and DHAP gets converted to Fructose 1,6-
bisphosphate by Aldolase
9) Fructose 1,6-bisphosphate gets dephosphorylated by Fructose-1,6-
bisphosphatase [hydrolysis of phosphate]
--- NEW STEP; replacement for PFK
--- major regularory steps
10) Fructose-6-phosphate gets isomerized to Glucose 6-phosphate
by phosphoglucose isomerase
11) Glucose 6-phosphate gets dephosphorylated to glucose by Glucose 6-
phosphatase [hydrolysis of phosphate]
New Steps/Enzymes in Gluconeogenesis
1) Pyruvate (3C) is converted to Oxaloacetate (4C) by Pyruvate
Carboxylase (requires ATP)
--- occurs in the mitocondria
--- several important steps involved see terms below
2) Oxaloacetate is phosphorylated to phosphoenolpyruvate (PEP)
by Phosphoenolpyruvate Carboxykinase (requires GTP)
--- oxaloacetate is shuttled into cytoplasm and a phosphate from GTP is added
9) Fructose 1,6-bisphosphate gets dephosphorylated by Fructose-6-phosphate
--- NEW STEP; replacement for PFK
--- major regularory steps
11) Glucose 6-phosphate gets dephosphorylated to glucose by Glucose 6-
phosphatase
Conversion of Pyruvate into Phosphoenolpyruvate (PEP)
In glycolysis, the conversion of PEP to Pyruvate was done in one step by Pyruvate
Kinase
--- pyruvate kinase is irreversible so it will not be used in gluconeogenesis
In gluconeogenesis, an intermediate between Pyruvate and PEP must be formed --
> oxaloacetate
Requires 2 Enzymes
pyruvate carboxylase (in mitochondria)
–-- converts pyruvate to oxaloacetate
phosphoenolpyruvate carboxykinase (in cytoplasm)
--- converts oxaloacetate to PEP

Gluconeogenesis Step 1: Pyruvate Carboxylase Reaction

Pyruvate (3C) + CO2 (1C) is converted into Oxaloacetate (4C)

--- building up --> REQUIRES ATP
--- Pyruvate carboxylase requires the vitamin biotin as a cofactor.
OCCURS IN THE MITOCONDRIA
Formation of Oxaloacetate

Requires energy from ATP and the addition of CO2 to go from 3C to 4C.
--- pyruvate carboxylase requires the vitamin biotin as a cofactor
THREE STAGES
Stage 1: Bicarbonate gets phosphorylated by ATP
Stage 2: biotin reacts with the phosphorylated bicarbonate to produce CO2-biotin
Stage 3: CO2-biotin transfers the CO2 to pyruvate to form oxoacetate
Biotin is used to carry a carboxylate group to a substrate (commonly seen in many
enzymes)
shows how energy from ATP is transformed to form this higher energy molecule

biotin
A vitamin used to carry a carboxylate (CO2) to an enzyme
ex. carries CO2 to pyruvate to convert it to oxaloacetate
Subunit of Pyruvate Carboxylase
Pyruvate Carboxylase consists of 4 identical subunits. This is one subunit
Each subunit has 4 domains:
1) Biotin Carboxylase domain
--- this is where the bicarbonate molecule becomes phosphorylated
2) Biotin carboxyl carrier protein domain
--- this is where biotin is found
--- biotin will swing to the Biotic Carboxylase domain, pick up the CO2 from the
phosphorylated bicarbonate, the swing over to the pyruvate carboxylase domain and
transfer to CO2 to the pyruvate
3) Pyruvate Carboxylase domain
--- this is where the pyruvate is found
--- when the CO2 is transferred from biotin, it will form oxaloacetate
4) Tetramerization domain
-- holds the other domains together

Getting Oxaloacetate from the mitochondria to the cytoplasm (where

phosphoenolpyruvate carboxykinase is located)
The formation of oxaloacetate by pyruvate carboxylase occurs in the mitochondria.
Problem
There is no carrier protein to get oxaloacetate out of the mitochondria
Solution
• Oxaloacetate is reduced to malate (paired with oxidation of NADH) and transported
into the cytoplasm
.• Once it is in the cytoplasm, malate is reoxidized to oxaloacetate (paired with reduction
of NAD+) [another opportunity to make NAD+]
.• PEP is then synthesized from oxaloacetate by phosphoenolpyruvate carboxykinase.

Gluconeogenesis Step 2: Phosphoenolpyruvate Carboxykinase

Oxaloacetate is shuttled into the cytoplasm and phosphorylated into
phosphoenolpyruvate (PEP).
--- requires ENERGY FROM GTP

net reaction for pyruvate to PEP in gluconeogenesis

required both ATP (step 1) and GTP (step 2)

--- lots of energy investment needed

Gluconeogenesis Step 9: Fructose 1,6 bisphosphatase

[hydrolysis of phosphate]
Enzyme that catalyzes the removal of the phosphate by water in fructose 1,6-
bipshosphate to fructose 6-phosphate
PKA is involved for the reverse direction, but that enzyme is irreversible
Gluconeogenesis Step 11: Glucose 6-phosphatase

[hydrolysis of phosphate]
Enzyme that catalyzes the removal of the phosphate by water in glucose 6-phosphate
to glucose
--- The generation of free glucose is an important control point. [regulatory site]
Generation of Glucose from Glucose 6-Phosphate
-- Glucose 6-phosphate is transported from the liver (site of gluconeogenesis) into the
lumen of the endoplasmic reticulum
-- Glucose 6-phosphatase, an integral membrane on the inner surface of the
endoplasmic reticulum, catalyzes the formation of glucose from glucose 6-phosphate.

How does glucose get out of the liver?

Glucose 6-phosphatase
- Glucose 6-phosphate is transported from the liver (site of gluconeogenesis) into the
lumen of the endoplasmic reticulum
Glucose 6-phosphatase, an integral membrane on the inner surface of the endoplasmic
reticulum, catalyzes the formation of glucose from glucose 6-phosphate.
Once glucose is formed, it is passed out of the ER so it can be reelased from the liver
cell

How many High-Transfer-Potential Phosphoryl Groups Are Spent in Gluconeogenesis?

6 (4 ATP and 2 GTP) needed to make 1 glucose

Recall glycolysis used 2 and gain 4 for net +2
Shows that a lot of energy is needed to make glucose

What barrier prevents glycolysis from simply running in reverse to synthesize glucose?
How is this barrier overcome in gluconeogenesis?
the 3 irreversible steps (1, 3, 10) have large negative delta G that won't allow the
reaction to go in reverse

Barrier is overcome by 4 other enzymes that are activated when neccesary

Guiding Principle of Glycolysis/Gluconeogenesis Regulation
When glucose concentration is high or the cell needs ATP, glycolysis is favored
When glucose concertation is low, gluconeogenesis is favored
Regulated Step of Glycolysis/Gluconeogenesis
Glycolysis
Step 1: Hexokinase
Step 3: Phosphofructokinase
Step 10: Pyruvate Kinase
Gluconeogenesis
Steps 1: Pyruvate Carboxylase
Step 2: Phosphoenolpyruvate carboxykinase
Step 9: Fructose 1,6-bisphophatase
Step 11: Glucose 6-phosphotase
Reciprocal Regulation of Phosphofructokinase and Fructose 1,6-bisphophatase
Catalyze the forward or reverse reaction of Fructose 6-phosphate to Fructose -1,6-
bisphosphate
--- most of the positive regulators for Phosphofructokinase are negative regulators for
Fructose 1,6-bisphophatase
Phosphofructokinase
Positive
F-2,6-BP
AMP
Negative
ATP
Citrate
H+
Fructose 1,6-bisphosphatase
Negative
F-2,6-BP
AMP
Positive
Citrate

Reciprocal Regulation of Pyruvate Kinase and pyruvate

carboxylase/phosphoenolpyruvate carboxykinase
Catalyze the forward or reverse reaction of Phosphoenolpyruvate to pyruvate
--- most of the positive regulators for Pyruvate Kinase are negative regulators
forpyruvate carboxylase/phosphoenolpyruvate carboxykinase
Pyruvate Kinase
Positive
F-1,6-BP
Negative
ATP
Alanine
Pyruvate Carboxylase
Negative
ADP
Positive
Acetyl CoA
phosphoenolpyruvate carboxykinase
ONLY negative
ADP

MEMORIZE THIS PIC

Fructose-2,6-bisphosphate (F-2,6-BP)
The key regulator of glucose metabolism in the liver -- Fructose 2,6-bisphosphate
stimulates phosphofructokinase and inhibits fructose 1,6-bisphosphatase
The presence of a large concentration of F-2,6-BP indicates that there is a high blood
glucose level and vice versa
The kinase that synthesizes fructose 2,6-bisphosphate (phosphofructokinase 2) and
the phosphatase that hydrolyzes this molecule (fructose bisphosphatase 2) are
located on the same polypeptide chain. Such an arrangement is called a bifunctional
enzyme.

Bifunctional Regulatory Enzyme Phosphofructokinase 2/Fructose 2,6-

Bisphosphatase
The enzyme that synthesize fructose 2,6-bisphosphate and the enzyme that hydrolyzes
the phosphate off the molecule are attached together (bifunctional enzyme)
It is one subunit, one polypeptide
Phosphofructokinase 2: enzyme that phosphorylates F6P (fructose-6-phosphate) into
fructose 2,6-bisphospate
--- kinase domain
Fructose 2,6-Bisphosphatase: the enzyme that hydrolyzes the phosphate off of
fructose 2,6-bisphospate converting it to F6P
--- phosphatase domain
How does it know when to run the kinase portion and when to run the
phosphatase portion?
Only one active site will be activated at a time, which is determined by blood glucose
level
When blood glucose is low, the hormone glucagon is secreted.
The glucagon signaling pathway leads to the phosphorylation of the bifunctional
enzyme, which inhibits the kinase and stimulates the phosphatase.

If you want to speed up the glycolysis pathway, the (kinase/phosphatase) region of the
F-2,6-BP bifunctional regulatory enzyme will be activated, which is the enzyme
___________
If you want to speed up the gluconeogenesis pathway,
the (kinase/phosphatase) region of the F-2,6-BP bifunctional regulatory enzyme will be
activated, which is the enzyme ___________
If you want to speed up the glycolysis pathway, the kinase region of the F-2,6-BP
bifunctional regulatory enzyme will be activated, which is the
enzyme Phosphofructokinase 2
If you want to speed up the gluconeogenesis pathway, the phosphatase region of the
F-2,6-BP bifunctional regulatory enzyme will be activated, which is the
enzyme Fructose 2,6-Bisphosphatase

Control of the Synthesis and Degradation of Fructose 2,6-bisphosphate

RECALL when there are high levels of Fructose 2,6-bisphosphate present, PFK will be
stimulated (sped up)
When glucagon is present (indicator of low blood glucose), it will phosphorylate the F-
26-BP Regulatory Enzyme which results in the inhibition of the Phosphofructokinase 2
--- the kinase part is no longer active; only the phosphatase part is active
When only the phosphatase part is active, it will dephosphorylate Fructose 2,6-
bisphosphate back to Fructose -6-phosphate effectively stopping glycolysis
When the F-26-BP Regulatory Enzyme is NOT phosphorylated, the phosphatase's
activity is blocked (blocks enzyme Fructose 2,6-Bisphosphatase) and only the kinase
part is active
--- this will phosphorylate Fructose -6-phosphate into Fructose 2,6-bisphosphate which
will go and stimulate glycolysis

What would be the effect on an organism's ability to use glucose as an energy source if
a mutation inactivated glucose 6-phosphatase in the liver?a. Nothing because you can
eat glucose.b. During the night, while fasting glucose levels would drop to dangerously
low levels.c. Glucose would be synthesized from glycerol from fats, so it would result in
weight loss.
. During the night, while fasting glucose levels would drop to dangerously low levels.
If cells synthesizing glucose from lactate are exposed to CO2labeled with 14C, what will
be the distribution of label in the newly synthesized glucose?a. all of the C in the new
glucose will be 14Cb. 3 of the 6 C in the new glucose will be 14Cc. C1 of the new
glucose will be 14Cd. C6 of the new glucose will be 14Ce. none of the C in the new
glucose will be 14

e. none of the C in the new glucose will be 14C

The ending compound of glycolysis is ___________, and the starting compound for the
citric acid cycle is ___________. The enzyme that facilitates the conversion between
the two is ___________.
pyruvate
Acetyl CoA
Pyruvate Dehydrogenase
Pyruvate Dehydrogenase
the complex (several enzymes) that converts pyruvate to acetyl-CoA (the essential
link between glycolysis and the citric acid cycle)
--- IRREVERSIBLE
--- occurs in the MITOCHONDRIA (citric acid cycle also occurs in the mitochondria)
The pyruvate dehydrogenase complex consists of 3 Enzymes and 5 Coenzymes
Key regulatory enzyme

mitocondria structure
Outer membrane
Inner membrane
Matrix
synthesis of acetyl CoA from pyruvate
The pyruvate dehydrogenase complex consists of 3 Enzymes and 5 Coenzymes
The synthesis of acetyl CoA from pyruvate consists of three steps:
1) a decarboxylation [E3]
2) an oxidation (removes two electrons, NAD+ gets reduced) [E1]
3) transfer to CoA [E2]
Enzymes in the Pyruvate Dehydrogenase Complex (and their cofactor/prosthetic groups

E1: pyruvate dehydrogenase

--- responsible for the oxidative decarboxylation of pyruvate
--- cofactor: TPP
E2: dihydrolipoyl transacetylase
--- transfers the acetyl group to CoA
--- cofactor: Lipoamide
E3: dihydrolipoyl dehydrogenase
--- regeneration of lipoamide
--- cofactor: FAD
The three enzymes of the pyruvate dehydrogenase complex are structurally integrated,
and the lipoamide arm (swinging arm) allows rapid movement of substrates and
products from one active site of the complex to another.

TPP (thiamine pyrophosphate)

Cofactor/prosthetic group on E1 (pyruvate dehydrogenase)

derived from vitamin B1

Lipamide
Lipoic acid (vitamin) + lysine
[coezyme] Cofactor/prosthetic group on E2 (dihydrolipoyl transacetylase)
Flexible Linkages (swinging arm) Allow Lipoamide to Move Between Different Active
Sites
Lipoamide, a coenzyme, is formed by the attachment of the vitamin lipoic acid to a
lysine residue in another enzyme in the complex, dihydrolipoyltransacetylase (E2).
location of FAD vs NAD+
Both are electron carriers, but
NAD+ is a cosubstrate found in solution
FAD is a cofactor/prosthetic group that is bound to the enzyme
Mechanism of Pyruvate Dehydrogenase Complex
each circle represents a different enzyme (E2 exists as a dimer)
--- within each complex, there are 45 copies of E1, 10 copies of E3, and 20 catalytic
sites on E2 (each catalytic site has a lipoamide arm)
Inactive State
The Lipoamide arm is in the oxidized form (sulfurs are bound to each other)
1) Pyruvate enter at E1
--- E1 removes a CO2 from pyruvate (decarboxylated)
--- Remaining two carbons (acetyl group) bind to the TPP cofactor
2) The Lipoamide arm from E2 moves into the E1 active site
3) The acetyl group is transferred to E2 the Lipoamide arm
--- lipoamide arm gets reduced
4) CoA enters E2 active site and acetyl group is transferred
--- acetyl CoA is formed
You've gotten what you wanted (acetyl CoA), the remaining 2 steps are to regenerate
the lipoamide arm (it was reduced in step 3)
--- it can't function in the reduced form
5) Lipomaide is oxidized by FAD and is reactivated
6) NAD+ picks up electrons oxides FADH2 to FAD

Regulation of Pyruvate Dehydrogenase

Pyruvate Dehydrogenase needs to be regulated because the formation of acetyl CoA is
irreversible in animal cells, so it needs to be regulated.
Regulation at E1
A kinase associated with the complex phosphorylates and inactivates E1.
A phosphatase, also associated with the complex, removes the phosphate and thereby
activates the enzyme.
Regulation by energy charge (allosteric regulation)
-- ATP, acetyl COA, and NADH inhibit the complex (indicate that there is enough
energy)
--- ADP and pyruvate stimulate the complex (indicating that energy needs to be
produced)
Pyruvate Dehydrogenase Regulation at E1
E1 is the regulatory point
E1 phosphorylated --> INACTIVE
E1 dephosphorylated --> ACTIVE
A kinase associated with the complex phosphorylates and inactivates E1.
A phosphatase, also associated with the complex, removes the phosphate and thereby
activates the enzyme
Regulation of Pyruvate Dehydrogenase (short)
Inhibited by
E1 PHOSPHORYLATED
ATP
acetyl CoA
NADH
Activated by
E1 DEPHOSPHORYLATED
ADP
Pyruvate
What are the advantages of having enzyme stay in a complex (rather than floating
around in space)?
1) reaction is facilitated by having the active sites in close proximity (don't need to wait
for the substrate to diffuse to the active site)
2) Reactants don't need to leave until the final product is made
--- negative: this limits side reactions
3) All the enzymes needed exist in the proper ratios
4) regulation is more efficient because the regulatory enzymes are also a part of the
complex.
Fates of Acetyl CoA
Two principle fates
1) citric acid cycle
2) incorporated into fatty acids