Glycolysis

Metabolism can be divided into various blocks, all interconnected. Each of them can be regulated.
In glycolysis, ATP is used to change ADP to ATP, and we used NAD to reduce to NADH. The main function
of glycolysis in muscle is to provide ATP. In the liver, glycolysis also generates ATP, but its main function
is to store excess glucose as fat.
NAD+ needs to be regenerated. The ATP is broken down into ADP by cellular processes. There are three
ways to regenerate NAD+. One is to change the end products and use the pathway to lactic acid, a
common pathway in muscle. The most common way is to send pyruvate into mitochondria, and use the
electron transport chain to convert NADH to NAD+. In yeast, pyruvate can be converted to ethanol, also
regenerating NAD+.
In cancer cells, even with oxygen present, there is a preference to use anaerobic glycolysis. Glycolysis
only produces 2 ATP for 1 glucose, while sending it to the mitochondria gains 38 ATP for 1 glucose. Since
cancer cells are very low efficiency, they require a lot of glucose. A FDG-PET scan can see the amounts of
glucose, usually tumors are linked to high concentrations.
Glucose can be imported into cells via active or passive transport. Active transport requires energy to
transport against a concentration gradient. Passive transport works as blood glucose is 5mM, and
glucose has lower concentration in cells due to metabolism.
In the small intestine lumen, there are tons of villi. Glucose concentration in the blood is higher than in
the intestine. Na+/K+ ATPase uses ATP to pump out glucose into the capillaries. As sodium is exported
out of the epithelial cells, sodium from the villi wants to enter, and attaches to glucose and comes in
together.
There is SGLUT-1, the sodium dependent glucose transporter. Others transport glucose (and some
transport galactose/fructose only, or as a combination) in various areas of the body. GLUT-1 is fairly
common, and GLUT-2 is a glucose sensor. GLUT-4 is responsive to insulin and will take up glucose.
There are so many glucose transporters because expression of different genes in different tissues allows
transport to be tailored to the needs of that tissue. Tissues often have more than one transporter for
glucose and their relative functions are controlled by the level of expression, and allows specificity for
the sugars.
Once glucose enters the cell, it becomes phosphorylated, to trap the glucose inside the cell, and to keep
the concentration of glucose in the cytoplasm low to allow more glucose to be taken up, allowing
glucose to be taken in with=out ATP expenditure.
There are three irreversible reactions in the glycolysis. There is always the first committed step. First,
glucose is phosphorylated. The second committed step involves the use of PFK, and the last committed
step is the conversion of PEP to pyruvate. There is energy investment as the process is started, with ATP
being used. There is directionality; without it ATP is used without more being produced. The process
begins with a 6-carbon molecule and results in two 3-carbon molecules.

The first step is phosphorylation. Hexokinase I and hexokinase II have very low Km, very high affinity, in
many tissues, that catalyzes the glucose phosphorylation. It is inhibited by high levels of glucose-6phosphoate. Glucokinase has a high Km, lower affinity, and is prevalent in liver and pancreas, not
inhibited by glucose-6-phosphate, is important when glucose concentrations are high, so glucose-6phosphate is eventually stored as glycogen. The liver clears glucose from the blood and stores it as
glycogen.
If glucose concentrations are low, hexokinase is used. As concentration of glucose increases, activity of
glucokinase increases to store glucose as glycogen.
GKRP (glucokinase regulatory protein) detects when glucose is low and glucokinase is not needed,
glucokinase is taken into the nucleus and disabled. When glucose is high, glucokinase is released to be
used.
PFK is the second irreversible reaction, a second phosphorylation, involving the conversion of ATP to
ADP. This commits cells to metabolize glucose. PFK is the most regulated, via kinases and allosteric
regulation. The activators of PFK are better described as deinhibitors of ATP because they reverse the
effect of inhibitory concentrations of ATP. PFK is inhibited by ATP and citrate, and activated by ADP,
AMP, cAMP, F6P, F2,6P.
The paradigm is that flux through glycolysis can increase 100x (the movement of glucose through its
pathway), but during vigorous exercise there is only a 10% drop in ATP concentration. Something else
has to control glycolytic flux. The inhibition of PFK is relieved by AMP. With no ATP, PFK is very active.
With some ATP, it is slowed. The addition of some AMP increases PFK activity.
The ATP concentration only drops 10% during exercise is partly due to the action of adenylate kinase
that rapidly equilibrates the ADP resulting from ATP hydrolysis with ATP and AMP.
In muscle, [ATP] is ~50 times [AMP] and ~10 times [ADP]. This situation will lead to a four-fold increase
in [AMP] for a 10% drop in [ATP]
2 ADP <-> ATP + AMP. K = [ATP][AMP]/[ADP]2 = 0.44
Assuming that [ATP] >> [AMP], and that AT = [AMP] + [ADP] + [ATP] and is constant, this is true.
AMP kinase is the main energy sensor in cells as [AMP] is the most changing. If AMP levels were
increasing, there needs to be more energy production.
The fourfold increase in [AMP] only increases PFK activity by ~9 fold. Substrate cycles are necessary to
increase PFK activity.
PFK is one way, the opposite FBPase goes the opposite direction. They were originally thought to be
futile cycles, but they were actually regulatory cycles. These cycles enables muscle to have a low rate of
glycolysis at rest, but respond quickly to a demand for energy. When the cells need energy, one
direction can be activated. AMP and F2,6P are activators for PFK and inhibitors of FBPase.
The assumption that a fourfold increase in [AMP] from adenylate kinase results in an increase in PFK
activity from 10% to 90% and a decrease in FBPase activity from 90% to 10% is valid. The maximum
activity of PFK is 10x higher than that of FBPase.

The flux at PFK when [AMP] is low is Jlow = Vf(low) = (10%x100) (90%X10) = 10 9 = 1
The flux at PFK when [AMP] is high is Jhigh = Vf(high_ = (90%x100) (10%x10) = 90 1 = 89
If there was no substrate cycle the increase in [AMP] would lead to a 9 fold increase in flux at PFK, but
with the substrate cycle a 89-fold increase in flux. A 10% change in [ATP] can lead to a ~90 fold change in
glycolysis through adenylate kinase and substrate cycle. The cycling of substrate is the price to pay to be
able to go from a resting state, where substrate cycling is maximum, to high activity.
F2,6P is a potent activator of PFK and inhibitor of FBPase. The [F2,6P] in cells depends on the balance
between its rate of synthesis by PFK-2 and degradation by FBPase-2. This is not a glycolic metabolite.
The liver is a source of metabolic control. The activity of PFK-2 and FBPase-2 are regulated by PKA
(protein kinase A). If blood glucose is dropping, the liver should be producing glucose and not
undergoing glycolysis as much. When [glucose] is dropping, [glucagon] increases, [cAMP] increase,
which activates PKA, which phosphorylates a PFK-2 isozyme which inhibits PFK-2, [F2,6P] drops, and PFK
is inhibited.
The heart has different PFK-2 and FBPase-2 isozymes, with phosphorylation on different sites. Activation
of PKA due to stress and [epinephrine] increase leads to activation of PFK-2, [F2,6P] and activates PFK.
In the muscles, when stressed, [epinephrine] increases, leading to [AMP] increase which activates AMP<
which activates PFK-2, [F2,6P] increase, and PFK activation. There are different PFK-2 and FBPase-2
isozymes, without phosphorylation cAMP-dependent control. Phosphorylation is instead done by AMPdependent protein kinase (AMPK).
Aldolase generates 2 3-carbon intermediates, GAP and DHAP. There are 2 classes of aldolase, class 1 and
class 2. Triose phosphate isomerase interconverts GAP and DHAP. There is an equilibrium, but
[DHAP]>>[GAP] since GAP continues along the glycolytic pathway.
Glyceraldehyde 3-phophate dehydrogenase generates 1,3-biphosphoglycerate, a high energy
intermediate, leading to generation of NADH.
Phosphoglycerate kinase leads to the generation of 2ATP. the dG for phosphoglycerate kinase is
negative, but in vivo the reaction occurs at equilibrium. This means Keq is very high. 1,3-BPG is rapidly is
rapidly passed between the two enzymes and does not accumulate to significant amount. It is difficult
for the reaction to reach 1,3-BPG, but one it is reached it is rapidly passed between the two enzymes.
Phosphoglycerate mutase converts 3-phosphoglycerate to 2-phosphoglycerate. A mutase catalyzes the
transfer of a functional group from one position to another on a molecule.
Enolase dehydrates 2-phosphoglycerate to phosphoenolpyruvate, a high-energy intermediate.
Pyruvate kinase converts phosphoenolpyruvate to pyruvate, involving generation of ATP. dG is 23kJ/mol, irreversal. There is allosteric and hormonal regulation. Allosteric is by ATP (which inactivates)
and fructose-1,6-bisphosphate (activates). Hormonal includes glucagon (activates), cAMP, protein kinase
A (inactivates). In muscle, there is no phosphorylation by protein kinase A.
The three committed steps are by FK, PFK1 and PK. Glucose is converted to 2 pyruvate. 2 ATP is used, 4
ATP is made, and 2 NADH is made. For each NADH, if there is oxygen, 3 ATP is generated.

NADH goes to the mitochondria. It is used to reduce pyruvate to lactate if glycolysis is faster than
oxidative phosphorylation.
Mitrochondrial oxidative phosphorylation is 19x more efficient than anerobic glycolysis. Anaerobic
glycolysis can produce ATP much more rapidly. Skeletal muscle has 2 types of fibers; slow twitch and
fast-twitch. Slow twitch have high mitochondrial content, while fast-twitch have low mitochondrial
content.