Generation of ATP from Glucose: Glycolysis



Glucose
is the universal fuel for human cells. Every cell type in the human is able
to generate adenosine triphosphate (ATP) from glycolysis, the pathway in which
glucose is oxidized and cleaved to form pyruvate. The importance of glycolysis in
our fuel economy is related to the availability of glucose in the blood, as well as
the ability of glycolysis to generate ATP in both the presence and absence of O2.
Glucose is the major sugar in our diet and the sugar that circulates in the blood
to ensure that all cells have a continuous fuel supply. The brain uses glucose
almost exclusively as a fuel.


Glycolysis
begins with the phosphorylation of glucose to glucose 6-phosphate
(glucose-6-P) by hexokinase (HK). In subsequent steps of the pathway, one glucose-
6-P molecule is oxidized to two pyruvate molecules with generation of two
molecules of NADH . A net generation of two molecules of ATP occurs
through direct transfer of high-energy phosphate from intermediates of the pathway
to ADP (substrate level phosphorylation).
Glycolysis occurs in the cytosol and generates cytosolic NADH. Because
NADH cannot cross the inner mitochondrial membrane, its reducing equivalents
are transferred to the electron transport chain by either the malate-aspartate
shuttle or the glycerol 3-phosphate shuttle . Pyruvate is then oxidized
completely to CO2 by pyruvate dehydrogenase and the TCA cycle. Complete


aerobic oxidation
of glucose to CO2 can generate approximately 30 to 32 moles
of ATP per mole of glucose.
When cells have a limited supply of oxygen (e.g., kidney medulla), or few or
no mitrochondria (e.g., the red cell), or greatly increased demands for ATP
(e.g., skeletal muscle during high-intensity exercise), they rely on anaerobic
glycolysis for generation of ATP. In anaerobic glycolysis, lactate dehydrogenase


oxidizes the NADH generated from glycolysis by reducing pyruvate to
lactate
. Because O2 is not required to reoxidize the NADH, the
pathway is referred to as anaerobic. The energy yield from anaerobic glycolysis
(2 moles of ATP per mole of glucose) is much lower than the yield from aerobic
oxidation. The lactate (lactic acid) is released into the blood. Under pathologic
conditions that cause hypoxia, tissues may generate enough lactic acid to
cause lactic acidemia.
In each cell, glycolysis is regulated to ensure that ATP homeostasis is
maintained, without using more glucose than necessary. In most cell types,


hexokinase (HK)
, the first enzyme of glycolysis, is inhibited by glucose
6-phosphate . Thus, glucose is not taken up and phosphorylated
by a cell unless glucose-6-P enters a ****bolic pathway, such as glycolysis or
glycogen synthesis. The control of glucose-6-P entry into glycolysis occurs at
phosphofructokinase-1(PFK-1), the rate-limiting enzyme of the pathway.
PFK-1 is allosterically inhibited by ATP and allosterically activated by AMP.
AMP increases in the cytosol as ATP is hydrolyzed by energy-requiring
reactions.






Overview of glycolysis and the TCA cycle.


Anaerobic glycolysis (shown in
blue). The conversion of glucose to lactate
generates 2 ATP from substrate-level phosphorylation.
Because there is no net generation of
NADH, there is no need for O
2, and, thus, the
pathway is anaerobic.





Glycolysis has functions in addition to ATP production. For example, in liver
and adipose tissue, this pathway generates pyruvate as a precursor for
fatty acid
biosynthesis. Glycolysis also provides precursors for the synthesis of compounds
such as amino acids and 5-carbon sugar phosphates.



GLYCOLYSIS

Glycolysis is one of the principle pathways for generating ATP in cells and is
present in all cell types. The central role of glycolysis in fuel ****bolism is
related to its ability to generate ATP with, and without, oxygen. The oxidation of
glucose to pyruvate generates ATP from substrate-level phosphorylation (the
transfer of phosphate from high-energy intermediates of the pathway to ADP) and
NADH. Subsequently, the pyruvate may be oxidized to CO
2 in the TCA cycle and
ATP generated from electron transfer to oxygen in oxidative phosphorylation.
However, if the pyruvate and NADH from glycolysis are converted to lactate
(anaerobic glycolysis), ATP can be generated in the absence of oxygen, via
substrate-level phosphorylation.
Glucose is readily available from our diet, internal glycogen stores, and the
blood. Carbohydrate provides 50% or more of the calories in most diets, and glucose
is the major carbohydrate. Other dietary sugars, such as fructose and galactose,
are oxidized by conversion to intermediates of glycolysis. Glucose is stored in cells
as glycogen, which can provide an internal source of fuel for glycolysis in emergency
situations (e.g., decreased supply of fuels and oxygen during ischemia, a low
blood flow). Insulin and other hormones maintain blood glucose at a constant level
(glucose homeostasis), thereby ensuring that glucose is always available to cells that
depend on glycolysis for generation of ATP.
In addition to serving as an anaerobic and aerobic source of ATP, glycolysis is an
anabolic pathway that provides biosynthetic precursors. For example, in liver and
adipose tissue, this pathway generates pyruvate as a precursor for fatty acid biosynthesis.
Glycolysis also provides precursors for the synthesis of compounds such as
amino acids and ribose-5-phosphate, the precursor of nucleotides.



The Reactions of Glycolysis

The glycolytic pathway, which cleaves 1 mole of glucose to 2 moles of the 3-carbon
compound pyruvate, consists of a preparative phase and an ATP-generating
phase. In the initial preparative phase of glycolysis, glucose is phosphorylated
twice by ATP and cleaved into two triose phosphates . The ATP expenditure
in the beginning of the preparative phase is sometimes called “priming the
pump,” because this initial utilization of 2 moles of ATP/ mole of glucose results
in the production of 4 moles of ATP/mole of glucose in the ATP-generating
phase.
In the ATP-generating phase, glyceraldehyde 3-phosphate (a triose phosphate) is
oxidized by NADand phosphorylated using inorganic phosphate. The highenergy
phosphate bond generated in this step is transferred to ADP to form ATP. The
remaining phosphate is also rearranged to form another high-energy phosphate
bond that is transferred to ADP. Because there were 2 moles of triose phosphate
formed, the yield from the ATP-generating phase is 4 ATP and 2 NADH. The result
is a net yield of 2 moles of ATP, 2 moles of NADH, and 2 moles of pyruvate per
mole of glucose.




Phases of the glycolytic pathway.





CONVERSION OF GLUCOSE TO GLUCOSE 6-PHOSPHATE

Glucose ****bolism begins with transfer of a phosphate from ATP to glucose to
form glucose-6-P . Phosphorylation of glucose commits it to ****bolism
within the cell because glucose-6-P cannot be transported back across the plasma
membrane. The phosphorylation reaction is irreversible under physiologic conditions
because the reaction has a high negative
G0. Phosphorylation does not,
however, commit glucose to glycolysis.
Glucose-6-P is a branchpoint in carbohydrate ****bolism. It is a precursor for
almost every pathway that uses glucose, including glycolysis, the pentose phosphate
pathway, and glycogen synthesis. From the opposite point of view, it also can be
generated from other pathways of carbohydrate ****bolism, such as glycogenolysis
(breakdown of glycogen), the pentose phosphate pathway, and gluconeogenesis
(the synthesis of glucose from non-carbohydrate sources).
Hexokinases, the enzymes that catalyze the phosphorylation of glucose, are a
family of tissue-specific isoenzymes that differ in their kinetic properties. The
isoenzyme found in liver and cells of the pancreas has a much higher Km than
other hexokinases and is called glucokinase. In many cells, some of the hexokinase
is bound to porins in the outer mitochondrial membrane (voltage-dependent anion
channels), which gives these enzymes first access to newly synthesized
ATP as it exits the mitochondria.




Phases of the glycolytic pathway.






CONVERSION OF GLUCOSE-6-P TO THE TRIOSE PHOSPHATES

In the remainder of the preparative phase of glycolysis, glucose-6-P is isomerized
to fructose 6-phosphate (fructose-6-P), again phosphorylated, and subsequently
cleaved into two 3-carbon fragments . The isomerization, which positions
a keto group next to carbon 3, is essential for the subsequent cleavage of the bond
between carbons 3 and 4.
The next step of glycolysis, phosphorylation of fructose-6-P to fructose 1,6-
bisphosphate (fructose-1,6-bisP) by phosphofructokinase-1 (PFK-1), is generally
considered the first committed step of the pathway. This phosphorylation requires
ATP and is thermodynamically and kinetically irreversible. Therefore, PFK-1 irrevocably
commits glucose to the glycolytic pathway. PFK-1 is a regulated enzyme in
cells, and its regulation controls the entry of glucose into glycolysis. Like hexokinase,
it exists as tissue-specific isoenzymes whose regulatory properties match variations
in the role of glycolysis in different tissues.
Fructose-1,6-bisP is cleaved into two phosphorylated 3-carbon compounds
(triose phosphates) by aldolase . Dihydroxyacetone phosphate
(DHAP) is isomerized to glyceraldehyde 3-phosphate (glyceraldehyde-3-P), which
is a triose phosphate. Thus, for every mole of glucose entering glycolysis, 2 moles
of glyceraldehyde-3-P continue through the pathway.

Glucose 6-phosphate ****bolism.





OXIDATION AND SUBSTRATE LEVEL PHOSPHORYLATION

In the next part of the glycolytic pathway, glyceraldehyde-3-P is oxidized and phosphorylated
so that subsequent intermediates of glycolysis can donate phosphate to
ADP to generate ATP. The first reaction in this sequence, catalyzed by glyceraldehyde-
3-P dehydrogenase, is really the key to the pathway . This
enzyme oxidizes the aldehyde group of glyceraldehyde-3-P to an enzyme-bound
carboxyl group and transfers the electrons to NAD
to form NADH. The oxidation
step is dependent on a cysteine residue at the active site of the enzyme, which forms
a high-energy thioester bond during the course of the reaction. The high-energy
intermediate immediately accepts an inorganic phosphate to form the high-energy
acyl phosphate bond in 1,3-bisphosphoglycerate, releasing the product from the
cysteine residue on the enzyme. This high-energy phosphate bond is the start of substrate-
level phosphorylation (the formation of a high-energy phosphate bond where
none previously existed, without the utilization of oxygen).
In the next reaction, the phosphate in this bond is transferred to ADP to form ATP
by 3-phosphoglycerate kinase. The energy of the acyl phosphate bond is high
enough (13 kcal/mole) so that transfer to ADP is an energetically favorable
process. 3-phosphoglycerate is also a product of this reaction.
To transfer the remaining low-energy phosphoester on 3-phosphoglycerate to
ADP, it must be converted into a high-energy bond. This conversion is accomplished
by moving the phosphate to the second carbon (forming 2-phosphoglycerate)
and then removing water to form phosphoenolpyruvate (PEP). The enolphosphate
bond is a high-energy bond (its hydrolysis releases approximately 14
kcal/mole of energy), so the transfer of phosphate to ADP by pyruvate kinase is
energetically favorable . This final reaction converts PEP to pyruvate.




SUMMARY OF THE GLYCOLYTIC PATHWAY



The overall net reaction in the glycolytic pathway is:
Glucose+2NAD
+ 2Pi +2ADP --->2Pyruvate+2NADH+4H2ATP+2H2O
The pathway occurs with an overall negative G0 of approximately –22 kcal.
Therefore, it cannot be reversed without the expenditure of energy.