GLYCOLYSIS CONCERNS

Although glycolysis is a nearly universal process, the fate of its

end product, pyruvate, may vary in different organisms or even

in different tissues.

In the presence of oxygen, the most common situation in

multicellular organisms and many unicellular ones, pyruvate is

metabolized to carbon dioxide and water through the citric acid

cycle and the electron transport chain.

In the absence of oxygen, fermentation generates a lesser

amount of energy; pyruvate is converted, or fermented, into

lactic acid in lactic acid fermentation or into ethanol in alcoholic

fermentation.

Lactic acid production takes place in skeletal muscle when

energy needs outpace the ability to transport oxygen.

Glycolysis is a catabolic pathway in the cytoplasm that is found

in almost all organisms—irrespective of whether they live

aerobically or anaerobically.

In eukaryotic cells, glycolysis takes place in the cytosol. This

pathway can be thought of as comprising three stages

Stage 1, which is the conversion of glucose into fructose 1, 6-

bisphosphate, consists of three steps: a phosphorylation, an

isomerization, and a second phosphorylation reaction.

The strategy of these initial steps in glycolysis is to trap the

glucose in the cell and form a compound that can be readily

cleaved into phosphorylated three-carbon units.

Stage 2 is the cleavage of the fructose 1,6-bisphosphate into

two three-carbon fragments. These resulting three-carbon units

are readily interconvertible.

In stage 3, ATP is harvested when the three carbon fragments

are oxidized to pyruvate.

Glycolysis involves ten individual steps, including three

isomerizations and four phosphate transfers. The only redox

reaction takes place in step [6].

[1] Glucose, which is taken up by animal cells from the blood

and other sources, is first phosphorylated to glucose 6-

phosphate, with ATP being consumed. The glucose 6-phosphate

is not capable of leaving the cell.

[2] In the next step, glucose 6-phosphate is isomerized into

fructose 6-phosphate.

[3] Using ATP again, another phosphorylation takes place, giving

rise to fructose 1,6-bisphosphate. Phosphofructokinase is the

most important key enzyme in glycolysis.

[4] Fructose 1,6-bisphosphate is broken down by aldolase into

the C3 compounds glyceraldehyde3-phosphate (also known as

glyceral3-phosphate) and glycerone3-phosphate

(dihydroxyacetone 3-phosphate).

[5] The latter two products are placed in fast equilibrium by

triosephosphate isomerase.

[6] Glyceraldehyde 3-phosphate is now oxidized by

glyceraldehyde-3-phosphate dehydrogenase, with NADH + H+

being formed. In this reaction, inorganic phosphate is taken up

into the molecule (substrate-level phosphorylation; and 1,3-

bisphosphoglycerate is produced. This intermediate contains a

mixed acid–anhydride bond, the phosphate part of which is at a

high chemical potential.

[7] Catalyzed by phosphoglycerate kinase, this phosphate

residue is transferred to ADP, producing 3-phosphoglycerate and

ATP. The ATP balance is thus once again in equilibrium.

[8] As a result of shifting of the remaining phosphate residue

within the molecule, the isomer 2-phosphoglycerate is formed.

[10] In the last step, pyruvate kinase transfers this residue to

ADP. The remaining enol pyruvate is immediately rearranged

into pyruvate, which is much more stable.

SOME HIGHLIGHTS

*1*Glucose is phosphorylated by ATP to form glucose 6-

phosphate. This step is notable for two reasons: (1) glucose 6-

phosphate cannot diffuse through the membrane, because of its

negative charges, and (2) the addition of the phosphoryl group

begins to destabilize glucose, thus facilitating its further

metabolism.

The glucose-induced structural changes are significant in two

respects. First, the environment around the glucose becomes

much more nonpolar, which favors the donation of the terminal

phosphoryl group of ATP. Second, the substrate-induced

conformational changes within the kinase enable it to

discriminate against H2O as a substrate. That means it blocks

the access of water (from the solvent), which might otherwise

enter the active site and attack (hydrolyze) the

phosphoanhydride bonds (esp., the γ phosphoryl group) of ATP

forming ADP and Pi. In other words, a rigid kinase would

necessarily also be an ATPase.

Like the other nine enzymes of glycolysis, hexokinase is a

soluble, cytosolic protein.

Hexokinase, like adenylate kinase and all other kinases, requires

Mg 2 + (or another divalent metal ion such as Mn2+) for activity.

The divalent metal ion forms a complex with ATP.

Kinetic studies of NMP kinases, as well as many other enzymes

having ATP or other nucleoside triphosphates as a substrate,

reveal that these enzymes are essentially inactive in the absence

of divalent metal ions such as magnesium (Mg2+) or manganese

(Mn2+), but acquire activity on the addition of these ions.

The metal is not a component of the active site. Rather,

nucleotides such as ATP bind these ions, and it is the metal ion-

nucleotide complex that is the true substrate for the enzymes.

Essentially all nucleoside triphosphates are present as NTP-

Mg2+ complexes.

(1) The magnesium ion neutralizes some of the negative charges

present on the polyphosphate chain, reducing nonspecific ionic

interactions between the enzyme and the polyphosphate group

of the nucleotide.

(2) The interactions between the magnesium ion and the oxygen

atoms in the phosphoryl group hold the nucleotide in well-

defined conformations that can be specifically bound by the

enzyme.

(3) The magnesium ion provides additional points of interaction

between the ATP-Mg2+ complex and the enzyme, thus

increasing the binding energy.

**In some cases, such as the DNA polymerases, side chains

(often aspartate and glutamate residues) of the enzyme can

bind directly to the magnesium ion. In other cases, the enzyme

interacts indirectly with the magnesium ion through hydrogen

bonds to the coordinated water molecules (Figure 9.50). Such

interactions have been observed in adenylate kinases bound to

ATP analogs.

Hexokinase is present in all cells of all organisms. Hepatocytes

also contain a form of hexokinase called hexokinase IV or

glucokinase, which differs from other forms of hexokinase in

kinetic and regulatory properties.

Two enzymes that catalyze the same reaction but are encoded

in different genes are called isozymes.

*2* The enzyme phosphohexose isomerase (phosphoglucose

isomerase) catalyzes the reversible isomerization of glucose 6-

phosphate, an aldose, to fructose 6-phosphate, a ketose.

The enzyme must first open the six-membered ring of glucose 6-

phosphate, catalyze the isomerization, and then promote the

formation of the five-membered ring of fructose 6-phosphate.

*3* Phosphorylation of Fructose 6-Phosphate to Fructose 1,6-

Bisphosphate, phosphofructokinase-1 (PFK-1) catalyzes the

transfer of a phosphoryl group from ATP to fructose 6-

phosphate to yield fructose 1,6-bisphosphate.

This enzyme is called PFK-1 to distinguish it from a second

enzyme (PFK-2) that catalyzes the formation of fructose 2,6-

bisphosphate from fructose 6-phosphate in a separate pathway.

The PFK-1 reaction is essentially irreversible under cellular

conditions.

Phosphofructokinase-1 is a regulatory enzyme, one of the most

complex known.

PFK1 is the major point of regulation in glycolysis. The activity of

PFK-1 is increased whenever the cell’s ATP supply is depleted or

when the ATP breakdown products, ADP and AMP (particularly

the latter), are in excess. The enzyme is inhibited whenever the

cell has ample ATP and is well supplied by other fuels such as

fatty acids. In some organisms, fructose 2,6-bisphosphate is a

potent allosteric activator of PFK-1.

The enzyme fructose 1,6-bisphosphate aldolase, often called

simply aldolase, catalyzes a reversible aldol condensation.

*4* Fructose 1,6-bisphosphate is cleaved to yield two different

triose phosphates, glyceraldehyde 3-phosphate (GAP) , an

aldose, and dihydroxyacetone phosphate (DHAP) , a ketose.

Although the aldolase reaction has a strongly positive standard

free-energy change in the direction of fructose 1,6-bisphosphate

cleavage, at the lower concentrations of reactants present in

cells, the actual free-energy change is small and the aldolase

reaction is readily reversible.

Only one of the two triose phosphates formed by aldolase,

glyceraldehyde 3-phosphate, can be directly degraded in the

subsequent steps of glycolysis.

*5* The other product, dihydroxyacetone phosphate, is rapidly

and reversibly converted to glyceraldehyde 3-phosphate by the

fifth enzyme of the sequence, triose phosphate isomerase.

This reaction is rapid and reversible. At equilibrium, 96% of the

triose phosphate is dihydroxyacetone phosphate.

*5* TIM catalyzes the transfer of a hydrogen atom from carbon

1 to carbon 2 in converting dihydroxyacetone phosphate into

glyceraldehyde 3-phosphate, an intramolecular oxidation-

reduction.

Two features of this enzyme are noteworthy. First, TIM displays

great catalytic prowess. Indeed, the k cat/K M ratio for

isomerization of glyceraldehyde 3-phosphate is 2 × 108 M-1 s-1,

which is close to the diffusion-controlled limit. In other words,

the rate-limiting step in catalysis is the diffusion-controlled

encounter of substrate and enzyme. TIM is an example of a

kinetically perfect enzyme. Second, TIM suppresses an

undesired side reaction, the decomposition of the enediol

intermediate into methyl glyoxal and inorganic phosphate.

The payoff phase of glycolysis includes the energy-conserving

phosphorylation steps in which some of the free energy of the

glucose molecule is conserved in the form of ATP.

One molecule of glucose yields two molecules of glyceraldehyde

3-phosphate; both halves of the glucose molecule follow the

same pathway in the payoff phase of glycolysis.

*6* The first step in the payoff phase is the oxidation of

glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate,

catalyzed by glyceraldehyde 3-phosphate dehydrogenase.

This is the first of the two energy-conserving reactions of

glycolysis that eventually lead to the formation of ATP.

The aldehyde group of glyceraldehyde 3-phosphate is oxidized,

not to a free carboxyl group but to a carboxylic acid anhydride

with phosphoric acid.

This type of anhydride, called an acyl phosphate, has a very

high standard free energy of hydrolysis.

So 1,3-Bisphosphoglycerate is an acyl phosphate.

Such compounds have a high phosphoryl-transfer potential; one

of its phosphoryl groups is transferred to ADP in the next step in

glycolysis.

The reaction is the sum of two processes: the oxidation of the

aldehyde to a carboxylic acid by NAD+ and the joining of the

carboxylic acid and orthophosphate to form the acyl-phosphate

product.

Much of the free energy of oxidation of the aldehyde group of

glyceraldehyde 3-phosphate is conserved by formation of the

acyl phosphate group at C-1 of 1,3-bisphosphoglycerate.

The acceptor of hydrogen in the glyceraldehyde 3-phosphate

dehydrogenase reaction is NAD+ , bound to a Rossmann fold.

The reduction of NAD+ proceeds by the enzymatic transfer of a

hydride ion (:H-) from the aldehyde group of glyceraldehyde 3-

phosphate to the nicotinamide ring of NAD+, yielding the

reduced coenzyme NADH. The other hydrogen atom of the

substrate molecule is released to the solution as H+ .

Glyceraldehyde 3-phosphate is covalently bound to the

dehydrogenase during the reaction. The aldehyde group of

glyceraldehyde 3-phosphate reacts with the --SH group of an

essential Cys residue in the active site, in a reaction analogous

to the formation of a hemiacetal, in this case producing a

thiohemiacetal. Reaction of the essential Cys residue with a

heavy metal such as Hg2+ irreversibly inhibits the enzyme.

IN OTHER WORDS

Let us consider the mechanism of glyceraldehyde 3-phosphate

dehydrogenase in detail.

In step 1, the aldehyde substrate reacts with the sulfhydryl

group of cysteine 149 on the enzyme to form a hemithioacetal.

Step 2 is the transfer of a hydride ion to a molecule of NAD +

that is tightly bound to the enzyme and is adjacent to the

cysteine residue. This reaction is favored by the deprotonation

of the hemithioacetal by histidine 176. The products of this

reaction are the reduced coenzyme NADH and a thioester

intermediate. This thioester intermediate has a free energy

close to that of the reactants.

In step 3, orthophosphate attacks the thioester to form 1,3-BPG

and free the cysteine residue. This displacement occurs only

after the NADH formed from the aldehyde oxidation has left the

enzyme and been replaced by a second NAD+. The positive

charge on the NAD+ may help polarize the thioester

intermediate to facilitate the attack by orthophosphate.

The favorable oxidation and unfavorable phosphorylation

reactions are coupled by the thioester intermediate, which

preserves much of the free energy released in the oxidation

reaction.

*7* The enzyme

phosphoglycerate kinase

transfers the high-energy

phosphoryl group from the

carboxyl group of 1,3-

bisphosphoglycerate to

ADP, forming ATP and

3-phosphoglycerate.

Notice that phosphoglycerate kinase is named for the reverse

reaction. Like all enzymes, it catalyzes the reaction in both

directions. This enzyme acts in the direction suggested by its

name during gluconeogenesis and during photosynthetic CO2

assimilation.

This Substrate-level phosphorylation (SLP) involves soluble

enzymes and chemical intermediates (1,3-bisphosphoglycerate

in this case).

The formation of ATP in this manner is referred to as substrate-

level phosphorylation because the phosphate donor, 1,3-BPG, is

a substrate with high phosphoryl-transfer potential.

Thus, the outcomes of the reactions catalyzed by

glyceraldehyde 3-phosphate dehydrogenase and

phosphoglycerate kinase are:

1. Glyceraldehyde 3-phosphate, an aldehyde, is oxidized to 3-

phosphoglycerate, a carboxylic acid.

2. NAD+ is concomitantly reduced to NADH.

3. ATP is formed from Pi and ADP at the expense of carbon

oxidation energy.

*8* The enzyme

phosphoglycerate mutase

catalyzes a reversible shift

of the phosphoryl group

between C-2 and C-3 of

glycerate; Mg2+ is

essential for this reaction.

Bisphosphoglycerate mutase catalyzes the conversion of 1,3-

bisphosphoglycerate to 2,3-bisphosphoglycerate, which is

converted to 3-phosphoglycerate by 2,3-bisphosphoglycerate

phosphatase (and possibly also phosphoglycerate mutase). This

alternative pathway involves no net yield of ATP from glycolysis.

However, it does serve to provide 2,3-bisphosphoglycerate,

which binds to hemoglobin, decreasing its affinity for oxygen

and so making oxygen more readily available to tissues.

In general, a mutase is an enzyme that catalyzes the

intramolecular shift of a chemical group, such as a phosphoryl

group.

This enzyme requires catalytic amounts of

2,3-bisphosphoglycerate to maintain an active-site histidine

residue in a phosphorylated form.

Examination of the first partial reaction reveals that the mutase

may function as a phosphatase it converts

2,3-bisphosphoglycerate into 2-phosphoglycerate. However, the

phosphoryl group remains linked to the enzyme. This

phosphoryl group is then transferred to 3-phosphoglycerate to

reform 2,3-bisphosphoglycerate.

*9* In the second glycolytic reaction that generates a

compound with high phosphoryl group transfer potential,

enolase promotes reversible removal of a molecule of water

(dehydration) from 2-phosphoglycerate to yield

phosphoenolpyruvate (PEP).

Although 2-phosphoglycerate and phosphoenolpyruvate contain

nearly the same total amount of energy, the loss of the water

molecule from 2-phosphoglycerate causes a redistribution of

energy within the molecule, greatly increasing the standard free

energy of hydrolysis of the phosphoryl group.

*10* The last step in glycolysis is

the transfer of the phosphoryl

group from phosphoenolpyruvate

to ADP, catalyzed by pyruvate

kinase, which requires K+ and

either Mg2+ or Mn2+ .

In this substrate-level phosphorylation, the product pyruvate

first appears in its enol form, then tautomerizes rapidly and

nonenzymatically to its keto form, which predominates at pH 7.

The overall reaction has a large, negative standard free energy

change, due in large part to the spontaneous conversion of the

enol form of pyruvate to the keto form.

The pyruvate kinase reaction is essentially irreversible under

intracellular conditions and is an important site of regulation.

Note that the energy released in the anaerobic conversion of

glucose into two molecules of pyruvate is -21 kcal mol-1

(-88 kJ mol-1).

In aerobic respiration, Electron transfer from NADH to O2 in

mitochondria provides the energy for synthesis of ATP by

respiration linked phosphorylation.

Glycolysis is tightly regulated in coordination with other energy-

yielding pathways to assure a steady supply of ATP. Hexokinase,

PFK-1, and pyruvate kinase are all subject to allosteric regulation

that controls the flow of carbon through the pathway and

maintains constant levels of metabolic intermediates.

The flux of glucose through the glycolytic pathway is regulated

to maintain nearly constant ATP levels (as well as adequate

supplies of glycolytic intermediates that serve biosynthetic

roles).

The required adjustment in the rate of glycolysis is achieved by

a complex interplay among ATP consumption, NADH

regeneration, and allosteric regulation of several glycolytic

enzymes—including hexokinase, PFK-1, and pyruvate kinase—

and by second-to-second fluctuations in the concentration of

key metabolites that reflect the cellular balance between ATP

production and consumption.

On a slightly longer time scale, glycolysis is regulated by the

hormones glucagon, epinephrine, and insulin, and by changes in

the expression of the genes for several glycolytic enzymes.

…MEDICAL CORRELATIONS…

Glucose uptake and glycolysis proceed about ten times faster in

most solid tumors than in noncancerous tissues. Tumor cells

commonly experience hypoxia (limited oxygen supply), because

they initially lack an extensive capillary network to supply the

tumor with oxygen. As a result, cancer cells more than 100 to

200 m from the nearest capillaries depend on anaerobic

glycolysis for much of their ATP production. They take up more

glucose than normal cells, converting it to pyruvate and then to

lactate as they recycle NADH.

The hypoxia-inducible transcription factor (HIF-1) is a protein

that acts at the level of mRNA synthesis to stimulate the

synthesis of at least eight of the glycolytic enzymes. This gives

the tumor cell the capacity to survive anaerobic conditions until

the supply of blood vessels has caught up with tumor growth.

2,3-bisphosphoglycerate binds to hemoglobin, decreasing its

affinity for oxygen and so making oxygen more readily available

to tissues.

Arsenite and mercuric ions react with the --SH groups of lipoic

acid and inhibit pyruvate dehydrogenase, as does a dietary

deficiency of thiamin, allowing pyruvate to accumulate.

Nutritionally deprived alcoholics are thiamin-deficient and may

develop potentially fatal pyruvic and lactic acidosis.

Patients with inherited pyruvate dehydrogenase deficiency,

which can be due to defects in one or more of the components

of the enzyme complex, also present with lactic acidosis,

particularly after a glucose load. Because of its dependence on

glucose as a fuel, brain is a prominent tissue where these

metabolic defects manifest themselves in neurologic

disturbances.

Deficiency of pyruvate kinase causes decreased production of

ATP from glycolysis. Red blood cells have insufficient ATP for

their membrane pumps, and a hemolytic anemia results,

although oxygen delivery to tissues is not necessarily affected.

As phosphoenolpyruvate accumulates, it is converted to 2-

phosphoglycerate, which leads to increased levels of 2,3-

bisphosphoglycerate in the red blood cells. The elevated levels

of 2,3-bisphosphoglycerate promote oxygen release from

hemoglobin in the tissues to an extent that is greater than in the

presence of normal 2,3-bisphosphoglycerate levels.

Inherited aldolase A deficiency and pyruvate kinase deficiency in

erythrocytes cause hemolytic anemia. The exercise capacity of

patients with muscle phosphofructokinase deficiency is low,

particularly on high-carbohydrate diets. By providing an

alternative lipid fuel, e.g., during starvation, when blood free

fatty acids and ketone bodies are increased, work capacity is

improved.

GLUT1 deficiency can have serious consequences. The GLUT1

transporter translocates glucose across the blood–brain barrier.

When one allele is defective, the rate of glucose entry into the

nervous system is insufficient for the cells’ needs, leading to

seizures, developmental delays, and microcephaly. The

treatment consists of a ketogenic diet, one high in fat, in order

to produce ketone bodies as an alternative energy source for

the nervous system.

An increase of lactate levels in the blood causes an acidosis

(lactic acidosis). This condition can result from hypoxia or

alcohol ingestion. Lack of oxygen slows down the electron

transport chain, resulting in increased NADH levels. High NADH

levels cause more than normal amounts of pyruvate to be

converted to lactate. High NADH levels from alcohol metabolism

also cause increased conversion of pyruvate to lactate. Thiamine

deficiency, which is common in alcoholics, decreases pyruvate

dehydrogenase activity, causing pyruvate to accumulate and

form lactate. Thiamine deficiency also slows down the TCA cycle

at the α-ketoglutarate dehydrogenase step. This and other

conditions that slow down the TCA cycle can also produce a

lactic acidosis.

Credit goes to----.

Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme

Usage subject to terms and conditions of license.

All right preserved.

Harper’s Illustrated Biochemistry, Twenty-Sixth Edition

LUBERT STRYER Biochemistry, Fifth Edition

Lehninger principles of biochemistry 4th ED.

BRS Biochemistry, Molecular Biology, and Genetics 6TH ED.

SUMMARIZED BY// ALY BARAKAT