Glycolysis

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GENERAL OVERVIEW

Principle route of metabolism for

Glucose, Fructose, Galactose & other dietary carbohydrates

Enzyme/transporters demonstrate Stereospecificity

for naturally occurring D-isomers

Glycolysis can take place WITHOUT O2

* Red

blood cells (RBCs), which lack mitochondria, are nearly completely reliant on

glycolysis as a source of energy. As such, many of the deficiencies of

glycolytic enzymes have a profound effect on RBC function.

TRANSPORT OF GLUCOSE INTO CELLS

-

NEEDS a

transport protein

-

Insulin

stimulates glucose transport into muscle and adipose cells by causing glucose

transport

proteins (GLUT-4) within cells to

move to the cell membrane.

-

Insulin

does not significantly stimulate the transport of glucose into tissues such as

liver, brain, and RBCs.

* Hereditary

deficiency of GLUT-1, an insulin-independent transporter, results in

decreased glucose in

the cerebrospinal fluid. Patients manifest with intractable seizures in infancy

and developmental delay.

REACTIONS OF GLYCOLYSIS

- Unlike glucose, which can diffuse through the

transporters on the cell membranes, glucose

6-phosphate is obligated to the intracellular compartment.

- Fructose-6-phosphateisphosphorylated by ATP,forming

fructose 1,6-bisphosphate and ADP. 1. Enzyme:phosphofructokinase -1(PFK-1) 2. The

first committed step of glycolysis.

- Fructose

1,6-bisphosphate is cleaved—with the enzyme aldolase—to form the triose

phosphates, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP).

- Absence of the A

isoform of aldolase (found in RBCs and muscle) has been reported. The disorder

presents with a nonspherocytic hemolytic anemia. Patients also have episodes of

rhabdomyolysis (destruction of muscle cells) following febrile illness.

* Note: Two moles of glyceraldehyde

3-phosphateare formed from 1 mole of glucose.

* Patients with

triose phosphate isomerase (TPI) deficiency have neonatal-onset hemolytic

anemia as well as progressive neurologic involvement. Children have progressive

hypotonia with eventual diaphragm paralysis that requires ventilation, as well

as cardiomyopathy.

- Glyceraldehyde

3-phosphate is oxidized by NAD+ and reacts with inorganic phosphate (Pi) to form

1,3-bisphosphoglycerate and NADH + H+.1. Enzyme: glyceraldehyde3-phosphatedehydrogenase

2. The aldehyde group of glyceraldehyde 3-phosphate is oxidized to a

carboxylic acid, which forms a high-energy anhydride with Pi.

-

1,3-Bisphosphoglycerate reacts with ADP—using the enzyme phosphoglycerate

kinase—to produce 3-phosphoglycerate and ATP.

- 2 -

Phosphoglycerateisdehydrated—using the enzyme enolase—to phosphoenolpyruvate

(PEP), which contains a high-energy enol phosphate

* The enzyme enolase

is inhibited by fluoride. To prevent ongoing glycolysis in a patient’s blood

samples collected for sensitive glucose tolerance tests, blood is collected in

tubes containing fluoride.

- Phosphoenolpyruvate reacts with ADP to form pyruvate

and ATP in the last reaction of glycolysis. 1. Enzyme: pyruvatekinase 2. Pyruvatekinase

is more active in the fed state than in the fasting state.

SPECIAL REACTIONS IN

RED BLOOD CELLS

- In RBCs,

1,3-bisphosphoglycerate can be converted to 2,3-bisphosphoglycerate (BPG), a

com- pound that decreases the affinity of hemoglobin for oxygen.

- BPG is

dephosphorylated to form inorganic phosphate and 3-phosphoglycerate, an

intermediate that reenters the glycolytic pathway.

- Fetal hemoglobin

(HbF), composed of two a subunits and two g subunits, has a lower affinity for

BPG than does HbA, and therefore, HbF has a higher affinity for O2. This

difference in maternal and fetal hemoglobin facilitates the unloading of O2 at the

maternal–fetal interface (i.e., the placenta).

REGULATORY ENZYMES OF

GLYCOLYSIS

Hexokinase is found

in most tissues and is geared to provide glucose 6-phosphate for ATP production

even when blood glucose is low.

- Hexokinase has a low

Km (Michaelis

constant) for glucose (about 0.1 mM). Therefore, it is working near its maximum

rate (Vmax),

even at fasting blood glucose levels (about 5 mM).

- Hexokinase is inhibited

by its product, glucose 6-phosphate. Therefore, it is most active when glucose

6-phosphate is being rapidly used.

Glucokinase is found

in the liver and actively functions at a significant rate only after a meal

(when blood glucose is high).

- Glucokinase has a high Km for glucose (about 6 mM). Therefore,

it is very active after a meal, when glucose levels in the hepatic portal vein

are high, and it is inactive during the post- absorptive state or fasting,

when glucose levels are low.

- Glucokinase is induced when insulin levels are

high.

* Maturity-onset

diabetes of the young (MODY) type 2 is an autosomal dominant disorder involving

mutations in the glucokinase (GCK) gene. Patients have nonprogressive

hyperglycemia that is usually asymptomatic at diagnosis and is usually managed

with diet alone.

- PFK-1 is an

allosteric enzyme regulated by several factors. It functions at a rapid rate in

the liver when blood glucose is high or in cells such as muscle when there is a

need for ATP.

* Phosphofructokinase

deficiency (a form of glycogen storage disease [type VII] in which glycogen

accumulates in muscles) results in inefficient use of glucose stores by RBCs

and muscles. Patients experience hemolytic anemia as well as muscle cramping.

- PFK-1 is activated by fructose 2,6-bisphosphate (F-2,6-BP),

an important regulatory mechanism in the liver

- PFK-2 acts as a kinase (in the fed state when it is

dephosphorylated) and as a phosphatase (in the fasting state when it is

phosphorylated). PFK-2 catalyzes two different reactions.

1. PFK-1 is activated

by adenosine monophosphate (AMP), an important regulatory mechanism in muscle

2. In muscle during

exercise AMP levels are high and ATP levels are low

3. Glycolysis is promoted

by a more active PFK-1, and ATP is generated.

4. PFK-1 is inhibited

by ATP and citrate which are important regulatory mechanisms in muscle

5. When ATP is

high the cell does not need ATP, and glycolysis is inhibited.

6. High levels of

citrate indicate that adequate amounts of substrate are entering the

tricarboxylic acid (TCA) cycle. Therefore, glycolysis slows down.

Pyruvate kinase

1.

Pyruvate

kinase is activated by fructose 1,6-bisphosphate and inhibited by alanine and

by phosphorylation in the liver during fasting when glucagon levels are high

2.

Pyruvate

kinase is activated in the fed state. Insulin stimulates phosphatases that

dephosphorylate and activate pyruvate kinase in the liver.

* Deficiency of

pyruvate kinase causes decreased production of ATP from glycolysis. RBCs have

insufficient ATP for their membrane pumps, and a hemolytic anemia results.

THE FATE OF PYRUVATE

Conversion to lactate

- Pyruvate can be

reduced in the cytosol by NADH, forming lactate and regenerating NAD+.

- NADH, which is produced by glycolysis, must be

reconverted to NAD+ so that carbons of glucose can continue to flow through

glycolysis.

- Lactate dehydrogenase (LDH) converts pyruvate to

lactate. LDH consists of four subunits that can be either of the muscle (M) or

the heart (H) type.

- Lactate is released by tissues (e.g., RBCs or exercising

muscle) and is used by the liver for glu- coneogenesis or by tissues such as

the heart and kidney, where it is converted to pyruvate and oxidized for

energy.

* The LDH reaction is reversible.

Conversion to acetyl coenzyme A (CoA)

1. Pyruvate can enter

mitochondria.

2. There it can be

converted by pyruvate dehydrogenase to acetyl CoA, which can enter the TCA

cycle.

Conversion to oxaloacetate (OAA)

1. Pyruvate can be

converted to OAA by pyruvate carboxylase.

- This reaction serves to replenish intermediates of

the TCA cycle as well as provide substrates for gluconeogenesis.

- The enzyme is activated by acetyl CoA.

Conversion to alanine

- Pyruvate can be

transaminated to form the amino acid alanine.

- The enzyme involved

is alanine aminotransferase, which requires pyridoxal phosphate as a cofactor.

Glucose 6-Phosphate

ATP ---> ADP

From glucose

Within a cell, glucose 6-phosphate is produced by phosphorylation of glucose on

the sixth carbon. This is catalyzed by the enzyme hexokinase in most cells,

Glucokinase in certain cells, most notably liver cells

- One molecule of ATP is consumed in this reaction

- Once Phosphorylated its stuck in the cell

- The phosphorylation adds a charged phosphate group so the glucose 6-phosphate

cannot easily cross the cell membrane.

Hexokinase (HK)

Deficiency

- Low 2,3-diphosphoglycerate

of the red cells causes high oxygen affinity of hemoglobin for oxygen. At the

same time it makes unloading of oxygen to tissues very difficult. This leads to

left shift of the

hemoglobin oxygen dissociation curve and tissue anoxia (or tissue

hypoxia)

- Lack of ATP formation due to failure of glycolytic pathways cause hemolytic

anemia

Generalization

Glycolysis deficiency in enzymes = -

All glycolytic enzymes share one aspect in common; their deficiency is

associated with

hemolytic anemia

Hexokinase/Glucokinase

- They differ in Km and Vmax, they are located in

different tissues and they are regulated

Hexokinase

- All other tissues to include Skeletal muscles,

Cardiac muscles

- Km= 0.05mM

- Low Vmax = Low Capacity

- Mutations to the enzyme can cause Hyper or Hypoglycemia

Glucokinase

- Liver

- Beta cells in the Pancreas

- Km= 5mM

- High Vmax = High Capacity

-Mutations to the enzyme can cause Hyper or Hypoglycemia

Phosphofructokinase

- Rate limiting enzyme for glycolysis and is highly

regulated.

Phosphofructokinase Deficiency

ATP----> ADP

- Glycogen storage disease

- The mutation impairs the ability of phosphofructokinase to phosphorylate

fructose-6-phosphate prior to its cleavage into glyceraldehyde-3-phosphate

which enters the energy generation phase of glycolysis, effectively limiting

energy production

- Impairs the ability of cells such as erythrocytes and rhabdomyocytes

(skeletal muscle cells) to use carbohydrates (such as glucose) for energy

Phosphofructokinase Deficiency Diagnosis

A diagnosis can be made through a muscle biopsy that

shows excess glycogen and then further tests that will show the levels of

phosphofructokinase enzyme.

Glyceraldehyde 3-phosphate dehydrogenase

NADH + H+ NAD+ Pi (Inorganic Phosphate)

- It catalyzes an oxidation-reduction reaction, only

oxidation-reduction reaction in glycolysis and produces energy rich NADH+H

- Arsenic metal ion competes with iP for this enzyme and inhibit the energy

yield from this reaction

- The sixth step of glycolysis and thus serves to

break down glucose for energy and carbon molecules

- Converts Glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate

- Releasing NADH + H+, NAD+ & Inorganic Phosphate

Glyceraldehyde 3 phosphate dehydrogenase deficiency

- GAPDH has been described to exhibit higher order

multifunctionality in the context of maintaining cellular iron homeostasis

- GAPDH can also be inhibited by arsenate, inhibiting glycolysis in red blood

cells and causing hemolytic anemia

Phosphorglycerate kinase

ADP à ATP

- It catalyzes the substrate-level phosphorylation

resulting in ATP production at substrate level

Phosphorglycerate kinase deficiency

- Phosphoglycerate kinase deficiency is a genetic

disorder that affects the body's ability to break down the simple sugar glucose

- Most people with phosphoglycerate kinase deficiency have either the hemolytic

form or the myopathic form

- Is caused by mutations in the PGK1 gene

- Mutations in the PGK1 gene reduce the activity of

phosphoglycerate kinase, which disrupts energy production and leads to cell

damage or cell death

Pyruvate kinase

ADP ---> ATP

- It catalyzes the substrate level phosphorylation

resulting in ATP production at substrate level and is one of the regulated

enzyme

Deficiency of this enzyme leads to hereditary hemolytic anemia.

Regulation of pyruvate kinase: It is activated by

fructose 1,6, BP (feed forward activation). It is inhibited by ATP, acetyl CoA,

long chain fatty acids and alanine. Insulin activates this enzyme by keeping it

in dephosphorylated state whereas glucagon keeps this enzyme inactive by

phosphorylating it.

Pyruvate kinase deficiency

- Pyruvate kinase deficiency is an inherited disorder that affects red blood

cells, which carry oxygen to the body's tissues

- People with this disorder have a condition known as chronic hemolytic anemia,

in which red blood cells are broken down (undergo hemolysis) prematurely,

resulting in a shortage of red blood cells (anemia)

- Pyruvate kinase deficiency is a common cause of a type of inherited hemolytic

anemia called hereditary nonspherocytic hemolytic anemia. In hereditary

nonspherocytic hemolytic anemia, the red blood cells do not assume a spherical

shape as they do in some other forms of hemolytic anemia.

- Pyruvate kinase deficiency is caused by mutations in the PKLR gene. The PKLR

gene is active in the liver and in red blood cells, where it provides

instructions for making an enzyme called pyruvate kinase

Glucokinase

Acts like a sensor for the Beta cells of the Pancreas

- Is present in hepatocytes and beta-cells of pancreas

- Present in liver and beta-cells of pancreas and has high Km and high Vmax for

glucose.

- This indicates, the enzyme will be active only at high glucose concentration

(like after food) and is a high capacity enzyme

- It is not feedback inhibited by its end product glucose 6-phosphate, but it

is induced by insulin

Glucokinase mutations

- Mutations cause mild fasting hyperglycemia, the hallmark of GCK-MODY;

homozygous inactivating mutations result in more severe hyperglycemia

presenting as permanent neonatal diabetes mellitus (PNDM)

- Other GCK mutations result in over-secretion of insulin and present with

hyperinsulinemic hypoglycemia

- Leads to a rare form of disease called

maturity-onset diabetes of the young (MODY)

- All due to ineffective insulin production or release by pancreatic beta cells

Phosphofructokinase-1

PFK-1

- Rate limiting enzyme - Control point for Glycolysis

- Is the master regulator of glycolysis and is the most important enzyme for

glycolysis as it determines if the glucose enters into glycolysis or some other

pathway, hence it is called rate limiting enzyme

- PFK-1 is inhibited by ATP and citrate, activated by AMP and fructose

2,6-bis-phosphate

- PFK is allosterically inhibited by ATP, so glycolysis is slowed when cellular

ATP concentrations are high. ATP binds to a site on PFK distinct from the

active site, causing a conformational change resulting in rotation of the

positions of Arg162 and Glu161

Insulin Stimulates and Glucagon Inhibits

ATP --> ADP --> AMP

AMP= Hunger signal for the cell, positively regulate PFK-1 there by increase in

glycolysis and hence the ATP generation

When cellular energy is limited, glycolysis should be unregulated. PFK is

allosterically activated by high levels of AMP. AMP overcomes the inhibitory

effect of ATP.

Aldolase enzyme

Three forms of it A, B & C

- Cleaves six carbon fructose 1,6-bisphosphate into three carbon intermediates

glyceraldehyde 3-phosphate and dihydroxyacetone phosphate

Deficiency of Aldolase B leads to disease called hereditary

fructose intolerance

Aldoses - Present in the liver and kidney

- Unlike fructose malabsorption, there are little or no gastrointestinal

symptoms like bloating and diarrhea.

- This is due to the problem lying with fructose breakdown once it is absorbed

into the body whereas fructose malabsorption is a problem within the gut

- Symptoms of hypoglycemia may also be present in hereditary fructose

intolerance due to the problem with aldolase B impairing gluconeogenesis

Glyceraldehyde 3-phosphate dehydrogenase

- Catalyze oxidation-reduction reaction, produce NADH+H

- NADH+H is oxidized by two major mechanisms to replenish NAD+ required for

glycolysis to continue

-

- Under anaerobic conditions pyruvate is diverted towards

lactate formation, the reaction is catalyzed by lactate dehydrogenase (LDH),

which oxidizes NADH+H produced during glycolysis step (glyceraldehyde

3-phosphate dehydrogenase) to provide NAD+ needed to continue glycolysis.

-

Formation of lactate is the major

fate of pyruvate in lens and cornea of the eye, kidney medulla, testes,

leukocytes and red blood cells (these tissues are poorly vascularized and/or

lack mitochondria)

-

In exercising skeletal muscle, NADH production exceeds the

oxidative capacity of respiratory chain, resulting in elevation of NADH/NAD+

ratio, favoring conversion of lactate from pyruvate

-

During intense exercise, lactate (lactic acid) accumulates

in muscle, causing a drop in intracellular pH, potentially resulting in muscle

cramps

Two major mechanisms for oxidizing NADH

- The NADH linked conversion of pyruvate to lactate (anaerobic glycolysis).

- Oxidation of NADH via respiratory chain (aerobic glycolysis). This requires

NADH+H shuttle mechanism to enter into mitochondria, as mitochondria is

impermeable to NADH+H

Mechanisms of arsenic poisoning

Arsenate can replace inorganic phosphate in the step of glycolysis that

produces 1,3-bisphosphoglycerate from glyceraldehyde 3-phosphate. This yields

1-arseno-3-phosphoglycerate instead, which is unstable and quickly hydrolyzes,

forming the next intermediate in the pathway

- Glycolysis proceeds but not ATP that was suppose to be generated by 1,3

Biphosphoglycerate

- In Arsenic poisoning 8 ATP's are lost

Bisphosphoglycerate mutase in

RBCs synthesize 2,3-BPG

- 2,3-BPG binds beta-chains of hemoglobin A (HbA) and decreases its affinity

for oxygen

- Helping with the unloading of O2 in the body

- 2,3-BPG shifts the oxygen dissociation curve towards right and

increase the release of oxygen from HbA in peripheral tissues, but still allows

100% saturation in the lungs

-One of the changes while adopting to high altitude is increased synthesis of

2,3-BPG to maintain tissue oxygenation.

- RBC's Use 2 ATP's

Diseases related to 2,3-BPG

- Hyperthyroidism = 2,3-BPG content in erythrocytes

by changes in the expression of phosphoglycerate mutase (PGM) and 2,3-BPG

synthase

- Iron deficiency anemia

This illness is characterized by a lack of iron, and

as 2,3-BPG needs this chemical element to be synthesized, BPG concentration

decreases and hemoglobin binds tightly to oxygen. As a result, oxygen release

to tissue is reduced.

- Chronic respiratory disease with hypoxia

Recently, scientists have found similarities between

low amounts of 2,3-BPG with the occurrence of high altitude pulmonary edema at

high altitudes.

Three enzymes that are irreversible in Glycolysis

- Glucokinase/hexokinase

- Phosphofructokinase

- Pyruvate kinase

Enzymes are induced by insulin and repressed by Glucagon

Glucokinase,

phosphofructokinase, pyruvate kinase

Substrate-level phosphorylation

2 of them in Glycolysis

- A type of metabolic reaction that results in the formation of adenosine

triphosphate (ATP) by the direct transfer and donation of a phosphoryl (PO3)

group to adenosine diphosphate (ADP) from a phosphorylated reactive

intermediate

How does Glucagon in hepatocytes stimulate gluconeogenesis?

- Glucagon inhibits this enzyme by phosphorylating it via G protein - cAMP

mediated activation of protein kinase A

- This leads to accumulation of phosphoenolpyruvate which takes a reverse

reaction towards glucose

In RBC's how does Glycolysis occur?

-

No mitochondria

- Anaerobic

2 Pyruvates --> 2 Lactates --> 2 ATP's

- RBC's use up glucose

- RBC's Consume NADH + H+ and their by product is NAD+ which is regenerated for

Glycolysis and used to produce ATP

Fluoride inhibits which enzyme of Glycolysis?

A. Hexokinase

B. Pyruvate kinase

C. Enolase

D. Phosphofructokinase

Fluoride acts primarily by inhibiting enolase in the glycolytic pathway.

Fluoride strongly inhibits the enzyme in the presence of inorganic phosphate.

The inhibitory species is the fluorophosphate ion, which when bound to

magnesium forms a complex with enolase and inactivates the enzyme

- If fluoride does not enter blood cells rapidly, then it cannot rapidly

inhibit the production of lactate, which is produced from pyruvate, the final

product of glycolysis