Module 12: Glycolysis

Glycolysis

• occurs in the cytosol

• extract energy oxidizing fuel molecules

• Generate ATP

• may generate reduced electron carriers and carbon skeletons that can be used in biosynthesis

• converge on a few intermediates

• multiple enzyme-catalyzed steps

• regulated

• primarily uses a few types of reactions

• happens in all cell types

• only source of metabolic energy for brain, kidney medulla, and rapidly contracting skeletal muscles, erythrocytes, sperm cells

How does glucose enter the cell?

Through glucose transporter protein (GLUT) transporter

How does glucose exit the cell?

As pyruvate either aerobically to the TCA Cycle or anaerobically as lactate

Stages of glycolysis: Reactions 1-5

Energy investment: high energy of ATP phosphoryl transfer used to generate low energy phosphoryl transfer compounds (only endergonic reactions!)

Stages of glycolysis: Reactions 6-10

Energy recovery: oxidation, coupled to phosphorylation using Phosphate and rearrangements convert these to high energy phosphoryl transfer compounds to make ATP (exergonic)

Step 1: Reactants to Products

Glucose and ATP —> Glucose-6-phosphate and ADP

- traps glucose because once it transforms, it is no longer glucose

Step 1: Enzyme

• Uses hexokinase enzyme to phosphorylate glucose (activate glucose)

  • hexokinase is nonspecific and can phosphorylate several types of sugars

• Mg2+ is a co-substrate (a type of coenzyme)

  • shields negative charges of phosphate group

  • large free energy change makes reaction irreversible

What is an inhibitor of hexokinase?

Glucose-6-phosphate

Step 1: Mechanism

• A phosphate group is transferred from an ATP to a glucose (coupled reaction because of ATP hydrolysis)

• Hydroxyl group on Carbon 6 performs a nucleophilic attack on phosphate in ATP to make it ADP

What kind of reaction is step 1?

• Priming reaction through phosphorylation 

• Also a regulating step 

Step 2: Reactant to product

Glucose 6-phosphate ←→ fructose-6-phosphate

• 6-carbon sugar becomes a 5-carbon sugar

Step 2: Enzyme

Uses phosphoglucose isomerase enzyme

Step 2: Mechanism 

- Involves general acid-base catalysis where an enzymatic acid, probably of a Lys residue, catalyzes ring opening

- Substrate binding

—> Acid-catalyzed ring opening: bond between O-C attacks an H+ atom on an acid, breaking the bond

—> base catalysis: base attacks the H+ on ‘2 carbon, double bond forms between carbon 1 and 2 from O-H bond

—> acid catalysis: double bond attacks another H+ from acid

—> base catalyzed ring closure: O- attacks ‘2 carbon to close the ring

—> product release

Step 3: Reactant to Product

Fructose 6-phosphate and ATP —> Fructose 1-6-bisphosphate

• A phosphate group is transferred from an ATP to a fructose 6-phosphate

Step 3: Enzyme

Uses PFK-1 (phosphofructokinase-1) enzyme

• when ATP is high, PFK is inhibited

• when ADP and AMP levels are high, PFK is stimulated

Step 3: Mechanism 

• A phosphate group is transferred from an ATP to a Fructose-6-phosphate (coupled reaction because of ATP hydrolysis)

• Hydroxyl group on Carbon 6 performs a nucleophilic attack on phosphate in ATP to make it ADP

What makes step 3 important?

• The committed step, 1-6 bisphosphate can only go through glycolysis

  • fructose 1-6-bisphosphate is an activator for Pyruvate kinase (step 10)

• rate-limiting step in glycolysis

• highly regulated step

  • several inhibitors and activators

Step 4: Reactant to product

Fructose-1,6-bisphosphate —> Dihydroxyacetone Phosphate and Glyceraldehyde-3-phosphate

6-carbon sugar is split into two 3-carbon sugars

Catalyzed by aldolase to cleave FBP to form two trioses

Step 4: Enzyme

catalyzed by aldolase

Step 4: Mechanism

substrate binding through Lys (nucleophilic attack)

—> protonated Schiff base formation through Asp (covalent catalysis)

—> aldol cleavage (base catalysis)

—> tautomerization and protonation (acid catalysis)

—> Schiff base hydrolysis where substrate is released

Step 5: Reactants to products 

The trioses formed from the last step (GAP and DHAP) are interconverted by an enediol intermediate and become ketoses, both Glyceraldehyde-3-phosphate

Step 5: Enzyme

triose phosphate isomerase

Step 6: Reactants to products

2 Glyceraldehyde 3-Phosphate + P_i <--> 1,3-biphosphoglycerate.

NAD+ <--> NADH

-The only redox reaction, carbonyl is being oxidized

-the first high energy intermediate

Step 6: Enzyme

Uses G3P dehydrogenase enzyme

NAD+ is a cofactor

Step 6: Mechanism

Substrate binding (nucleophilic attack)

—> active site thiol addition (covalent catalysis)

—> dehydrogenation (oxidation)

—> phosphate binding (nucleophilic attack)

—> product release and NADH/NAD+ exchange

What kind of reaction is step 6?

Oxidative

What is an inhibitor for step 6?

NADH

Step 7: Reactants to Products

1,3-bisphosphoglycerate + ADP —> 3-phosphoglycerate + ATP

Step 7: enzyme 

Uses phosphoglycerate kinase enzyme

What is step 7 classified as?

Pay-off step because we gain 2 ATP

Step 8 Reactant to product

3-phosphoglycerate ←→ 2-phosphoglycerate

Step 8: Enzyme

phosphoglycerate mutase

• Mutase: an enzyme that catalyzes the transfer of a functional group from one position to another

Step 9: Reactant to Product

2-phosphoglycerate —> 2 phosphoenolpyruvates

Step 9: Enzyme

enolase

Step 9: Product

2 phosphoenolpyruvate (PEP), a phosphorylated molecule with higher transfer potential than ATP

Step 10: Reactants to products

2 PEP + 2 ADP --> 2 Pyruvate + 2 ATP

Step 10: Enzyme 

Uses pyruvate kinase enzyme

Step 10 Inhibitors and Activators

ATP is an inhibitor

activation by FBP and PEP

Net production of Glycolysis

2 ATP

2 NADH

2 Pyruvate

Why does glycolysis occur in steps?

Not all energy is dissipated as heat, rather it is stored in carrier molecules ATP and NADH. Energy can then be extracted in usable amounts

Fate of Pyruvate with oxygen (aerobic)

it becomes Acetyl CoA

it goes through the TCA cycle in the mitochondria

it becomes CO2 and H2O

Fate of pyruvate without oxygen: Alcoholic Fermentation

• process to become ethanol and CO2

• enzymes: pyruvate decarboxylase and alcohol dehydrogenase

Homolactic fermentation

• it is converted to lactate by its gain of electrons form NADH (reduced to NAD+) 

2 Pyruvate + 2 NADH —> 2 lactate + 2NAD+

• lactate dehydrogenase

• redox reaction

• used to regenerate NAD+

  • Low NAD+ stops glycolysis 

Regulation steps in Glycolysis

Very negative delta G

• 1st step —> hexokinase

• 3rd step —> Phosphofructokinase 

• 10th step —> pyruvate kinase

PFK

Substrates = fructose-6-phosphate and ATP can only bind to substrate site

Allosteric activators = AMP, ADP, Fructose-2-6-biphosphate

• can only bind to regulatory site

Allosteric inhibitors = ATP, Citrate, PEP

• can only bind to regulatory site

PFK Allostery

ATP stabilizes T state

F6P and ADP stabilizes R state

• Glutamate flips to the R state and becomes positive

• This attracts F6P because it is negative 

Substrate Cycle

Fructose 1-6-bisphosphate —> Fructose-6-phosphate

Fructose 1-6-BisPhosphatase is the enzyme

• phosphatases catalyze the removal of a phosphate group

Gluconeogenesis

formation of glucose from noncarbohydrate sources in the liver
- maintains low glucose levels

Obesity

cause by the failure to maintain the input and output of energy, mainly from fructose, also from substrate cycling

Fructose

- can be converted to glycogen as well
- liver absorbs almost all the fructose in our diet so our muscles rarely receive it
- appears to have a greater tendency to be metabolized into triglycerides for energy storage relative to glucose

Fructose skips the committed step and goes straight to the 5th step as glyeraldehyde-3-phosphate through glucokinase