Ch 14 Glycolysis

What are the four major pathways of glucose utilization?

• Storage

• Glycolysis

• Pentose Phosphate Pathway

• Synthesis of Structural Polysaccharides

When glucose is converted for storage purposes what 3 molecules are the most common product?

• Glycogen

• Starch

• Sucrose

When glucose is converted by glycolysis via oxidation what molecule is the product?

Pyruvate

When glucose is converted by the pentose phosphate pathway via oxidation what is the product?

Ribose 5-phosphate

When glucose is converted for the synthesis of structural polymers what are the products?

• Extracellular matrix

• Cell wall polysaccharides

Explain how glucose can be used for storage

Can be stored in the polymeric form (starch, glycogen) when there’s plenty of excess energy

Explain how glucose is used by glycolysis

Generates energy via oxidation of glucose for short term energy needs

Explain how glucose is used by the pentose phosphate pathway

Generates NADPH via oxidation of glucose used for detoxification and biosynthesis of lipids and nucleotides

Where can structural polysaccharides of glucose be found?

Cells walls of bacteria, fungi, and plants

Under what conditions can glycolysis occur, aerobic and/or anaerobic?

Both aerobic and anaerobic

What living organisms use the glycolysis pathway?

All known living organisms

What are 3 reasons why glucose makes an excellent fuel?

1. Yields a good amount of energy upon oxidation

2. Can be efficiently stored in the polymeric form

3. Many organisms and tissues can meet their energy needs on glucose only

What was one of the first pathways to be identified and understood?

Glycolysis

Why is glucose a particularly versatile biochemical precursor in bacteria?

Bacteria can use glucose to build the carbon skeleton of:

• All the amino acids

• Membrane lipids

• Nucleotides in DNA and RNA

• Cofactors needed for metabolism

Glycolysis is the pathway in which a sequence of enzyme-catalyzed reactions by which glucose is converted into what?

Pyruvate

How can pyruvate be used?

• Pyruvate can be further aerobically oxidized

• Pyruvate can be used as a precursor in biosynthesis

What is are the two possible products pyruvate is converted to under anaerobic conditions?

Ethanol or Lactate

The oxidation-free energy when converting glucose to pyruvate is captured and used by what other reactions?

Synthesis of ATP and NADH

What are the two phases of glycolysis?

1. Preparatory phase

2. Pay off phase

What are the 4 steps of the preparatory phase?

1. Glucose → Glucose 6-phosphate (First priming rxn)

2. Glucose 6-phosphate → Fructose 6-phosphate

3. Fructose 6-phosphate → Fructose 1,6-bisphosphate (Second priming rxn)

4. Fructose 1,6-bisphosphate → Glyceraldehyde 3-phosphate + Dihydroxyacetone phosphate (Cleavage of 6C sugar phosphate to two 3C sugar phosphates)

What can be said about the energy changes in the preparatory phase of glycolysis?

• ATP is invested / consumed

• Free energy content of intermediates is increased

What are the 6 steps of the pay off phase?

5. Dihydroxyacetone phosphate → Glyceraldehyde 3-phosphate

6. Glyceraldehyde 3-phosphate → 1,3-Bisphosphoglycerate (oxidation and phosphorylation)

7. 1,3-Bisphosphoglycerate → 3-Phosphoglycerate (1st ATP forming rxn)

8. 3-Phosphoglycerate → 2-Phosphoglycerate

9. 2-Phosphoglycerate → Phosphoenolpyruvate

10. Phosphoenolpyruvate → Pyruvate (2nd ATP forming rxn)

How much ATP total is generated in glycolysis?

4 ATP

How much ATP does glycolysis use?

2 ATP

What is the net gain of ATP from glycolysis?

2 ATP

What are the three main reactions we were told to remember?

1. Lysis of glucose 6-C chain to 3-C chain

2. Phosphorylation of ADP to ATP

3. Transfer of hydride to NAD+ → NADH

What is the full written equation of reactants and products of glycolysis?

Glucose + 2NAD+ +2ADP + 2Pi → 2 Pyruvate + 2NADH + 2H+ + 2ATP + 2 H2O

What are hexokinases? How many kinds do humans have?

Enzyme that transfer the terminal phosphate of ATP to the substrate which in this case is hexoses. Humans have 4 different genes encoding for different hexokinases (I to IV)

Enzymes that act on a common substrate are called what?

Isozymes

What is the rationale for step 1 phosphorylation of glucose?

• Traps glucose inside the cell by making molecule charged

• Lowers intracellular glucose concentration to allow further uptake of glucose

What enzyme is responsible for step 1 of glycolysis in eukaryotes? And in bacteria?

Hexokinase in eukaryotes

Glucokinase in prokaryotes

How does Mg facilitate the nucleophilic attack of oxygen to C6 of glucose?

Shields the negative charges on ATP

What is step 1 of glycolysis mainly regulated by?

Substrate inhibition

What kind of conversion does step two of glycolysis entail?

Aldose → Ketose

What is the rationale for step 2 of glycolysis?

• C1 of fructose is easier to phosphorylate by phosphofructokinase (PFK) in step 3

• Allows for symmetrical cleave by aldolase in step 4

An aldose → ketose reaction occurs via what type of intermediate?

Enediol intermediate

Isomerization in step 2 of glycolysis is catalyzed by what active site residue using what catalytic method?

A glutamate residue via general acid/base catalysis

What is the first committed reaction? What step? What does that mean?

Step 3, virtually irreversible under cellular conditions, fructose 1,6-bisphosphate is committed to become pyruvate and yield energy

Do other pathways use Glucose-6-phosphate, Fructose-6-phosphates, and Fructose 1,6-bisphosphate?

Glucose-6-phosphate - Yes

Fructose-6-phosphates - Yes

Fructose 1,6-bisphosphate - No, channeled only to glycolysis pathway

What is the rationale for step 3 of glycolysis?

• Further activation of glucose

• Allows for 1 phosphate per 3 carbon sugar after step 4

What steps of glycolysis use the energy of ATP?

1 and 3

How is phosphofructokinase-1 regulated?

Regulated by ATP, fructose-2,6-bisphosphate, and other metabolites. If [ATP] is high_, glucose is not burned

What is the reverse reaction type of step 4?

Aldol condensation

What is the rationale of step 4 of glycolysis?

Cleavage of a six-carbon sugar into two three-carbon high energy phosphate sugars

In step 4 concentration of which product is kept low to drive the reaction forward?

Glyceraldehyde 3-phosphate (GAP)

What is the rationale for step 5 of glycolysis?

• Allows glycolysis to proceed to payoff phase by a single chemical pathway

What is the purpose of step 5? And what enzyme is used?

Convert all DHAP to GAP by triose phosphate isomerase

What is the key different between the 2 sugars produced in step 4?

1 Aldose and 1 Ketose

What type of enzyme is used to convert an aldose to a ketose or vice versa?

Isomerase

In triose phosphate isomerase conversion of DHAP (ketose) to GAP (anldose) occurs via what mechanism? What residues are involved?

1. DHAP acted upon by Glu as a base forming C=C and His as a acid donating to the C2 ketone

2. Enediol intermediate forms and deprotonated His acts as base on OH of C1

3. Tautomerization collapses into GAP

What is the result of the preparatory phase?

Glucose is converted to 2 molecules of Glyceraldehyde, used 2 ATP molecules and generated NO ATP

How many ATP molecules are invested prior to the pay off phase?

2

What powers formation of a compound having high phosphoryl-transfer potential, Delta Gp?

Oxidation of an aldehyde

What 2 types of reactions occur in step 6?

Oxidation and phosphorylation

What molecule is the result of the first step of the payoff phase? Describe its energy level and why thats relevant

1,3-Bisphosphoglycerate (BPG). First molecule in glycolysis with an energy higher than ATP which is capable of substrate level phosphorylation forming ATP as a result.

Is ATP or another NTP always required for phosphorylation?

No, 3 molecules with higher energy levels than NTPs

What is the rationale for step 6 of glycolysis?

• Generation of high-energy phosphate compound

• Incorporates inorganic phosphate

• Allows production of ATP

What is the first energy yielding step of glycolysis? Why type of energy is yielded?

Step 6 via oxidation energy

In step 6 is NAD oxidized or reduced?

Reduced NADH

What enzyme is used in step 6? Which residue plays a key role by forming what type of intermediate? How is this enzyme regulated?

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). An active site Cys residue forms a high-energy thioester intermediate. The Cys is subject to inactivation by oxidative stress which slows glyolysis.

What two reactions are coupled to allow step 6 to occur? Why is this necessary?

1. Oxidation of an aldehyde to carboxylic acid by NAD+

2. Acyl-phosphate formation

Needed to make phosphorylation of GAP energetically favorable

How many molecules of NAD+ are used in step 6?

2

What are the substeps of step 6?

1. Substrate binding to GAPDH

2. Covalent Catalysis w/ Cys residue

3. Oxidation by NAD+

4. Replacement of NADH w/ 2nd NAD+

5. Incorporation of Pi by breakage of thioester

6. Product released from GAPDH as 1,3-BPG

What is the rationale for step 7 of glycolysis?

Substrate-level phosphorylation make ATP

Is 1,3-bisphosphoglycerate a high energy compound? Is it capable of converting ADP to ATP?

Yes and yes

What are kinases?

Enzymes that transfer phosphate groups between ATP and various substrates

Why is Step 7 of glycolysis reversible?

Reversible because of coupling to GAPDH reaction (step 6)

What enzyme is used in step 7 of glycolysis?

Phosphoglycerate kinase

What are the three outcomes of steps 6 & 7 of glycolysis?

1. G-3P (an aldehyde) → 3-phosphoglycerate (a carboxylic acid)

2. NAD+ reduced to NADH

3. ATP is formed from Pi and ADP due to carbon-oxidation energy

What is the rationale for step 8 of glycolysis?

Be able to form high energy phosphate compound

What are mutases?

Enzymes that catalyze the (apparent) migration of functional groups

What enzyme is used in step 8? What active site residue is postranslationally modified to what?

Phosphoglycerate mutase. His residue is modified to phosphohistidine.

What is the mechanism of step 8?

Phosphoryl transfer from phosphohistidine. Follow by second His acting as general base on C-2 (OH). Phosphoryl transfer from C-3 to first His. Second His acts as a general acid catalyst

What is the rationale for step 9 of glycolysis?

Generate a high-energy phosphate compound. 2-Phosphoglycerate is not a good enough phosphate donor because 2 negative charges in PG are fairly close but loss of phosphate from 2-PG would give a secondary alcohol with no futher stabilization.

For steps 8 & 9 is reactant concentration kept low or high to drive the reaction forward?

High (thus product concentration is kept low)

What enzyme is used in step 9 of glycolysis?

Enolase

What is the rationale for step 10 of glycolysis?

• Substrate level phosphorylation to make ATP

• Net production of 2 ATP per glucose

• Loss of phosphate from PEP yields an enol that tautomerizes into a ketone

How does tautomerization drive the reaction 10 towards ATP formation?

Effectively lowers the concentration of the reaction product

What enzyme is used in step 10 of glycolysis? What is required for this enzymes activity?

Pyruvate kinases requires divalent metals (Mg++ or Mn++) for activity

How is step 10 regulated?

Regulated by ATP, divalent metals, and other metabolites

In the summary of glycolysis what was used and what was made?

Used:

• 1 Glucose

• 2 ATP

• 2 NAD+

Made:

• 2 pyruvate

• 4 ATP

• 2 NADH

What must happen to NADH in order for glycolysis to continue?

NADH must be reoxidized to NAD+

What does regulation of glycolysis ensure? (2 things)

1. Ensure proper use of nutrients

2. Ensure production of ATP only when needed

What are the 2 possible fates of pyruvate in anaerobic pathways? Through process are these products produced? What is the purpose of these products?

2 Pyruvate is converted to 2 Lactate or 2 Ethanol and CO2 which are used to regenerate NAD+ so glycolysis can continue

Does fermentation produce a net change in the oxidation state of the sugars?

No

What is the oxidation state of a molecule?

The H:C ratio

What enzymes is used to convert pyruvate to L-Lactate?

Lactate dehydrogenase

Is the reduction of pyruvate to lactate a reversible rxn?

Yes

Lactate builds up where in the body due to what for how long? How does this affect the body?

Builds up in the muscle due to strenuous exercise for generally less than 1 minute. The acidification of muscle prevents its continuous strenuous work. Lactate can be transported to the liver and converted there to glucose. Requires a recovery time.

Do animals undergo lactic acid fermentation?

Yes

What enzyme(s) is used in the conversion of pyruvate to ethanol? And what if any cofactors?

1. Pyruvate decarboxylase (Mg2+ & TPP)

2. Alcohol dehydrogenase (Zn++ & NADH)

What type of microorganism undergoes ethanol fermentation?

Yeast

Is the reduction of pyruvate to ethanol a reversible reaction?

No

Do humans have pyruvate decarboxylase?

No