Lecture Three

What is the pentose phosphate pathway (PPP)

• Glucose enters glycolysis and is converted into glucose-6-phosphate (G6P)

• G6P doesn’t HAVE to continue through glycolysis and can be redirected into the PPP which is also known as the hexose monophosphate shunt

• The cell does this because sometimes it does not primarily need ATP, it may need NADPH and Ribose-5-phosphate

• Instead of breaking down glucose to make ATP, the cell redirects G6P to produce NADPH and R5P

Why would the cell need NADPH and Ribose-5-phosphate

• NADPH: provides reducing power for biosynthesis and antioxidant defense 

• Ribose-5-phosphate (R5P): Provides the sugar backbone for nucleotides which are needed for DNA and RNA synthesis

Two phases of PPP: 

1. Oxidative Phase:

   1. Produces NADPH 

   2. Produces CO₂

   3. Converts G6P → Ribulose-5-P

   4. Irreversible overall 

   5. Supports antioxidant defense and biosynthesis 

2. Nonoxidative Phase: 

   1. Rearranges carbon skeletons

   2. Does not directly produce NADPH 

   3. Interconverts 3-, 4-, 5-, 6-, and 7- carbon sugars

   4. Reversible reaction 

   5. Supplies R5P or returns carbons to glycolysis

Oxidative Phase

Making NADPH, first phase

• Glucose-6-phosphate (6C) 

  • NADP+ → NADPH 

• 6-phosphogluconate (6C) 

  • NADP+ → NADPH 

  • CO2 Released

• Ribulose-5-phosphate (5C)

Produces 2 NADPH, 1 CO2, 1 ribulose 5-phosphate per G6P 

Why is NADPH so important

• NADPH provides electrons for reductive biosynthesis and antioxidant agents

• It is needed to help synthesize fatty acids and sterols

Glutathione

Cells need NADPH to protect themselves

Problem: During processes like mitochondrial respiration, cells can generate reactive oxygen species (ROS)

Ex) Superoxide radicals (O₂.-) which is very reactive and can damage lipids in cell membranes, proteins, and DNA → potentially causing mutation

• the cell will need to neutralize them

How does the cell neutralize reactive oxygen species

• superoxide radical → Hydrogen peroxide (H2O2) → Water (2H2O)

• Problem: glutathione gets oxidized while helping neutralize hydrogen peroxide, so the cells need a way to regenerate the reduced glutathione which is where NADPH comes in

Glutathione Cycle

1. Glutathione Peroxidase: uses reduced glutathione (GSH) to neutralize hydrogen peroxide

   1. H2O2 + 2GSH → 2H2O + GSSG

   2. Hydrogen peroxide becomes water and reduced GSH becomes oxidized (GSSG)

2. Glutathione Reductase: Regenerates reduced glutathione using NADPH

   1. GSSG + NADPH + H+ → 2GSH + NADP+

      1. NADPH is oxidized to NADP+

      2. GSSG gets reduced to 2GSH

      3. The cell can now reuse GSH to neutralize more hydrogen peroxide

How does the glutathione cycle connect to the PPP

• Oxidative phase of PPP produces NADPH

• NADPH helps regenerate GSH

• Regenerated GSH can help neutralize more H2O2 and protect against oxidative damage

What if the cell cannot make enough NADPH

• G6P dehydrogenase catalyzes the first NADPH producig reaction of the PPP, if G6PD activity were reduced:

  • less NADPH produced

  • Glutathione reductase has less NADPH available

  • less oxidized glutathione can be regenerated into GSH

  • cells become more vulnerable to oxidative damage

What is the purpose of nonoxidative phase

Imagine the cell has already made the NADPH it needs but now it has a lot of the 5C sugars from the PPP but it doesn't need to make many nucleotides. Rather than wasting energy and resources, it can rearrange their carbons into molecules that will reenter glycolysis

How does carbon shuffling work

• Transketolase: Transfers a 2 carbon unit from one sugar to another 

• Transaldolase: Transfers a 3 carbon unit from one sugar to another 

• The total number of carbons is conserved!

Transketolase: 5C + 5C ⇌ 3C + 7C

Transaldolase: 7C + 3C ⇌ 4C + 6C 

Transketolase: 5C + 4C ⇌ 3 C + 6C

Overall: three 5C sugars can be rearranged into 2 6C sugars and one 3C sugar 

Where do the products of carbon shuffling go

• The 6C sugar is fructose-6-phosphate, which can be converted into G6P

• The 3C sugar is glyceraldehyde-3-phosphate, which can enter glycolysis or be converted through additional reactions to help regenerate hexose phosphates

Why does the cell recycle carbons back into G6P

Allows the cell to keep generating NADPH without accumulating unnecessary ribose-5-phosphate

Regulation of PPP by NADPH

• Glucose-6-phosphate dehydrogenase catalyzes the first reaction of the oxidative PPP

• NADPH negatively regulated G6PD

• High NADPH → G6PD inhibited → oxidative PPP flux decreases

• High NADPH/increased NADP → oxidative PPP flux increases

How can nonoxidative PPP products be recycles towards G6P

• Transketolase and transaldolase rearrange 5C sugars into F6P and G3P

• F6P can be converted into G6P via phosphoglucose isomerase

• G3P can enter additional reactions to help regenerate hexose phosphates

• This allows carbons to be reused when the cell needs more NADPH than ribose-5-phosphate

G6PD deficiency and hemolytic anemia

A mutation in G6PD can cause hemolytic anemia, where red blood cells burst and are destroyed. 

This happens because G6PD is responsible for the first NADPH producing reaction of oxidative PPP. RBCs are dependent on the PPP because they don't have mitochondria, and rely on PPP-generated NADPH to maintain a reduced glutathione. 

• Less G6PD can cause less NADPH to regenerate GSH, making the cell less capable of neutralizing reactive oxygen species. ROS will damage red blood cell proteins and membranes causing hemolytic anemia. 

Carbon Shuffling in non-oxidative PPP

1. Ribulose-5-phosphate can become two different sugars via two reactions: 

   1. Reaction A: Isomerization → Ribulose-5-P → Ribose-5-P

      1. Ribulose 5-phosphate isomerase converts a ketose into an aldose 

   2. Reaction B: Epimerization → Ribulose-5-P → Xu5P via Ribulose 5-phosphate 3-epimerase

      1. Stays as a ketose but the configuration around carbon three changes 

• This is important since the cells need different types of sugars to perform carbon shuffling 

2. Transketolase: The first carbon-shuffling reaction 

   1. Xylulose 5-phosphate + Ribose-5-phosphate ⇌ G3P + S7P (7C) 

      1. Xu5P is a ketose donor and gives away one 2-carbon fragments

      2. Its remaining 3C become G3P

      3. Ribose-5-phosphate is the aldose acceptor, receiving the 2C fragment, and becoming 7C S7P

   2. Utilizes TPP cofactor

3. 3. Transaldolase: second carbon-shuffling reaction 

   1. S7P + G3P ⇌ Fructose-6-phosphate + Erythrose-4-phosphate 

      1. S7P (ketose so loses) donated 3C, leaving E4P 

      2. G3P (aldose so gains) accepts 3C forming F6P

   2. Doesn’t require TPP. Instead, an active-site forms covalent intermediates to stabilize during the carbon transfer

4. Transketolase: Third carbon-shuffling reaction 

   1. Xu5P + E4P ⇌ G3P + F6P

TPP Role

• Transketolase requires the thiamine pyrophosphate (TPP) 

• It helps the enzyme transfer a two-carbon unit between sugars

How does the transketolase reaction work

• R5P + Xu5P ⇌ S7P + G3P 

1. TPP performs a nucleophilic attack on the carbonyl group of the Xu5P, allowing the TPP to interact with the ketose sugar and participate in the transfer of the two carbon unit 

2. Xu5P loses two carbons which becomes G3P, this is released from the active site 

   1. 2C stay attached to the TPP 

3. Ribose-5-phosphate enters the active site and gains the two carbons forming S7P

• This reaction is reversible, and it can catalyze this reaction in both directions

Formation of S7P

• The Ribose-5-phosphates aldehyde carbonyl group turns into an alcohol group 

• This is because the histidine acts as a proton donor, protonated this OH

ATCase enzyme in nucleotide synthesis

1. Make the ring: ATCase participates in the process of building the pyrimidine ring 

2. Add the sugar component: the sugar component comes from the ribose-5-phosphate produced through the PPP 

°PPP provides the sugar component needed for nucleotide synthesis, while ATCase is involved in making the pyrimidine ring

Impact of electrons leaking out of the ETC

When an electron reacts with an Oxygen, it forms a superoxide which is an ROS and can damage proteins, membrane lipids, and DNA

Allergic reactions and G6PD

1. Someone has reduced G6PD activity 

2. Their red blood cells produce less NADPH through the PPP 

3. They have a reduced ability to regenerate GSH 

4. Certain antibiotics can cause oxidative stress 

5. Their red blood cells become damaged and undergo hemolysis 

This is an adverse reaction to taking an antibiotic and assume they are allergic but it could be a result of oxidative damage due to G6PD deficiency

First reaction of oxidative PPP

Standard step: G6P → 6-phosphoglucono-δ-lactone, and creating NADH + H+ from NADP+

PPP and the Glutathione cycle relationship

related but not the same

• Oxidative PPP generated NADPH

• Glutathione reductase uses the NADPH to generate GSH

• The glutathione peroxidase will use GSH to neutralize the H2O2

So the PPPs role is to supply the NADPH, and the glutathione cycle just uses that NADPH to maintain antioxidant defense.

Why is NADPH the best regulator

Why is NADPH the best regulator: 

• High NADPH → G6PD inhibition 

• NADPH/NADP+ balance reflects the cell’s need for reducing power 

• The PPP produces multiple products, including NADPH and ribose-5-phosphate, which serve different cellular needs.

• Regulating the PPP solely through sugar-product accumulation would not accurately reflect NADPH demand.

  • Example: The cell may need high NADPH but little ribose-5-phosphate.