PPP & Free Radical Biochem

2 phases of PPP

1. oxidative phase

2. non-oxidative phase

during oxidative phase, 2 molc of ________ are produced

NADPH

in non-ox phase of PPP, pentose phosphate can be converted back to _________

glucose-6-phosphate

purposes of PPP

1. produce pentose phosphates from glucose-6-phosphate

2. generate NADPH

pentose phosphates are critical for …

cell replication (because they are used to make the nucleotides that are the building blocks of DNA)

in what cells is PPP highly active?

cells that divide rapidly, such as bone marrow, skin and intestinal epithelium

NADPH serves as an electron donor for ___________ pathways

synthetic

• such as FA and synthesis steroids

NADPH is req for reduction of ROS by _________

glutathione

reductive detoxification

process by which glutathione reduces oxygen radicals

glutathione reduced form

GSH

glutathione

peptide made of GLU, CYS, and GLY

• under normal conditions, most exists in reduced state (GSH)

when needed, GSH can donate ________ to specific molc/substances

single electron

what happens when 2 GSH molc are oxidized (donate an electron)?

disulfide bond forms bw them to form glutathione disulfide (GSSG)

glutathione peroxidase

decreases amt of H2O2 in cell & decreases formation of hydroxyl radicals

glutathione reductase

GSSG is reduced back to 2 GSH

• always active, keeps cellular conc of GSH high

glucose-6-phosphate dehydrogenase

responsible for regeneration of NADPH from NADP+

why must NADPH be regenerated from NADP+ to maintain activity of ____________

glutathione reductase

2 substrates of glucose-6-phosphate dehydrogenase

1. glucose-6-phosphate

2. NADP+

products of glucose-6-phosphate dehydrogenase

1. 6-phosphoglucono-δ-lactone

2. NADPH

3. H+

enzyme that catalyzes first step of PPP

glucose-6-phosphate dehydrogenase

favism

disease characterized by a deficiency in glucose-6-phosphate dehydrogenase activity

o This disease is an X-linked recessive disease, - all boys born with a single copy of the gene will have the disease, but all girls must have both copies of the gene to have the disease.

o Most people with this disease are normally asymptomatic but can develop symptoms if they take certain drugs, ingest certain foods, or become ill.

o The disease will result in hemolytic anemia due to the inability of the RBCs to reduce (detoxify) reactive oxygen species

fava beans exacerbate effects of ____________ deficiency

glucose-6-phosphate dehydrogenase

effect of glucose-6-phosphate dehydrogenase deficiency

cell can’t reduce NADP+ to NADPH

without NADPH, glutathione reductase is incapable of reducing ______ to _________

GSSG / glutathione

without GSH, glutathione peroxidase can’t act on _________ to detoxify it

hydrogen peroxide

what happens when hydrogen peroxide builds up within the cell?

can be converted to more potent reactive oxygen species → damage critical cell structures such as cytosolic proteins & membrane lipids

effect of ROS buildup in RBCs

accumulation of Heinz bodies, which are aggregates of cross-linked hemoglobin that form on the cell membrane and subject the cell to mechanical stress as it weaves through small capillaries.

lipid peroxides

lipid radicals, make membrane less flexible

effect of Heinz body imposed stressed + increased ROS in RBC

hemolysis & ultimately hemolytic anemia

first step of PPP is catalyzed by _____________

glucose-6-phosphate dehydrogenase

2 substrates of glucose-6-phosphate dehydrogenase

1. glucose-6-phosphate

2. NADP+

products of glucose-6-phosphate dehydrogenase

1. 6-phosphoglucono-lactone

2. NADPH

substrates of gluconolactonase

1. 6-phosphoglucono-lactone

2. H2O

product of gluconolactonase

6-phosphogluconate

substrates of 6-phosphogluconate dehydrogenase

1. 6-phosphogluconate

2. NADP+

products of 6-phosphogluconate dehydrogenase

1. ribulos-5-phosphate

2. NADPH

3. CO2

substrate of phosphopentose isomerase

ribulose-5-phosphate

product of phosphopentose isomerase

ribose-5-phosphate

general goal of non-ox phase of PPP

regenerate glucose-6-phosphate

2 enzymes in non-ox phase of PPP

1. transketolase

2. transaldolase

transketolase

cat rxn that transfers 2C from 1 sugar to another

transaldolase

cat rxn that transfers 3C from 1 ugar to another

overall outcome of non-ox phase of PPP

6 five carbon sugars can be converted to 5 six carbon sugars

key reg enzyme of PPP

glucose-6-phosphate dehydrogenase

major regulator of PPP

NADPH

• strong inhib of glucose-6-phosphate

insulin regulation of glucose-6-phosphate DH

induce transcription of enzyme

free radicals tend to have a ___________ life span due to their reactivity

very short

why are free radicals so chemically reactive?

unpaired electron in outer shell

list 3 fairly stable free radicals

1. molecular oxygen

2. NO

3. Vitamin E

molecular oxygen

contains two unpaired electrons in the outer shell of each oxygen atom.

• These two electrons participate in the covalent double bond that links the two

oxygen atoms together, thus stabilizing the overall molecule.

NO

contains a single electron in the outer shell of the nitrogen atom.

• Nitric oxide is an important bioactive molecule but is known to have an extremely short half-life.

Vitamin E

exogenous antioxidant molecule, which has the ability to accept single electrons.

when are free radicals formed?

both normal & pathological processes

is hydrogen peroxide a free radical?

NO

• but it is a reactive oxygen species

process of molecular oxygen → ROS

o The first electron accepted by molecular oxygen converts the oxygen to superoxide (O2-); superoxide is a free radical.

o The next electron accepted converts superoxide to hydrogen peroxide (H2 O2 ).

o The third electron will convert hydrogen peroxide to the hydroxyl radical (•OH), generating a single water molecule in the process.

o The final electron added will convert the hydroxyl radical to a second molecule of water

role of free radicals in inflammatory response

free radicals are deliberately produced to assist with attacking and killing invading or necrotic cells.

reactive oxygen species

reactive free radicals that extract electrons (with hydrogen ions) from neighboring compounds in order to become more stable (more reduced).

o ROS initiate a chain reaction of free radical generation.

o hydroxyl radical is the most potent, because it is capable of initiating reactions that form lipid peroxides and organic radicals.

most potent ROS

hydroxyl radical

• capable of initiating reactions that form lipid peroxides and organic radicals

superoxide radical is highly ________ but has limited _________ so it is unable to…

reactive / solubility / diffuse far from point of gen

why is hydrogen peroxide an ROS?

can generate a hydroxyl radical

• thru combination w superoxide anion in Haber-Weiss rxn

• Fenton rxn

fenton rxn

transition metals such as iron (Fe2+ ) and copper (Cu2+ ) catalyze the formation of the hydroxyl radical from hydrogen peroxide in a non- enzymatic reaction

can hydrogen peroxide diffuse thru memb?

yes- lipid soluble

what happens when hydrogen peroxide diffuses thru memb?

generates hydroxyl radicals at localized Fe2+ and Cu 2+ -containing sites, such as at the electron transport chain

how do you minimize Fenton rxn from occurring?

restrict location of transition metals in the cell

nitric oxide

oxygen-containing free radical req for life but also very toxic

• functions as a NT and hormone

• stimulates vasodilation in vascular endothelium

at high concentrations, nitric oxide combines with molecular oxygen or superoxide to form …

other reactive and toxic species called reactive nitrogen oxygen species (RNOS)

nitric oxide stimulates ________

vasodilation

• in vascular endothelium

3 isozymes of NO

1. neuronal NOS (nOS)

2. inducible NOS (iNOS)

3. endothelial NOS (eNOS)

enzyme that synthesizes NO

nitric oxide synthase

substrates of NO synthase

1. Arginine

2. NADPH

3. H+

4. O2

products of NO synthase

1. citrulline

2. NO

3. NADP+

can NO diffuse thru cell memb?

yes- Once inside the cell, NO binds a specialized receptor that activates a signal transduction cascade

direct toxic effects of NO

involve NO binding to iron-containing proteins (not its receptor)

NO binds ____-containing compounds that have single e- available

iron

major destructive sites of NO

• Complexes I-III of the electron transport chain (ETC)

• hemoglobin

• ETC cytochromes (cytochrome b-c1 and cytochrome Q).

normally, NO is kept low and there is very little damage. under what conditions can low levels of NO cause serious damage?

if ATP production is impaired due to ox phos diseases or lack of oxygen

how is peroxynitrite (ONOO-) formed?

NO combines nonenzymatically w superoxide

is peroxynitrite a free rad?

no— strong oxidizing agent that is STABLE & DIRECTLY TOXIC

can peroxynitrite diffuse thru cell memb?

yes— interacts w a wide range of cellular targets

peroxynitrite can be broken down to _______

nitrogen dioxide NO2

peroxynitrite can be broken down into NO2, which can initiate ___________

lipid peroxidation

peroxynitrite products can nitrate ___________ in proteins and DNA, forming ________

aromatic rings / nitrotyrosines and nitroguanosines

damage caused by peroxynitrite

• enzyme inhibition

• mitochondrial lipid peroxidation

• inhibition of ETC → energy deprivation

• single/double-stranded DNA breaks

• DNA base mod

major routes of ROS gen in cell

1. NADPH oxidase

2. mitochondrial activity (ETC)

3. cytochrome P450 enzyme activity

exogenous mech of ROS gen

• Some drugs (such as chemotherapeutic agents)

• radiation

• some environmental pollutants can also provide enough energy to break water molecules into the hydroxyl radical and a hydrogen atom.

phagocytic cell resp to infection/other stimuli

rapidly consume oxygen in what is known as the respiratory burst

respiratory burst is a major source of …

• superoxide

• H2O2

• hydroxyl radical

• hydrochlorous acid (HOCL)

• RNOS

purpose of respiratory burst products

destroy invading microorganisms, tumor cells, and other cells the body targets for destruction & removal

NADPH oxidase

cat transfer of an e- from NADPH to molecular oxygen to form superoxide