Ox Phos

• What is the stable product at the terminal step of oxidative phosphorylation?

• How much energetic efficiency is harvested by ATP?

• The stable product at the terminal step of oxidative phosphorylation is H₂O

• ATP harvest's energetic efficiency ˜ 40%

Ox Phos

1. Much of the energy released from oxidation of ___, ___ & ___is saved in the form of high energy e^-s
2. What are the universal e^- acceptors?
3. What releases the e^-s so that they are free to combine w/ O₂ to form water?
4. What is this process called?

1. Much of the energy released from oxidation of CH₂Os, fats & aas is saved in the form of high energy e^-s
2. The universal e^- acceptors are NAD⁺ & FAD (β-oxid gives us FADH₂ & NADH)
3. NADH & FADH₂ releases the e^-s so that they are free to combine w/ O₂ to form water
4. This process is called Oxidation

Ox Phos

1. Energy released from e^- carriers is coupled to proteins in the  ____ to drive an ____ rxn
2. What is the rxn?

1. Energy released from e^- carriers is coupled to proteins in the mitochonrial inner membrane to drive this endergonic rxn
2. The rxn is: ADP + Pi → ATP (phosphorylation)

Note: coupling both processes (1 exergonic & 1 endergonic = Oxidative Phosphorylation

Ox Phos

• The layout of the mt allows for what?

• The layout of the mt allows for very high O₂ uptake; Adenine necessarily very stable structure

Note: Uric acid is a product → gout

Ox Phos

1. Which mt membrane is freely permeable to small mlcs & ions?
2. Which mt membrane is impermeable to most small mlcs & ions, including H⁺
3. What is contained in the matrix?

1. The outer mt membrane is freely permeable to small mlcs & ions
2. The inner mt membrane is impermeable to most small mlcs & ions, including H⁺ [Note: contains comlexes I-IV, ADP-ATP translocase & ATP synthase]
3. Matrix contains - PDH complex, TCA enzymes, Fatty-acid β-oxidation enzymes, aa oxidation enzymes, DNA & ribosomes, ATP, ADP, Mg²⁺, Ca²⁺, K⁺

Ox Phos

1. What is the energy yield from the oxidation of NADH?
2. FADH₂

1. The energy yield from the oxidation of NADH is 52.6 kcal/mol [Note: this is over 7 times the free energy content of the phospho-anhydride bonds of ATP
2. FADH₂ is 40 kcal/mol

Ox Phos

1. e^-s from NADH + H⁺ are accepted by ___ at the start of the respiratory chain
2. e^-s also come from ____ by way of FADH₂; these e^-s are accepted by ___ rather than by NADH-Q reductase

1. e^-s from NADH + H⁺ are accepted by NADH-Q reductase at the start of the respiratory chain
2. e^-s also come from succinate by way of FADH₂; these e^-s are accepted by succinate dehydrogenase rather than by NADH-Q reductase

Note: huge ΔG @ complex IV - cytochrome oxidase; O₂ is the ultimate energy source

Ox Phos

• What is the order of reduction potential (lowest to highest) for redox couples of the ETS?

Note: reduction potential = tendency to accept e^-s

Order of the reduction potential for redox couples of the ETS

1. NAD⁺/NADH (strongest reductant) E₀ = -0.32
2. FMN/FMNH₂
3. Cytochrome c Fe³⁺/Fe²⁺
4. 1/2 O₂/H₂O (water is reduced O₂) E₀ = +0.82

Ox Phos

• Approx. how much energy (in volts) can be harvested btw reduced e^- carriers NADH → O₂

• 1.2 volts can be harvested btw NADH → O₂ [intermediates in between]

Ox Phos

1. When ___ accepts e^-s, some can escape leading to ___ which require detoxification.

1. When CoQ accepts e^-s, some can escape leading to reactive oxy species (ROS) which require detoxification

Ox Phos

1. Which complex only sees Protons (H⁺) & never touches e^-s
2. Which complexes are involved in the proton pump?
3. Which complex is not part of the proton pump & what's the significance of this?

1. Complex V only see H⁺s & never touches e^-s
2. Complexes I, III, IV are involved in the proton pump (note: system spans the membrane, H⁺s are pumped into the intermembrance space)
3. Complex II is not part of the pump & b/c the mt flavoproteins (including succinate deh & mt α-glycerophosphate deh) bypass complex I they transfer their e^-s directly to ubiquinone. That's the reason for FADH₂ →  2(1.5)ATP vs. 3(2.5)ATP

Ox Phos

1. What are the 2 prosthetic groups of NADH dehydrogenase?
2. What passes to these groups?
3. These flavoproteins are often assoc w/ what?
4. What occurs btw flavin & its non-heme iron?
5. What bonds are present here?

1. The 2 prosthetic groups fo NADH deh are FMN (flavin mononucleotide) & FAD (flavin adenine dinucleotide)
2. 2 e^-s & 2 H⁺s pass to these groups
3. Flavoproteins are often assoc w/ proteins having FeS centers/clusters
4. A single e^- transfer btw flavin & its non-heme iron
5. Cys- bonds (the e^- carrier heme iron is complexed to S of a cysteinyl side chain) 

Ox Phos

1. How does protein-bound Fe fxn in e^- transfers?
2. How does this differ from Hb heme?

1. Protein bound Fe (Fe-porphyrin) fxns in e^- transfers by Δing its oxidation state btw Fe²⁺ & Fe³⁺  (note: makes it a reversible e^- carrier)
2. Hb heme has a constantly bivalent Fe

Ox Phos

1. In cytochromes __ & __ heme is attached to S (from cysteine) of the proteins
2. In cytochromes __ & __ heme is attached by non-covalent means to membrane lipids

1. In cytochromes c & c₁ heme is attached to S (from cysteine) of the proteins
2. In cytochromes a & a₃ heme is attached by non-covalent means to membrane lipids (note: strong hydrophobic force pulls it in)

Ox Phos

1. This mlc is also referred to as Coenzyme Q (CoQ) has no permanent assoc w/ ____ and has a long hydrophobic tail (___) embedded in membrane
2. How many H⁺s does this mlc carry? 
3. It also acts in single e^- transfers but it forms a ____ enabling e^- to escape there
4. Of what complex is it considered a part?

1. Ubiquinone is also referred to as Coenzyme Q (CoQ) has no permanent assoc w/ apoprotein  and has a long hydrophobic tail (isoprenoid) embedded in membrane
2. It carries 2 H⁺s
3. It also acts in single e^- transfers but it forms a free-radical intermediate enabling e^- to escape there (note: can create a toxic chemical)
4. It's considered a part of complex III

Ox Phos

1. Which 4 participants in the ETC are freely diffusible?
2. Which one must be present in the mt matrix?
3. Which one is a peripheral membrane protein on outer surface of inner membrane?
4. Which one is only on the membrane?
5. Which one is freely diffusible across membranes, receives H⁺s & e^-s on matrix side of inner membrane

1. NADH, ubiquinone, cytochrome c & molecular O₂ 
2. NADH must be present in the mt matrix (note: PDH, IDH, α-KD, malate deh all make NADH)
3. Cytochrome C is a peripheral membrane protein on outer surface of inner membrane (soluble)
4. Ubiquinone is on the membrane only
5. Molecular O₂ is freely diffusible across membranes, receives H⁺s & e^-s on matrix side of inner membrane

Ox Phos

1. How many lg multi-protein complexes in the respiratory chain are there?
2. Which ones have Fe-S clusters?
3. Which ones are where protons are extruded?
4. What is the purpose of the pumps?

1. There are 4 lg multi-protein complexes in the ETC
2. The 1st 3 (I, II & III) have Fe-S clusters - accept & then pass on e^-s
3. Complexes I, III, & IV - H⁺s are extruded (pumped up a gradient) across the membrane into the intermembrane space
4. These 3 pumps concentrate power for ATP = chemiosmotic theory

Ox Phos - 1st pump

NADH-Q reductase complex I aka NADH deh / NAD⁺ reductase

• Transfers e^-s from ___ (+ H⁺) to ___ (Co Q)
• e^-s pass from ___ to ___, then through succession of __ centers to ___

• Transfers e^-s from NADH (+ H⁺) to ubiquinone (CoQ)
• e^-s pass from NADH to FMN, then through succession of Fe-S centers to CoQ

Note: Fe-S clusters → non-heme iron (Fe⁺³), cysteine side chain, assoc w/ flavoproteins (FMNs) & complexes I, II, III

Ox Phos - 2nd pump

Cytochrome c reductase complex III

• What does this pump contain?
• What's the pathway?
• What are 2 other characteristics?

• This pump contains cytochrome b, Fe-S protein & cytochrome c
• (e^- from Ubiquinone) Cytochrome b → Fe-S → Cytochrome c₁ → Cytochrome c
• Proton pump, Fe-S clusters

Note: Cytochrome c = freely diffusible peripheral membrane protein

Ox Phos - 3rd pump

Cytochrome oxidase complex

1. (from cytochrome c) → Fe/__ (extremely high O₂ affinity - final e^- acceptor → cytochrome __ → 2H⁺ to __ formation & the other 2H⁺ through _____
2. How many hemes & Cu atoms are there?
3. What is clamped tightly btw heme a₃ & Cu?
4. When is the mlc from Q2 released? 
5. What's special about this pump?

1. (from cytochrome c) → Fe/Cu (extremely high O₂ affinity - final e^- acceptor → cytochrome a → 2H⁺ to H₂O formation & the other 2H⁺ through proton pump
2. There are 2 hemes & 2 Cu atoms (each heme near a Cu)
3. O₂ is clamped tightly btw heme a₃ & Cu during its reduction to H₂O
4. O₂ released only after complete production of H₂O
5. Special because extremely high O₂ affinity (note: under hypoxic or anoxic condition would strip O₂ from everything)

Ox Phos

1. For every 2 e^-s transferred, __ H⁺s are expelled
2. Intermembrane space is the __ side
3. Matrix is the __ side

1. For every 2 e^-s transferred, 4 H⁺s are expelled
2. Intermembrane space is the P side
3. Matrix is the N side

Ox Phos - Complex II

• Its ___ is not in direct path of e^- transfer
• Proposed fxn: reduce "leakage" of e^-s away from __ & out of system (ROS, H₂O₂ & O^-₂₎
• Mutations in II subunits or ___ binding site (not lipid-like CoQ) → higher __ production, deranged ______ & tissue damage

• Its heme b is not in direct path of e^- transfer
• Proposed fxn: reduce "leakage" of e^-s away from CoQ & out of system (ROS, H₂O₂ & O^-₂₎
• Mutations in II subunits or ubiquinone binding site (not lipid-like CoQ) → higher __ production, deranged succinate oxidation & tissue damage (hereditary paraganglioma: tumors in head, neck, carotid body [O₂ sensor]

Note: Succinate → Fumarate

ETC

Where is the ETC located?

The ETC is located in the inner mitochondrial membrane.

It is the final common pathway for which e^-s derived from different fuels of the body flow to oxygen.

ETC

If site-specific inhibitors of electron transport are in place what happens to the electron carriers before & after the block?

The electron carriers before the block are fully reduced whereas those located after the block are oxidized

Ox Phos - Chemisosmotic theory?

1. After protons have been transferred to the cytosolic side of the inner mt membrane via the ETC, where do they go?
2. How do they get there?

1. The chemiosmotic theory proposes that after protons have been transferred to the cytosolic side of the inner mt membrane, they reenter the mt matrix
2. By passing through a channel in the membrane-spanning domain (F₀) of complex V

Ox Phos - chemiosmotic theory

1. What happens as protons pass through F₀?
2. What is the result of this process?

1. Protons passing through F₀ drives rotation of F₀ causing conformational Δes in the extra-membranous F₁ domain that activates its catalytic activity
2. This process results in the synthesis of ATP from ADP + P_i & at the same time, the dissipation of the pH & electrical gradients (drives ATP Synthase - complex V)

Ox Phos - uncoupling proteins (UCPs)

1. Where are UCPs located?
2. What do UCPs create?
3. How is the energy released and what's the name of this process?

1. UCPs occur in the inner mt membrane
2. UCPs create a proton leak, they allow protons to reenter the mt matrix w/o energy being captured as ATP
3. The energy is released as heat, and the process is called nonshivering thermogenesis

Note: Uses H⁺ gradient to produce heat → loss of gradient = no ATP)

Ox Phos - uncoupling proteins (UCPs)

1. What's the other name for UCP1?
2. For what is it responsible?
3. ___ → (lethal) no ATP but s/he can be hyperthermic
4. __ → competitive inhibition of ADP/P_i transporter & P_i binding site on ATP synthase (No P_i = No ATP!)
5. __ → deposit @ the basal ganglia in infants

1. UCP1 is also known as thermogenin
2. Thermogenin is responsible for the activation of fatty acid oxidation & heat production in brown adipocytes (natural uncoupler found in infants)
3. Pentachlorophenol→ (lethal) no ATP but s/he can be hyperthermic (elevates NADH oxidation & O₂ consumption)
4. Arsenic poisoning (Arsenate)→ competitive inhibition of ADP/P_i transporter & P_i binding site on ATP synthase (No P_i = No ATP!)
5. Bilirubin → deposit @ the basal ganglia in infants w/ hyperbilirubinemia, uncoupling @ resulting brain damage early in life

Ox Phos - Overview

• Complex I → NADH-Q-Reductase (4H⁺)
• Complex II → Succinate Deh (no H⁺) [FADH₂ same enzyme from the TCA cycle]
• Complex III → Cytochrome c Reductase (4H⁺)
• Complex IV → Cytochrome c Oxidase (4H⁺ -- 2H⁺ / H₂0)
• ATP synthase → uses H⁺ gradient to convert ADP to ATP
• Complexes contained w/i inner mt membrane
• H₂0 → stable product at terminal step [O₂ is the final e^- acceptor!]

NADH

• Pyruvate Deh
• Isocitrate Deh
• α-ketoglutarate Deh
• Malate Deh

FADH₂

• Succinate Deh (Complex II)

ETC - transfer of e^-

• Complex I (Complex II) → Ubiquinone (Co Q) → Complex III → Cytochrome C → Complex IV

Ubiquinone

• Act in single e^- transfers → e^- can often escape = formation of ROS (i.e. O₂ gains an e^- and forms Superoxide
• Lipid like highly diffusible (move laterally in membrane)
• Long hydrophobic tail - embedded in membrane

NADH/FADH₂

• H⁺ gradient is the source for ___ complex → ___ transferred through Complex, I, III, IV → all 3 complexes contribute to H⁺ gradient → net result is ___

• ___ starts w/ Complex II → transferred through Complex II, III, IV → only III & IV contribute (proton pump) → net result is ___

• H⁺ gradient is the source for ATP synthase complex → NADH (e^-) transferred through Complex, I, III, IV → all 3 complexes contribute to H⁺ gradient → net result is 3 ATP via 1 NADH mlc

• FADH₂ starts w/ Complex II → transferred through Complex II, III, IV → only III & IV contribute (proton pump) → net result is 2 ATP via 1 FADH₂ mlc

Ox Phos - Complex II (Succinate Deh)

• (from FADH₂) → __ → FMN → Fe-S clusters
• No ___
• Reduce # of e^-s that escape from ___

• (from FADH₂) → FAD → FMN → Fe-S clusters
• No Proton pump (H⁺)
• Reduce # of e^-s that escape from Ubiquinone
• Transfers e^- to ubiquinone

Ox Phos - ATP synthase, Complex V

• Use of H⁺ gradient to phosphorylate __ to __ (one revolution = __)
• H⁺ gradient ONLY when e^-s pulled off __ → ONLY pulled off if __ is available

3 Steps

1. Binding substrates (__)
2. Phosphorylation (__)
3. Release of __

• Some ATP intercepted in intermembrane space by ___

• Use of H⁺ gradient to phosphorylate ADP to ATP (one revolution = 3 ATP)
• H⁺ gradient ONLY when e^-s pulled off NADH/FADH₂ → ONLY pulled off if O₂ is available (no O₂ = no e^-s = no H⁺ gradient = NO ATP SYNTHESIS)

3 Steps

1. Binding substrates (ADP & P_i)
2. Phosphorylation (ATP)
3. Release of ATP (exported to cytosol)

• Some ATP intercepted in intermembrane space by nucleoside diphosphate kinase converts w/ broad specificity other nucleoside triphosphates

Ox Phos - ATP synthase

• No __ ctrl → __ is the controlling variable
• ADP/ATP antiporter → __
• Reversible - ATP synthase ↔ __

• No Allosteric ctrl → ADP is the controlling variable (P_i & NADH not limiting)
• ADP/ATP antiporter → obligatory transporter (can't have one w/o the other) [remember...mt inner membrane basically impermeable to ions (need a pump/transporter)]
• Reversible - ATP synthase ↔ mt ATPase (breakdown of ATP for membrane transport of H⁺) [reversal of ATP-depend membrane transport of H⁺s (as seen in the lysosome)]

Ox Phos - Inhibitors

Inhibitors

• __ → inhibits flow of e^-s from Fe-S complexes to ubiquinone
• __ → blocks ETC by binding to Fe⁺³ in cytochrome a/a₃ (complex IV)
• __ → inhibit e^- flow through NADH-Q complex
• __ → blocks e^- flow through the QH₂ - cytochrome c reductase complex

Inhibitors

• Rotenone → inhibits flow of e^-s from Fe-S complexes to ubiquinone
• Cyanide → blocks ETC by binding to Fe⁺³ in cytochrome a/a₃ (complex IV)
• Barbiturates → inhibit e^- flow through NADH-Q complex
• Antimycin-A → blocks e^- flow through the QH₂ - cytochrome c reductase complex

Ox Phos failure

1. ___ → low O₂ availability
2. ___ → absence of O₂ (systemic or local)
3. ___ → damage due to lack of O₂ (local)

1. Hypoxia → low O₂ availability
2. Anoxia → absence of O₂ (systemic or local)
3. Ischemia → damage due to lack of O₂ (local)

Note: loss of fxn due to lack of ATP always precedes cell death

Ox Phos - Regulation

• What are the inhibitors of Ox Phos?

• What are the activators of Ox Phos?

• The inhibitors of Ox Phos are: NADH, ATP or products of key rxns (all represent high energy charge)

• The activators of Ox Phos are: P_i, AMP, ADP (all represent low energy charge)

Note: effect regulatory steps only

Ox Phos - Mutations

• Can affect ATP production → tissue health
• Mt __ → brain & muscle incur 1° damage [__ (inherited from mother)]
• Leber's Hereditary Optic Neuropathy (LHON) →CNS, optic n., vision loss in early adulthood (due to aa substition in Complex __) [e^- transfer from __ → CoQ can't proceed efficiently, ATP yield much reduced (similar to Rotenone)]

• Mt encephalopathies → brain & muscle incur 1° damage [x-linked (inherited from mother)]
• Leber's Hereditary Optic Neuropathy (LHON) →CNS, optic n., vision loss in early adulthood (due to aa substition in Complex I) [e^- transfer from NADH → CoQ can't proceed efficiently, ATP yield much reduced (similar to Rotenone)]

HMP

Two central fxns:

1. production of ___
2. formation of ___

1. production of NADPH (2 for each mlc of G-6-P oxidized) → FYI (3rd from malic enzyme) [used for reductive biosynthesis]
2. formation of ribose-5-phosphate (purine synthesis) [DNA, RNA, certain coenzymes]

HMP

active in tissues that routinely synthesize FAs or steroids (occurs in cytosol)

• ___: synthesis of palmitoyl-CoA requires 14 NADPHs

• ___: requires 14 NADPHs

• Fatty acid synthesis: synthesis of palmitoyl-CoA requires 14 NADPHs

• Cholesterol synthesis: to make 1 cholesterol requires 14 NADPHs

HMP - extremely active in RBC

• Heme-Fe⁺² maintained = __

• Protection again ROS = __

• Heme-Fe⁺² maintained = NADH (uses methemoglobin reductase)

• Protection again ROS = NADPH (reducing agents)

HMP - 2 sections

1. ___: make ribulose-5-P
2. ___: interconversion of varying sugar Ps, generation of NADPH; potential to re-enter as ___

1. Oxidative: make ribulose-5-P [irreversible]
2. Non-oxidative: interconversion of varying sugar Ps, generation of NADPH; potential to re-enter as G-6-P

Note: Isomerase → ribose-5-P

Epimerase → Xylulose-5-P → transketolase/transaldolase

HMP - Oxidation of G-6-P

• Which enzyme does the oxidation?
• G-6-P + __ → 6-P-gluconolactone + __ + H⁺
• Potent ___ inhibition by NADPH; when demand for NADPH is low
• The major ____

• G-6-P deh
• G-6-P + NADP⁺ → 6-P-gluconolactone + NADPH + H⁺
• Potent allosteric inhibition by NADPH; when demand for NADPH is low
• The major regulatory site

HMP - Oxidative decarboxylation of 6-P-gluconate

• What is the enzyme used?
• 6-P-gluconate + __ → __ + NADPH + H⁺ + CO₂
• __ is the potent inhibitor of this step

• The enzyme used is 6-P-gluconate deh
• 6-P-gluconate + NADP⁺ → ribulose-5-P + NADPH + H⁺ + CO₂
• NADPH is the potent inhibitor of this step

Note: similar to decarboxylation of isocitrate in TCA cycle

HMP - non-oxidative portion

• 3 __ near-equilibrium rxns

• Ctrl by supply & demand no __

• Transaldolase → Transketolase  → ___ → Transaldolase

• 3 reversible near-equilibrium rxns

• Ctrl by supply & demand no allosterism

• Transaldolase (3C) No TPP → Transketolase (2C) TPP! → Epimerase (Ribulose -5-P → Xyulose-5-P) → Transaldolase

Note: TPP = thiamin pyrophosphate

• transketolase (TPP) → 2 C' transfers - TPP = Schiff Base - lysine residue
• transaldolase (no TPP) → 3 C' transfers (able to be intermediates for glycolytic pathway - G-3-P, F-6-P)

HMP - clinical correlation

TPP deficiency → Wernicke-Korsakoff Syndrome

• mutation that reduces (to <10) affinity for ___ in Transketolase
• vitamin/coenzyme deficit can make normal, essential equilibrium rxn ___
• Normal fxn has been lost; abnormal → ___

• mutation that reduces (to <10) affinity for Vit B-1 (TPP) in Transketolase
• vitamin/coenzyme deficit can make normal, essential equilibrium rxn rate limiting
• Normal fxn has been lost; abnormal → serious disease

Note: malnourished alcoholics → amnesia, partial paralysis, irreversible

HMP - G-6-P

What happens to G-6-P entering the shunt?

Most of the G-6-P entering the shunt is recycled, while 1/6 is convered to CO₂ & P_i

Note: insulin enhances G6PD gene expression = ↑ flux in well fed state

HMP - tissue

• Which tissues require generous amnts of NADPH

• Liver: 20-30% __ production may arise from HMP activity (NADPH reduces ___ → GSH)

• RBCs, liver, mammary gland, testis, adrenal cortex, all sites of FA or steroid synthesis

• Liver: 20-30% CO₂ production may arise from HMP activity (NADPH reduces GSSG → GSH)

HMP - NADPH

• NADP⁺/NADPH ratio is 1:10

Uses:

1. indirectly maintains ribonucleotide reductase in reduced form (to convert NDP → dNDP) by reducing thioredoxin reductase
2. Reduces oxidized Glutathione (GSSG) back to Glutathione (GSH) via glutathione reducatase (re-establishes cysteine bonds)

-GSH = key reducing agent of H₂O₂

-NADPH + GSH → recycle Ascorbate (Vit C)

  3. Provides the reducing power for  

      Cyt P-450 mono-oxygenases

      (reduces ROS)

  4. Synthesis of NO (nitric oxide)

      - via NO-synthase (Ca²⁺),   

      arginine

  5. Reduces molecular O₂ in 

      phagocytic cells - forming

      Superoxide

HMP - Glutathione

NADPH reducing power 2° from glutathione

1. on sulfhydryl groups of __ residues (come from __)
2. __ derivatives (later in vitamins)
3. major defense against __
4. can reduce oxidized __ groups on other cmpds non-enzymatically
5. non-ribosomal __ origin

1. on sulfhydryl groups of cysteine residues (come from methionine)
2. folate derivatives (later in vitamins)
3. major defense against oxidative stress
4. can reduce oxidized thio groups on other cmpds non-enzymatically
5. non-ribosomal hepatic origin

HMP - Glutathione (Heinz)

• In the RBC excessive __ oxidation can produce SS-bonds in __ proteins
• This causes mlc to lose ___ structure → form ___
• This makes the cell less pliable → __ removal → __ → splenic destruction

• In the RBC excessive -SH oxidation can produce SS- bonds in cytosolic proteins
• This causes mlc to lose 3° structure → form Heinz bodies (insoluble masses)
• This makes the cell less pliable → RBCs removal → hemolysis → splenic destruction

Note: Heinz bodies → pts w/ G-6-P deh deficiency

HMP - Glutathione, peroxides

RBC

•  glutathione __ requires __ as a cofactor
• 2GSH + __ → __ + H₂O
• glutathione __ reestablishes 2 -SH groups
• GSSG + __ + H⁺ → 2__ + NADP⁺

RBC

•  glutathione peroxidase requires Se as a cofactor
• 2GSH + H₂O₂ → GSSG + H₂O
• glutathione reductase reestablishes 2 -SH groups
• GSSG + NADPH + H⁺ → 2GSH+ NADP⁺ (≈ unidirectional rxn; GSH/GSSG ≈ 500:1)

HMP - glutathione, liver

Peroxides

1. reduces organic peroxides to their __
2. reverses __ peroxidation

• glutathione __ attaches glutathione to toxic mlcs, making them more __

1. reduces organic peroxides to their alcohols
2. reverses lipid peroxidation

• glutathione-s-transferase attaches glutathione to toxic mlcs, making them more soluble

HMP - glutathione, clinical

• What is broken down by cytochrome P-450 into a highly toxic intermediate which glutathione acts on to make a non-toxic product?
• What happens in an overdose?

• Acetaminophen is broken down by cytochrome P-450 into a highly toxic intermediate which glutathione acts on to make a non-toxic product
• In an overdose (acetminophen poisoning), glutathione reserves are rapidly exhausted in the liver, toxic intermediates build up → liver failure

HMP - glutathione

• Dehydroascorbate [oxidized Ascorbic acid (Vitamin C)] is continually recycled by acquiring e^-s & H⁺s from reduced __ which in turn is brought back to the reduced form by __ from the shunt

• Dehydroascorbate [oxidized Ascorbic acid (Vitamin C)] is continually recycled by acquiring e^-s & H⁺s from reduced glutathione (GSH) which in turn is brought back to the reduced form by NADPH from the shunt

HMP - Cytochrome P-450

• mlc O₂ used but only __ bound to cytochrome's heme
• this creates a very reactive __
• can steal H from hydrocarbon, __ can escape
• __ runs e^- transfers
• Heme oscillates btw __ & __
• __ group established

• mlc O₂ used but only single O₂ bound to cytochrome's heme
• this creates a very reactive perferryl ion FeO³⁺
• can steal H from hydrocarbon, 1 O can escape
• FAD runs e^- transfers
• Heme oscillates btw Fe²⁺& Fe³⁺
• OH group established

Note: R-H + NADPH (H⁺) + O₂ → R-OH + H₂O + NADP⁺

HMP - NO·

• Ach → Ca²⁺ influx →NO· synthase [__ + O₂ + NADPH + H⁺] → citrulline + NO· + __→ NO diffuses to smooth m. cells → cGMPsynthesis → m. relaxation
• __ stimulates NO· synthase (makes NO· gas from arginine)
• NO· easily crosses plasma membrane, immediately causes relaxation or __ of smooth m. 
• relaxation caused by __ synthesized by NO· - depend guanylate cyclase
• Which drug was developed based on the prolonged action of NO· due to the inhibition of cGMP breakdown?

• Ach → Ca²⁺ influx →NO· synthase [L-arginine + O₂ + NADPH + H^+] → citrulline + NO· + NADP⁺ → NO diffuses to smooth m. cells → cGMPsynthesis → m. relaxation
• Ca²⁺ stimulates NO· synthase (makes NO· gas from arginine)
• NO· easily crosses plasma membrane, immediately causes relaxation or vasodilation of smooth m.
• relaxation caused by cyclic-GMP synthesized by NO· - depend guanylate cyclase
• Viagra was developed based on the prolonged action of NO· due to the inhibition of cGMP breakdown

HMP - G-6-P deh

• No G-6-P-deh → no __ = no GSH = __ = hemolytic anemia

• No G-6-P-deh → no NADPH = no GSH = Heinz bodies in RBC = hemolytic anemia

Note: Hemolytic anemia in neonatal period = high amnt of bilirubin from Heme destruction  = neonatal jaundice

HMP - G-6-P deh deficiencies

• Favism, observed in pts. w/ genetic deficiency in G-6-P deh in RBC
• __ from fava beans is strong oxidant → RBC lysis → kidney failure → death
• antimalarial (quininoid) drugs → severe hemolytic anemia, cause?
• deficiency has partially beneficial effect against __

• Favism, observed in pts. w/ genetic deficiency in G-6-P deh in RBC
• divicine from fava beans is strong oxidant → RBC lysis → kidney failure → death
• antimalarial (quininoid) drugs → severe hemolytic anemia = G-6-P deh deficient
• deficiency has partially beneficial effect against Plasmodium falciparum (sensitive to oxidative damage)

Note: deficiencies of G-6-P deh are X-linked

Oxygen toxicity

• Superoxide: by-product of Ox Phos; enzyme(s) __

• Hydrogen peroxide: enzyme(s)

• Hydroxyl radical: enzyme(s)

• Superoxide: by-product of Ox Phos (escapes at level of CoQ); enzyme(s) Superoxide Dismutase

• Hydrogen peroxide: Catalase (contains heme & Fe²⁺), H peroxidase & Glutathione peroxidase (peroxisomes) [H₂O₂ + Cl^- → hypochlorous acid (HOCl of bleach)

• Hydroxyl radical: readily produced from H₂O₂; DNA repair mechanisms

Note: a radical is a mlc w/ a single unpaired e^- in an orbital

Anti-oxidants besides NADPH

• Fat soluble: vitamins __ & __, α-tocopherol

• H₂O soluble: vitamin __ & glutathione

• __: against lipid peroxidation

• Fat soluble: vitamins A & E, α-tocopherol

• H₂O soluble: vitamin C & glutathione

• Bilirubin: against lipid peroxidation (oxidation of FAs in the membrane)

Superoxide Dismutase

• Cytosol: cofactor pair is __ & __ to capture the substrate O_2·^-

• Mitochondrion: cofactor pair is __ & __

• What disease is due to defective cytoplasmic superoxide dismutase?

• Cytosol: cofactor pair is Zn²⁺ & Cu⁺ to capture the substrate O_2·^-

• Mitochondrion: cofactor pair is Mn²⁺ & Cu⁺

• Amyotrophic Lateral Sclerosis (ALS = Lou Gehrig's disease) is due to defective cytoplasmic superoxide dismutase

Superoxide Anions

• __ oxidase in plasma membrane of phagocytes catalyzes reduction of mlc O₂ to form O₂·^-

1. NADPH reduces __
2. FADH₂ is re-oxidized by Fe³⁺ form of __
3. Reduced Fe²⁺ cyt b reduces _{__} to O₂·^-

• NADPH oxidase in plasma membrane of phagocytes catalyzes reduction of mlc O₂ to form O₂·^-

1. NADPH reduces FAD
2. FADH₂ is re-oxidized by Fe³⁺ form of cyt b
3. Reduced Fe²⁺ cyt b reduces O₂ to O₂·^-

Note: from lowest to highest reduction potential

Non-glucose sugars

• Sucrose is made up of what 2 sugars?
• What enzyme(s) does fructose metabolism in muscle?
• liver?

• Sucrose = Fructose + Glucose
• Fructose metabolism in muscle: hexokinase converts fructose to F-6-P but only when it's not saturated w/ glucose to phosphorylate [HK's K_m for glucose is low, & for fructose is high]
• In liver: fructokinase (ATP → ADP) converts fructose to F-1-P, will use it just like glucose

• What is responsible for fructose transport into the cell?

• Glut-5 is responsible for fructose transport into the cell (NOT under the ctrl of insulin)

Note: can still get glucose from sucrose & galactose so in DM pts. still have to watch sugar intake

F-1-P

• F-1-P can not immediately enter __ or __
• F-1-P → __ (DAP) (direct uptake) & __ (glyceraldehyde) (no uptake)
• What enzyme(s) split F-1-P into these 2 products?

• F-1-P can not immediately enter glycolysis or gluconeogenesis
• F-1-P → triosephosphate (DAP) (direct uptake) & unphosphorylated triose (glyceraldehyde) (no uptake)
• Aldolase B splits F-1-P into these 2 products

Aldolase B - liver

• Km_F-1-P is higher than the Km_FBP, what's the consequence of this?
• Hereditary fructose intolerance or fructose poisoning can occur as a result of __

• the consequence of the higher Km is that at times F-1-P can accumulate
• Hereditary fructose intolerance or fructose poisoning can occur as a result of Aldolase B deficiency

Aldolase B - liver

• Dihydroxyacetone phosphate (DAP) can enter glycolysis after equilibration → __

• Glyceraldehyde requires conversion: glyceraldehyde + ATP → ADP + __

• Dihydroxyacetone phosphate (DAP) can enter glycolysis after equilibration → glyceraldehyde-3-P

• Glyceraldehyde requires conversion: glyceraldehyde + ATP → ADP + glyceraldehyde-3-P

F-6-P

• Why does muscle metabolize fructose faster than glucose?

• Muscle metabolizes fructose faster than glucose because HK's F-6-P synthesis can bypass the G-6-P to F-6-P step in glycolysis (speed up acetyl-CoA & ATP production)

Fructose metabolism disorders

• Which deficiency is relatively benign?
• Which deficiency leads to hereditary fructose intolerance?
• What's the tx?

• Fructokinase deficiency is relatively benign
• Aldolase B deficiency leads to hereditary fructose intolerance [AR; ties up PO₄ in F-1-P (Phosphate sequestration); diminished energy charge
• Tx: remove all fructose (& sucrose) from diet

F-6-P to Glucosamine

• F-6-P + __ → Glucosamine-6-P
• Glucosamine-6-P ends up as UDP-N-acetylglucosamine when __ is added

• F-6-P + Glutamine → Glucosamine-6-P (note: nitrogen from Glu; irreversible rxn)
• UDP-N-acetylglucosamine when UMP is added (note: UMP of UTP is joined to a sugar-1-P)

F-6-P covalent attachment of modified sugars to proteins

• O-linked to __ or __
• N-linked to __
• Location for O? N?
• Sugars addded by for O? N?

• O-linked to serine or threonine
• N-linked to asparagine
• Location for O - mainly Golgi; for N - mainly ER
• Sugars added by activating groups for O; by dolichol for N

M-6-P

• From where does most of the cell's M-6-P usually come?
• What is the main fxn of M-6-P?

• Most of cell's M-6-P is created by isomerization of F-6-P (reversible)
• The main fxn of M-6-P is a tag for lysosomes (I-cell)

Sorbitol metabolism

• Glucose + __ + H⁺ → __ + NADP⁺
• What enzymes for this rxn?

• Glucose + NADPH + H⁺ → sorbitol + NADP⁺
• Aldose Reductase

glucose → sorbitol → fructose

1. glucose to sorbitol via aldose reductase & NADPH
2. sorbitol to fructose via sorbitol deh & NADH

Note: used in seminal vesicles - energy for sperm; also see in lens, retina, Schwann cells, RBCs (hyperglycemia is a problem for these tissues)

Galactose metabolism

• Lactose → Galactose + glucose; what enzyme is used?
• What's special about galactose (similar case as w/ fructose?

• the enzyme used is β-galactosidase (Lactase)
• Galactose like fructose does not need insulin

Galactose metabolism - liver

• Galactose + ATP →Galactose-1-P + ADP; enzyme used?
• Further metabolism requires what?
• What enzymes is req'd?
• Deficiency of this enzyme results in what?

• enzyme used to phosphorylate galactose is galactokinase
• further metabolism requires a transfer of UMP from glucose to galactose = UDP-galactose
• Gal-1-P uridyl transferase is req'd
• Deficiency in this enzyme cause Classic Galactosemia (AR) resembles Hered Fru Intol (remove milk from diet)

UDP-galactose

• Lactose synthase is composed of 2 proteins, what are they?
• A & B combine to produce this enzyme fxn?
• What hormone inhibits protein B?
• activates it?

• Lactose synthase is composed of A (galactosyltransferase) & B (α-lactalbumin)
• UDP-galactose:glucose galactosyltransferase
• Progesterone inhibits protein B
• Prolactin activates protein B

Glucuronic Acid

• Glucose modified to form glucuronic acid via __
• This pathway synthesizes __

• Glucose modified to form glucuronic acid via UDP-glucose (many EC products)
• This pathway synthesizes Vitamin C (humans lack terminal enzyme)

UDP-Glucuronate

• intermediate to form glucuronic acid
• for __ to be excreted it needs solubilization w/ glucuronate
• Deficiency of this enzyme leads to jaundice

• for bilirubin to be excreted it needs solubilization w/ glucuronate
• deficiency of bilirubin-glucuronyl-transferase → jaundice

Conjugation of Bilirubin

• (spleen) __ + NADPH → Bilirubin
• (blood) - Bilirubin bound to __
• (liver) - Bilirubin + __ → Bilirubin diglucuronide - via bilirubin-glucuronyl-transferase
• Excretion

• (spleen) Biliverdin + NADPH → Bilirubin
• (blood) - Bilirubin bound to albumin
• (liver) - Bilirubin + (2) UDP-glucuronate → Bilirubin diglucuronide - via bilirubin-glucuronyl-transferase
• Excretion

FA oxid

TAG depots → release of FAs

1. Irreversible synthesis of __
2. Phosphorylation of __ "unmasks" lipid droplet
3. Phosphorylation (PKA) activates __ → breaks down TAGs to (3) FFAs & glycerol

1. Irreversible synthesis of cAMP (starts multiplicative cascade)
2. Phosphorylation of perilipin "unmasks" lipid droplet (open up = makes it more polar)
3. Phosphorylation (PKA) activates Hormone-sensitive Lipase (HPL) → breaks down TAGs to (3) FFAs & glycerol (w/ help of perilipin)

Hormonal ctrl of lipolysis

• What is the 1° hormone that ctrls lipolysis?
• T or F: glucocorticoids, growth hormone & thyroid hormones all facilitate lipolysis by direct stimulation of cAMP formation

• NE is the 1° hormone that ctrls lipolysis
• F: glucocorticoids, GH & TH facilitate lipolysis via induction of lipolytic proteins

• Which disease is a result of excess glucocorticoids?
• What's the mechanism?
• Distinguishing features of disease

• Cushing's disease is a result of excess glucocorticoids
• The mechanism: glucocorticoids augment lipolytic effects of NE & E
• "buffalo hump"; thin extremities (unequal response to lipolytic effects of these hormones)

• Does insulin initiate or inhibit lipolysis?
• Mechanism?
• What is the effect of glucagon?

• Insulin inhibits lipolysis
• Insulin inhibits HSL via de-phosphorylation
• Glucagon stimulates lipolysis

Note: high insulin → TAGs synth'd after meal

Liver v. adipocytes

• liver releases particulate lipid as  ___
• adipocytes release it as __ & __
• What transports FFAs in the blood?
• What happens when this substance is low?
• Why does glycerol have to go to the liver?

• liver releases particulate lipid as  lipoproteins
• adipocytes release it as FFAs & glycerol
• plasma albumin is a non-covalent carrier of FFAs (can't be used by RBCs or brain)
• low plasma albumin curtails ability of blood to take FFAs to their destination
• glycerol has to go to the liver (to be phosphorylated there & reenter metabolism) b/c adipocytes lack glycerokinase

• Where do FAs go to get oxidized?
• What must happen before oxidization?
• FAs get activated to __
• What drives the thioester formation?

• FAs must enter the mitochondrion to get oxidized
• Activation must happen before oxidation
• FAs get activated to fatty acyl-CoA (via Fatty acyl-CoA synthetases (ligases) (short & long chain)
• ATP → AMP + PPi drives thioester formation

• How do FAs w/ 12 C or less get into the cell?
• Transport of long chain FAs into the mt matrix requires what?
• What is the origin of this substance?
• What provides it?

• FAs w/ 12 C or less freely diffuse in the the mt matrix
• Transport of long chain FAs into the mt matrix requires carnitine
• Carnitine comes from 2 essential aas → lysine & 3 methionines (from SAM) (Trimethyllysine)
• Liver & kidneys provide carnitine to muscles (97%), heart & other tissues

• FA is transferred from acyl-CoA to critical __
• What is the catalyst for this rxn?

• FA is transferred from acyl-CoA to critical -OH of carnitine
• This rxn is catalyzed by Carnitine-palmitoyl transferase (CPT) aka carnitine acyltransferase (CAT)

Note: there are 2 CPTs (I & II) on the outer (I) & inner (II) mt membranes

• β-oxidation: each 4 step cycle removes __ from a 16-18 C polymer of acetate units
• What is essential?
• in each cycle β-carbon is oxidized; the yield is __
• What other 2 things are formed that become substrates in the respiratory chain?

• β-oxidation: each 4 step cycle removes 2-C units from a 16-18 C polymer of acetate units
• Sufficient CoASH is essential
• in each cycle β-carbon is oxidized; the yield is 1 acetyl CoA (via thiolytic cleavage)
• 1 NADH & 1 FADH₂ are formed to become substrates in the respiratory chain

1. Oxidation via DH
   
   1. Fatty acyl CoA → Enoyl CoA
   2. Product?
2. __ via Hydrolase
   
   1. Enoyl CoA → 3-hydroxyacyl CoA
3. Oxidation via DH
   
   1. 3-hydroxyacyl CoA → 3-ketoacyl CoA
   2. Product?
4. Thiolysis via Thiolase
   
   1. 3-ketoacyl CoA → Fatty acyl CoA (-2C') + __]

1. Oxidation via DH
   
   1. Fatty acyl CoA → Enoyl CoA
   2. [FADH₂]
2. Hydration via Hydrolase
   
   1. Enoyl CoA → 3-hydroxyacyl CoA
3. Oxidation via DH
   1. 3-hydroxyacyl CoA → 3-ketoacyl CoA
   2. [NADH]
4. Thiolysis via Thiolase
   
   1. 3-ketoacyl CoA → Fatty acyl CoA (-2C') + Acetyl CoA
   2.  *last cycle produces 2 Acetyl CoAs

Note: 16C' FA (palmitate) = 7 cycles = 7 FADH₂ = 7 NADH = 8 Acetyl CoA

ATP pay off

• 1 Acetyl CoA = __ ATPs
• 1 FADH₂ = __ ATPs
• 1 NADH = __ ATPs
• __ ATPs are used to activate FA
• Oxidation of FA to H₂O requires much O₂
  • Respiratory Quotient (RQ) for FA = 0.7
  • RQ for CH₂O = 1
  • RQ = CO₂ released / O₂ consumed
• What would be the 1° fuel source for RQ = .85?
• Compare complete oxidation of 1 glucose to 1 palmitate

• 1 Acetyl CoA = 12 ATPs
• 1 FADH₂ = 2 ATPs
• 1 NADH = 3 ATPs
• 2 ATPs are used to activate FA
• Oxidation of FA to H₂O requires much O₂
  • Respiratory Quotient (RQ) for FA = 0.7
  • RQ for CH₂O = 1
  • RQ = CO₂ released / O₂ consumed
• For RQ = .85 both FAs & CH₂Os would be the 1° fuel source ?
• 38 ATPs vs. 129 ATPs (high energy store, can last a long time)

Regulation of FA utilization

• 1° at level of __ & LPL (lipoprotein lipase)
• 2° __ high when FA synthesis is active - inhibits CPT I
  • reciprocal activation & __
• 3° CPT I in liver subject to long term regulation; activity __ in prolonged fast

• 1° at level of HSL & LPL (lipoprotein lipase)
• 2° Malonyl-CoA high when FA synthesis is active - inhibits CPT I
  • reciprocal activation & inhibition
• 3° CPT I in liver subject to long term regulation; activity ↑ in prolonged fast

Note: No direct allosteric ctrl;

CPT = carnitine-acyl transferase; CPT II is NOT regulated

• Why is their no allosteric ctrl of β-oxidation?

• there is no allosteric ctrl b/c each successive removal of an acetyl-CoA is a simple repetition of 4 steps
  
  • allosteric reg'n at step just preceding 1st oxidation

• What 4 types of FAs requires specialized rxns to be oxidized?
• What happens if a defect is present?

1. Very long (22-C & up)
2. Branched
3. Odd-numbered
4. Unsaturated FAs

• if defect is present → serious metabolic disease

VLCFAs

• Location of degradation?
• What is essential to the process?
• How far does this pathway go?

• peroxisomes is where VLCFAs are degraded
• H₂O₂ is essential to the process (1st rxn catalyzed by flavoprotein)
• This pathway proceeds only as far as C₄ & C₆ acyl CoAs
• 2 key points: 100s/cell & ½ life avg ≈ 2days

• What disease is a result of defective import of enzymes into peroxisomes?
• Characteristics of this disorder?

• Zellweger's syndrome: defective import of enzymes into peroxisomes
• High forehead, broad nose, widely spaced eyes

Aberrant α-oxid of branched FAs

Normally

1. Activation by __ 
2. α-carbon is __ in O₂-ascorbate & Fe²⁺ requiring rxn
3. __ forms an aldehyde shorter by 1 C
4. __ to a carboxylic acid w/ no group on its β-carbon; normal β-oxidation can run smoothly

1. Activation by CoASH (always)
2. α-carbon is hydroxylated in O₂-ascorbate & Fe²⁺ requiring rxn
3. Decarboxylation forms an aldehyde shorter by 1 C
4. Oxidation to a carboxylic acid w/ no group on its β-carbon; normal β-oxidation can run smoothly

Branched FAs - clinical correlation

• What disease is a defect in this rxn: phytanoyl-CoA hydroxylase (α-hydroxylase) → β-oxidation can't run; α-carbon can't be hydroxylated?
• What's the tx?

• Refsum's disease; AR; severe neuro disorder
  • normally α-oxid of phytol & phytanic acid; oxid @ α-carbon creates -OH; defect @this step allows phytanic acid to accumulate
• tx: restrict phytanate-rich green vegetables & milk & meat from ruminants

Unsaturated FAs

Double bonds must be modified before β-oxidation

• the double bonds in our natural FAs are in __ configuration
• Proper oxidation of unsaturated FAs requires isomerization to an intermediate __ shape

• the double bonds in our natural FAs are in cis configuration
• Proper oxidation of unsaturated FAs requires isomerization to an intermediate trans-shape

Odd-chain FAs

3-rxn process

1. Carboxylation
   
   1. __ is a glucogenic precursor/substrate (unlike acetyl CoA)
   2. requires __
2. Racemization
3. Mutation
   
   1. Methomalonyl CoA mutase
   2. What's the co-factor?

1. Carboxylation (1st gets converted to an even # when gets to 3 C's)
   
   1. propionyl-CoA (to D-methylmalonyl CoA)  is a glucogenic precursor/substrate (unlike acetyl CoA)
   2. requires biotin (coenzyme)
2. Racemization (D- to L-methylmalonyl CoA)
3. Mutation (L-methylmalonyl CoA → succinyl CoA)
   1. Methomalonyl CoA mutase
   2. cobalamin - Vit B12 (one of only 2 rxns that require it)

• defects in β-oxidation compromise muscle & liver mostly, why?

• defects in β-oxidation compromise muscle & liver mostly b/c they derive most of their energy from FAs, esp in the fasting state

• What is the most common deficiency of β-oxidation enzymes?
• What's the effect?
• When does this present itself?
• What's the recommendations?

• most common is medium chain (C₅-C₁₂) acyl-CoA dehydrogenase deficiency (1st rxn of β-oxidation)
• FAs of 6 or more C in length can't be catabolized to acetyl-CoA (longterm effect: lipid deposition & fatty liver)
• Usually in the 1st 2 years of life; usually during fasting hypoglycemia
• Recommend: low fat diet & oral supplements of carnitine

Note: MCAD has been id'd as the cause of some cases originally reported as SIDS or Reye syndrome

Carnitine deficiency

• common cause of carnitine depletion is __
• if liver affected: carnitine deficiency leads to __
• Why low ketones?
• Why might you see fatty degeneration of the liver?

• common cause of carnitine depletion is acyl-CoA dehydrogenase deficiency
• if liver affected: carnitine deficiency leads to hypoketotic  (low ketone) hypoglycemia during extended fasting (usually CPT-1 deficiency)
• Low ketones b/c FAs major source of acetyl-CoA are not being degraded
• excess acyl-CoA that cannot be transported into the mt is diverted to TAG synthesis

Note: CPT-II deficiency occurs 1° in cardiac & skeletal m.

Defective Hepatic β-oxidation

• Gluconeogenesis - requires steady supplies of __ & GTP
  
  • __ only source of energy for fasting liver
  • Any defect in β-oxidation diminishes gluconeogenesis
• ketogenesis
  • impaired as the supply of __ is reduced

• Gluconeogenesis - requires steady supplies of ATP & GTP
  
  • FA oxidation only source of energy for fasting liver
  • Any defect in β-oxidation diminishes gluconeogenesis
• ketogenesis
  • impaired as the supply of acetyl-CoA is reduced

FA synthesis

• Where does FA synthesis take place?
• Where are FAs stored?

• FA synthesis takes place 1° in the liver
• FAs are stoed in specific cell types esp. liver, heart & muscle

Unsaturated FAs

• Double bonds of polyunsaturates are __ carbons apart
• What's btw them?
• Arachidonic acid has how many double bonds?
• Humans can't insert new double bonds beyond which Carbon?

• Double bonds of polyunsaturates are 3 carbons apart
• A single methylene group lies btw them
• Arachidonic acid has 4 double bonds
• Humans can't insert new double bonds beyond Carbon 9

Unsaturated FAs

• β-oxidation: the 1st oxid (using FAD) creates a __ double bond
• after activation, for proper oxid to proceed, __ must be Δed to __ 
• Double bonds (unsaturated FAs) __ the melting point

• β-oxidation: the 1st oxid (using FAD) creates a trans double bond
• after activation, for proper oxid to proceed, our dietary unsaturated cis FAs must be Δed to trans configuration
• Double bonds (unsaturated FAs) decrease the melting point

Essential FAs

• What are the 2 essential FAs?
• Which one gives rise to arachidonic acid?
• What happens if it's lacking in the diet?

• Linoleic (sunflower, corn, linseed) & α-linolenic (salmon, trout, canola)
• linoleic acid gives rise to arachidonic acid
• if it's lacking in the diet then arachidonic acid becomes essential

FA synthesis

• How do we generate cytosolic Acetyl-CoA?
• In what tissue(s) does this occur?
• When does this occur?

• Citrate moves from mt to cytosol (essential b/c Acetyl CoA can't cross the inner mt membrane) where it's cleaved by ATP-dependent citrate lyase → acetyl CoA & OAA
• This occurs in the liver (can't leave in heart m.)
• This occurs when
  
  • the cell can divert extra citrate to the cytosol (i.e. after high CH₂O meal)
  • TCA cycle is partially inhibited 1° at isocitrate deh by high ATP levels (i.e. liver can afford it)

Acetyl-CoA

• Acetyl-CoA → (committed) Malonyl CoA; what enzyme?
• What's the coenzyme?
• To what does it bind?
• What is the active state?
• Activators? Inhibitors?

• Acetyl-CoA → (committed) Malonyl CoA; enzyme is Acetyl CoA carboxylase 
• the coenzyme is biotin (CO₂ & ATP also req'd)
• Biotin binds to a critical lysine in a Schiff base
• the enzyme is in the active state when it's de-phosphorylated (insulin = activator; glucagon & epi = phosphorylation = inactivator)
• Activators: citrate; Inhibitor: malonyl-CoA (also palmitoyl CoA)

Note: this carboxylation is both the rate-limiting & regulated step in FA synthesis

FA synthesis

• After Malonyl CoA (3C') formation: Malonyl ACP + Acetyl ACP (decarboxylation) → 4C' → __ → __ → __ → Butyryl (4C') + Malonyl ACP...etc
• What is essential to ACP?

• After Malonyl CoA (3C') formation: Malonyl ACP + Acetyl ACP (decarboxylation) → 4C' → reduction (NADPH) → dehydration → reduction (NADPH) → Butyryl (4C') + Malonyl ACP...etc
• Pantothenic acid (vit B5) [also used in the fabrication of coenzyme ASH], creates thioester bonds

ACP = acyl carrier protein

FA Synthesis

Seemingly opp. of β-oxidation

1. Carboxylation of malonyl-CoA (add'n of 3-C to be decarboxylated so actually add'n of a 2-C unit
2. Decarboxylation
3. __ w/ NADPH
4. __
5. Reduction w/ NADPH

1. Carboxylation of malonyl-CoA (add'n of 3-C to be decarboxylated so actually add'n of a 2-C unit
2. Decarboxylation
3. Reduction w/ NADPH
4. Dehydration
5. Reduction w/ NADPH

Synthesis of Palmitate (16-C)

• How many NADPH are used per cycle?
• How much ATP is used?

• 2 NADPH are used per cycle (14 NADPH in total for Palmitate)
• 1 ATP is used  (citrate to acetyl-CoA

Malate shuttle

• OAA + NADH + H⁺ → malate + NAD⁺
• there are 2 fates for malate what are they?

1. enter mt for TCA fxn or malic enzyme (ME)
2. Malate + NADP⁺ → Pyr + CO₂ + 3rd rxn producing → NADPH + H⁺ (in theory ME could fill 33-50% of FAs need for NADPH)

Conversion to TAG

In liver 4 key lipogenic enzymes ↑ in well fed states & ↓ in fasting state

1. __ (acetyl-CoA → malonyl-CoA)
2. __ (citrate →OAA + acetyl-CoA)
3. __ (NADPH)
4. fatty acid synthase (FAS)

1. acetyl-CoA carboxylase (acetyl-CoA → malonyl-CoA)
2. ATP citrate lyase (citrate →OAA + acetyl-CoA)
3. G-6-P deh (NADPH)
4. fatty acid synthase (FAS)

Note: FAS is overexpressed in some (breast, ovarian) CAs; FAS inhibitors slow tumor growth & induce apoptosis

TAG

• After synthesis, FAs are 1st __ & 2nd __ to TAG
• What is the main source of glycerol for the liver & the sole source for adipocytes?

• After synthesis, FAs are 1st activated & 2nd esterified to TAG
• Glycolysis is the main source of glycerol for the liver (also possesses glycerokinase, GK) and sole source for adipocytes (no GK)

Elongation of FAs

• Where is chain elongation most active?
• What is the 2nd site?

• chain elongation is most active in the ER
• in the mt (2 C's at a time) works w/ sat'd & unsat'd FAs

Desaturation of FAs

• Addt'n double bonds may be introduced btw 1st double bond & carboxy group but not beyond which C?
• What's the significance of this?

• Addt'n double bonds can't be added beyond the 9th C
• Because of this, linoleic & liniolenic acid are essential in the human diet

Arachidonic acid

• The production of arachidonic acid from linoleic acid requires what process?

• The process of producing arachidonic acid from linoleic acid involves 2 desaturations  separated by one elongation step (1 elongation adds 2 C's to the chain)
• linoleic acid (18:2) → desaturation (18:3) via desaturase → elongation (20:3) → desaturation (20:4) via desaturase → arachidonic acid (20:4)

Oxid of poly-unsaturated FAs

• the more unsaturated a FA → the more it can be __ → forms reactive aldehyde group → can cause membrane damage
• what accumulates?

• the more unsaturated a FA → the more it can be peroxidized → forms reactive aldehyde group → can cause membrane damage
• lipofuscin accumulates ("age pigment" in lysosomes)

Note: lipid peroxidation also implicated in atherosclerosis

Ketones

• Where does ketogenesis occur?

• From 3 acetyl-CoAs you can create what soluble 4-C mlc?

• Only in liver mt

• you can create β-hydroxybutyrate (and retain CoASH inside the mt)

Ketones

• __ gets reduced to β-hydroxybutyrate by NADH
• __ gets formed from spontaneous decarboxylation of acetoacetate
• Why can't liver utilize the ketones that it produces?
• Which tissues use ketones?

• Acetoacetate gets reduced to β-hydroxybutyrate by NADH
• Acetone gets formed from spontaneous decarboxylation of acetoacetate
• Liver lacks ketoacid transferase (aka acetoacetate:succinyl CoA CoA transferase) so it can't utilize ketones
• All tissues except liver & RBCs use ketones (brain after app 3-5 days of starvation)

Ketone utilization

• β-hydroxybutyrate enters the cell and gets oxidized to NADH + H⁺ + acetoacetate
• Acetoacetate (CoASH acceptor) + __ → acetoacyl-CoA + __
• Acetoacyl-CoA + CoASH → __
• Where does this take place?

• Acetoacetate (CoASH acceptor) + succinyl-CoA → acetoacyl-CoA + succinate (activated form) [ enzyme: acetoacetate:succinyl CoA CoA transferase]
• Acetoacyl-CoA + CoASH → 2 acetyl-CoA (ketothiolase is the enzyme)
• this takes place in the mt

Note: acetyl-CoA now enters the TCA cycle & forms citrate

• What is the rate-limiting enzyme of ketogenesis?
• What stimulates HMG-CoA synthase?
• High glucagon:insulin ratio; how does glucagon stimulate ketogenesis?

• HMG-CoA synthase is the rate-limiting enzyme of ketogenesis from acetyl-CoA
• HMG-CoA synthase is stimulated by insulin deficiency → lipid degradation
• glucagon stimulates ketogenesis by limiting formation of malonyl-CoA

During fasting & starvation, several enzymes are induced to prolong survival, name 4 examples

1. 4 gluconeogenic enzymes
2. various transaminases
3. CPT-I
4. HMG-CoA synthase
   

Ketones - clinical correlation

• What's the relationship btw ketones & Diabetes?

• in DM type I  you have an accumulation of keto acids → the pH of the blood is substantially decreased (≈6.8)

Ketones - salient points

• Source of ketone carbon: __
• Synthesis favored when lipids predominate as fuel: in what kind of state should the body be?
• Production is __ & __
• Ctrl of synthesis - at level of __
• Distribution via plasma as __
• Utilization in all mt-bearing tissues but __
• Oxidation - TCA cycle - needs donation of __
• Highly soluble due to low __
• High levels can sometimes produce __

• Source of ketone carbon: acetyl-CoA (3)
• Synthesis favored when lipids predominate as fuel: in what kind of state should the body be? starvation
• Production is mt & hepatic
• Ctrl of synthesis - at level of HMG-CoA synthase
• Distribution via plasma as β-hydroxybutyrate
• Utilization in all mt-bearing tissues but liver
• Oxidation - TCA cycle - needs donation of CoASH (succinyl-CoA)
• Highly soluble due to low pKa
• High levels can sometimes produce lethal acidosis

Eicosanoids

• these 20-C mlcs are synthesized from __
• they are released from the __ of the cell membrane
• from what mlc is arachadonic acid released?

• these 20-C mlcs are synthesized from arachidonic acid
• they are released from the inner leaflet of the cell membrane
• arachidonic acid is released from PL-A2 (position 2 of phosphatidyl inositol)

There are 2 pathways for  arachidonic acid → eicosanoids, name them.

1. Cyclooxygenase pathway: produces prostanoids, including the prostaglandins, prostacyclin & thromboxanes
2. Lipoxygenase pathway: Produces the leukotrienes (bronchial constriction)

• Prostaglandins are synthesized from which essential FA?
• Which enzyme is used?
• This is a bi-fxnal enzyme what are the 2 fxns?

• Prostaglandins are synthesized from linoleic acid
• the enzyme used is Prostaglandin synthase (PGH synthase, PGS)
• cyclooxygenase & peroxidase (requires glutathione)

Note: prostaglandins synthesized in almost all tissues (via COX)

What is the difference btw Prostaglandin I & thromboxanes?

Prostaglandin I (aka prostacyclin) has a 5-membered oxygen containing ring

Thromboxanes have 6-membered oxygen containing ring

• Which prostaglandin is from intact endothelium and prevents platelet aggregation & thrombus formation?

• Which thromboxane acts on platelets & vascular smooth m. & is antagonized by PGI₂?

• PGI₂ is from intact endothelium and prevents platelet aggregation & thrombus formation

• TXA₂ acts on platelets & vascular smooth m. & is antagonized by PGI₂

Cyclo-oxygenase (COX) = Prostaglandin Synthase (PGS)

• Which COX is constitutive?
• Which COX is inducible?
• What common medication blocks COX-1?

• COX-1(PGS-1) is constitutive: enzyme of gastric mucosa
• COX-2 (PGS-2) is inducible: response to inflammation; activated monocytes & macrophages; suppressed by glucocorticoids
• Aspirin blocks COX-1 → doesn't block peroxidase/leukotrienes → contraindicated in asthma

Note: uncompetitive inhibition, critical serine residue

What are the 3 reasons that ketones are impt sources of energy for the peripheral tissues?

1. they are soluble in aq soln (i.e. don't need to be incorporated into lipoproteins or carried by albumin
2. produced in the liver when the amnt of acetyl CoA present exceeds the oxidative capacity of the liver
3. they are used in proportion to their conc in the blood by extrahepatic tissues (spare glucose during fasting)

Prostaglandins

• Which 2 prostaglandins are potent vasodilators?
• How do they carry out their fxn?
• How are these 2 used clinically?

• PGE & PGI are 2 prostaglandins that are potent vasodilators
• They work via 2nd messengers, i.e. ↑ in cAMP (tx-induced vasoconstriction ↓ cAMP & ↑ intracellular Ca²⁺
• They are used in surgery on small children w/ pulmonary stenosis to maintain the patency of the ductus arteriosus

Prostaglandins

• Which 2 prostaglandins induce uterine contraction?
• What hormone also participates in normal induction of labor?
• What's the difference btw the two?

• PGE₂ & PGF_2α are 2 prostaglandincs that induced uterine contractions
• Oxytocin also participates in normal induction of labor
• Unlike oxytocin, prostaglandins cause the uterus to contract powerfully @ all times → can be used to induce abortion

Inflammation

• Which 2 eicosanoids are local mediators of inflammation?
• What are the signs of inflammation?
• What happens if inflammation response is too extreme?

• PGE₂ & TXA₂ are local mediators of inflammation
• Rubor (redness), calor (heat) & dolor (pain)
• If inflammation response is too extreme, inhibitors (eg. aspirin, steroids) are often chosen

Eicosanoids

• These eicosanoids are powerful contrictors of bronchial & intestinal smooth m. 
• Are they more or less stable than other eicosanoids?
• Clinical implication?

• Leukotrienes are powerful constrictors of bronchial & intestinal smooth m.
• Leukotrienes are more stable (longer ½ life)
• LTC₄ (glutathione), LTD₄ & LTE₄ are implicated in the protracted bronchoconstriction in the asthmatic pt

Anti-inflammatory drugs

• What 2 types of anti-inflammatory drugs are used to block synthesis of eicosanoids?

The 2 types of anti-inflammatory drugs that are used to block synthesis of eicosanoids are:

1. Glucocorticoids (SAIDs) block all production of PL-A2 (eg. cortisol)
2. Non-steroidal anti-inflammatory drugs (NSAIDs) block constitutive COX-1 (acute cases COX-2 blocked) [ eg. aspirin, ibuprofen]

Note: Aspirin is contra-indicated in asthma; broncho-constriction is induced by leukotrienes not prostaglandins

aspirin diverts arachidonate → Leukotriene synthesis

Phospholipids

• What is the central phospholipid mlc?
• Where in the cell are phospholipids assembled?

• Phosphatidic acid → necessary for all other phospholipids
• Phospholipids are synthesized & assembled in the smooth ER

Note: packaged in vesicles for secretion via exocytosis

Group 1 Phospholipids

• The phosholipids base their structure on __
• What feature do they possess?
• What are the 3 origins of this mlc?

• The phospholipids base their structure on Phosphatidic acid
• These phospholipids possess a glycerol spine
• The 3 origins are: DHAP, glycerol-3-P & diacylglycerol *
  

*addt'n of fatty acyl group always requires it to be in the activated state - acyl-CoA

Group 2 Phospholipids

• Instead of glycerol these phospholipids contain __

• Linking a FA via an amide bond to -NH₂ of above mlc forms a __

• Instead of glycerol these phospholipids contain Sphingosine

• Linking a FA via an amide bond to -NH₂ of sphingosine forms a ceramide

Note: Sphingomyelin is a type of sphingolipid consisting of phosphorylcholine + ceramide

only phospholipid w/o a glycerol spine

Phosphatidic Acid is a central precursor mlc for which mlcs?

• TAGs (glycerol + 3 FAs
• Cardiolipin
• Phosphatidyl Inositol
• PAF
• Plasmalogens

• Which mlc = 2 phosphatidic acid mlcs esterified to glycerol
• How many FA tails are there?
• What part of the cell is affected?

• Cardiolipin = 2 phosphatidic acid mlcs esterified to glycerol
• there are 4 FA tails
• makes the inner mt membrane very dense

Plasmalogens

• __ → nerve tissue
• __ → heart muscle, lecithin, surfactant
• __ → apoptosis
• Unsaturated FA at C-1 w/ __ link

• PA-ethanolamine → nerve tissue
• PA-choline → heart muscle, lecithin, surfactant (DPPC)
• PA-serine → apoptosis
• Unsaturated FA at C-1 w/vinyl ether link

Note: PA-serine → PA-choline (3 methyl groups from essential Methionine via S-adenosylmethionine (SAM-ATP + methionine)

Platelet-activating factor (PAF)

• An ether glycerophospholipid
• What does it have at C-1 & C-2
• What are its roles?

• saturated FA @ C-1; acetyl group @ C-2
• triggers thrombotic rxns & inflammation responses

Phosphatidyl-choline (PA-choline)

• What are the 2 forms of synthesis?
• Translocation of __ from cytosol to ER  can activate lung surfactant

1. PA-serine + SAM
2. Choline + ATP → phosphocholine → (CTP) CMP + phosphocholine → CDP-choline (+diacylglycerol)  → phosphatidyl-choline

• Translocation of CTP-phosphocholine cytidylyltransferase from cytosol to ER  can activate lung surfactant

Note: Resp distress syndrome seen in newborns of diabetic mothers (deficient glucocorticoids)

Phosphatidyl Inositol (PIP2)

• Fxn?
• What's on C-1? C-2?
• Initial step of signalling: __ causes release of __
• Clinical correlation?
• What is the role of glycosylated phosphatidyl inositol (GPI anchor)

• Fxns in membrane-anchoring & signal transduction
• on C-1: stearate; on C-2: arachidonate (PLP-A2)
• Initial step of signalling: PLP-C causes release of IP3 (& DAG) from PIP2
• Lithium inhibits inositol phosphatase, preventing inositol's release, blocks resynthesis of phosphatidyl inositol, bisphosphate
• GPI-anchors proteins to the membrane's surface (N-terminal end of GPI protein)

Phospholipases (PL)

• egs. pancreatic juice, toxins & venoms
• PLP-A2 → __
• PLP-C → __
• sphingomyelinase degrades __

• PLP-A2 → arachidonic acid
• PLP-C → IP3/DAG
• sphingomyelinase degrades sphingomyelin

Sphingomyelin

• Where is sphingomyelinase found?
• How is sphingomyelin degraded?
• Clinical correlation - defective sphingomyelinase?

• Sphingomyelinase is found in the lysosomes
• Sphingomyelin  (1. remove phosphorylcholine) → ceramide (2. ceraminidase) → FA + sphingosine (blocks PKC (IP3/DAG)
• If sphingomyelinase defective can't degrade sphingomyelin → Niemann-Pick (AR)
  • Type A - neurodegenration, early death
  • Type B - no neural involvement
  • Ashkenazi pop

Glyco(sphingo)lipids

• No __ - Ceramide backbone
• Where are they located?

1. __ → simplest (no charge)
   1. ceramide + gal/glu
   2. Galactocerebroside → most common in __
2. __ → most common/complex (acidic)
   1. (CMP) __/sialic acid addt'n @ C-terminal
3. __ → strong negative charge
   1. Galactocerebroside + __ (via PAPS) = sulfatide

• No Phosphate - Ceramide backbone
• Located on plasma membrane's outer leaflet

1. Cerebrosides → simplest (no charge)
   1. ceramide + gal/glu
   2. Galactocerebroside → most common in glycocalyx
2. Gangliosides → most common/complex (acidic)
   1. (CMP) NANA/sialic acid addt'n @ C-terminal [only mononucleotide activator known]
   2. Botox
3. Sulfatides → strong negative charge
   1. Galactocerebroside + SO₄ (via PAPS*) = sulfatide

*PAPS - 3'-phosphoadenosine-5' phosphosulfate = 3ATPs; ≈ SAM (1 ATP)

• Where are glyco(sphingo)lipids synthesized?
• All sugars but __ must be in UDP-activated state
• 2 enzymes are used, what are they?

• glycosphingolipids are synthesized in the golgi
• All sugars but NANA must be in UDP-activated state
• Glycosyl transferases (O-linked) or Sulfotransferases (use PAPS; S from methionine)

Note: "last on = first off"

Sphingolipidoses

• General class of deficiencies is?
• Mode of inheritance?
• Tay-Sach's disease is a deficency in __
• Niemann-Pick = ?
• __ is the most common of the sphingolipidoses, mental retardation absent, β-Glucosylceramidase deficiency
• __ α-galactosidase deficiency

• Deficiencies: specific lysosomal hydrolases
• All AR except Fabry's disease (X-linked)
• Tay-Sach's disease is a deficiency in Hex-A, GM₂ (gangliosides) build-up (cherry-red macula)
• Niemann-Pick = sphingomyelinase deficiency; sphingomyelin builds up
• Gaucher's disease is the most common of the sphingolipidoses, mental retardation absent, β-Glucosylceramidase deficiency, Glucosylceramide builds-up
  
• Fabry's disease - X-linked, α-galactosidase deficiency, trihexosylceramide (globosides) builds up
  

GAG structure

• chains of repeating __ units containing these 2 types of sugars

1. an __ sugar (eg. glucuronic acid)
2. __ sugar (eg. N-acetylglucosamine)

• chains of repeating disaccharide units containing these 2 types of sugars

1. an acidic sugar (eg. glucuronic acid)
2. amino sugar (eg. N-acetylglucosamine)

GAGs - amino sugars

• carboxyl groups -vely charged @ physiological pH values
• __ groups are strongly negative
• negative charges are the physical basis for the __ of GAGs

• carboxyl groups -vely charged @ physiological pH values
• sulfate groups are strongly negative (keratan, dermatan, chondroitin, heparin sulfate)
• negative charges are the physical basis for the resilience of GAGs (eg. cartilage)

Name that GAG

• This GAG is the most abundant in the body
• This GAG is the most heterogeneous
• This GAG is unsulfated and is the only one not covalently bond to proteins 
• This GAG serves as an anti-coagulant & is an IC component of mast cells
• This GAG is found in basement membranes

• Chondroitin 4- & 6-sulfates are the most abundant in the body
• Keratan Sulfates are the most heterogeneous
• Hyaluronic Acid is unsulfated and is the only one not covalently bond to proteins(serine residues)
• Heparin serves as an anti-coagulant & is an IC component of mast cells
• Heparan Sulfate is found in basement membranes

• What is the 1st stem amino sugar?
• What 2 univ cmpds are used?

• The 1st stem amino sugar is glucosamine-6-P
• The 2 univ cmpds are F-6-P & Glutamine (-NH₂) source

Note: this leads to NANA activated by CMP (CTP = source)

• What is the initial substrate for the uronic pathway (vit C synthesis)?
• Which substrate makes less polar cmpds more soluble?

• UDP-glu is the initial substrate for the uronic pathway (vit C synthesis)?
• UDP-glucuronate is the substrate that makes less polar cmpds more soluble?

Synthesis of carbohydrate chains

• step 1: sugars are joined to the -OH group of threonine or __
• UDP-xylose (5-C) + core protein → core protein-xylose + UDP
  
  • what's the enzyme?
• 2 __ added (step 2)
• add __ from PAPS
• How does the synthesis of GAGs differ from that of glycogen?

• step 1: sugars are joined to the -OH group of threonine or serine
• UDP-xylose (5-C) + core protein → core protein-xylose + UDP
  
  • xylosyl-transferase
• 2 gal's added (step 2)
• add -SO₄ from PAPS (step 4)
• GAGs are produced for export from the cell, so produced in the Golgi rather than the cytoplasm

Note: UDP-activation essential for each sugar attachment

PAPS

• When does the sugar get sulfated?
• From where do the -SO₄ groups come?
• Synthesis of PAPS uses how many ATP equivalents?
• Defects in sulfation → __

• The sugar gets sulfated after it has been attached to the nascent carbohydrate chain
• -SO₄ groups come from PAPS
• Synthesis of PAPS uses 3 ATP equivalents
• Defects in sulfation → chondrodystrophies

Degradation of GAGs

• Where does degradation occur?
• Degradative enzymes destined for the lysosomes bear what tag?
• What must precede degradation? Why?

• Degradation occurs in the lysosomes
• Degradative enzymes destined for the lysosomes bear a Mannose-6-P tag
• Phagocytosis must precede degradation because GAGs have extracellular fxns

• What's the name for the class of diseases involving GAGs?
• Mode of inheritance?
• What's the common enzyme deficiency?

• Mucopolysaccharidoses is the name for the class of diseases involving GAGs
• Mode of inheritance is Autosomal Recessive
• Enzyme deficiency: exo-glucosidases

Name the Mucopolysaccharidosis

• α-L-iduronidase; AR; corneal clouding; "coarse" feature
• iduronate sulfatase; X-linked; no corneal clouding
• β-glucuronidase; affects degradation of dermatan & heparan sulfate; hepatosplenomegaly
• involves 4 enzyme steps that remove N-sulfated or N-acetylated glucosamines from heparan sulfate

• Hurler's: α-L-iduronidase; AR; corneal clouding; "coarse" feature
• Hunter's: iduronate sulfatase; X-linked; no corneal clouding
• Sly's: β-glucuronidase; affects degradation of dermatan & heparan sulfate; hepatosplenomegaly
• Sanfilippo's: involves 4 enzyme steps that remove N-sulfated or N-acetylated glucosamines

• almost all of our secreted globular proteins are part of this group
• N-linked have this aa btw the protein & carbohydrate
• O-linked have this aa
• both N- and O-linked share the same core __

• almost all of our secreted globular proteins are glycoproteins
• N-linked have asparagine btw the protein & carbohydrate
• O-linked have serine or threonine
• both N- and O-linked share the same core pentasaccharide

Used for our ABO blood group proteins

• this oligosaccharide does not elicit antibodies in most humans
• type A is __
• type B is __

• the O oligosaccharide does not elicit antibodies in most humans
• type A is O+GalNac
• type B is O+Gal

• Major exception to the O-linked aa pairing is what?
• On what is the formation of this -OH group dependent?

• Major exception to the O-linked aa pairing is the linkage btw galactose or glucose and the -OH of hydroxylysine in collagen
• the formation of this -OH group is vitamin C-dependent (deficiency = Scurvy)

• Where does the O-linked oligosaccharide fxn?
• N-linked are associated w/ high __ content & this membrane anchor__

• the O-linked oligosaccharide fxns in the glycocalyx
• N-linked are associated w/ high mannose content & this membrane anchor dolichol

• UDP is for __ & __
• GDP is for __ & __
• CMP is for __
• All attachments are __

• UDP is for glucose & galactose
• GDP is for mannose & fucose
• CMP is for NANA
• All attachments are covalent

O-linked synthesis

• Glycoproteins are synthesized in the __, travel through the __ & later released or incorporation into __ or __

• Glycoproteins are synthesized in the RER, travel through the Golgi & later released or incorporation into membrane or lysosome

N-linked synthesis

• Synthesized in the __, instead of glycosylation a lipid-linked oligosaccharide is fashioned consisting of __ attached through a __ linkage
• The oligosaccharide is transferred from dolichol to?
• What are the terminal/final groups?

• Synthesized in the RER, instead of glycosylation a lipid-linked oligosaccharide is fashioned consisting of dolichol attached through a pyrophosphate linkage
• The oligosaccharide is transferred from dolichol to an asparagine side group of the protein
• Fucose or NANA are the terminal/final groups

1. It can become part of the outer aspect of the plasma membrane
2. It can become translocated to the lysosomes (man-6-P receptors)

Note: the man-6-P receptors are recycled numerous times for re-use

I-cell disease

• absence of __ → substrates can't be __ accumulate → likely cause is the inability to add __ → incorrect targeting & delivery

• absence of lysosomal hydrolases  → substrates that can't be destroyed accumulate → likely cause is the inability to add -PO₄ phosphorylate mannosyl residues (no man-6-P tag)→ incorrect targeting & delivery

Degradation of glycoproteins

• __ attack from the outside → inside
• removal of groups is in what order? consequences?
• oligosaccharidoses, mode of inheritance? partially degraded cmpds in __? after cell death these appear in urine

• exoenzymes attack from the outside → inside
• removal of groups is last attached is 1st removed (= glycosphingolipid degd'n scheme); if one step can't fxn the next are stymied
• oligosaccharidoses, AR, partially degraded cmpds in lysosomes after cell death these appear in urine

GAGs vs. Glycoproteins

• This one is 1° carbohydrate w/ a small amnt of protein
• This one is 1° protein w/ a small amnt of carbohydrate
• This one can bind lg amnts of H₂O; forms the basis for ground substance & is a component of mucous secretions

• GAGs are 1° carbohydrate w/ a small amnt of protein
• Glycoproteins are 1° protein w/ a small amnt of carbohydrate
• GAGs can bind lg amnts of H₂O; forms the basis for ground substance & are a component of mucous secretions

CHL metabolism

• What 3 things are req'd?
• What is the precursor?
• What is the rate-limiting step?
• What is the 1st steroid product?
• How many ATP & NADH are used per mlc?
• How many carbons in cholesterol?

• Requires: ↑ energy state, ↑ NADPH, ↑ insulin (↓ CHL)
• Precursor: (2) Acetyl CoA
• Rate-limiting step: HMG CoA Reductase
  • active in the un-phosphorylated state
• 1st steroid product: Lanosterol (30 C')
• 3ATP & 4 NADH per CHL mlc
• CHL = 27 C' mlc

Note: 1-OH (3C') group; 2 methyl groups (18C'/19C'); & branched hydrocarbon chain (17C')

• On what does the absorption of CHL depend?
• How does Acetyl CoA become available for CHL synthesis?
• What uses the same process?

• The absorption of CHL depends on bile salts & dietary intake
• Acetyl CoA has been shuttled to the cytosol via citrate 
• ketones use the same shuttle

• What is the 1st committed step of CHL synthesis?
• What is req'd NADPH or NADH?

• Mevalonic acid synthesis is the 1st committed step 
• 2 NADPH are req'd

1st committed step → 3 phosphorylations, 1 decarboxylation convert mevalonate 1st to IPP (5C') then DPP (5C')

6 such 5C' units form 30-C' __ (first one to be __) which is then cyclized to __ (1st __ product)

6 such 5C' units form 30-C' Squalene  (first one to be non-phosphorylated) which is then cyclized to Lanosterol (1st steroid product)

• Regulation of CHL synthesis via __ at gene level, __ is the target
• Inhibition at the transcriptional level: ↑CHL = proteases → __
• Inhibition at the post-transcriptional level → covalent modification, i.e. __

• Regulation of CHL synthesis via feedback inhibition at gene level, HMG-CoA reductase is the target
• Inhibition at the transcriptional level: ↑CHL = proteases → breakdown HMG-CoA reductase
• Inhibition at the post-transcriptional level → covalent modification, i.e. phosphorylation

• Insulin/Glucagon which one favors production of HMG-CoA reductase? 
• What's the effect of the other hormone?
• Rapid CHL delivery & uptake has what effect on trx rates?

• Insulin (storage hormone) stimulates HMG-CoA reductase production
• Glucagon inhibits synthesis of lipids → low energy intake; HMG-CoA reductase gets phosphorylated (inhibited) by an AMP-activated PK
• Rapid CHL delivery & uptake → lower trx rates

Note: Ser is phosphorylated

• What drugs can be used to lower CHL production? fxn?

• Statins → reversible competitive inhibitors of HMG-CoA reductase

• What are the precursors to isoprenoids?
• What are our principal isoprenoids?

• The precursors to isoprenoids are IPP & DPP
• Our principle isoprenoids are: Ubiquinone, Dolichol, side chain of Heme a & Farnesyl/geranyl groups

• Farnysl is 15-C mlc that has what purpose in the cell?
• What enzyme is used?
• How is it a target in medicine?

• The purpose of Farnysl is to anchor proteins into the membrane
• The enzyme used is farnysl transferase
• It is an attractive target for inhibition in CA chemotherapy (RAS); inhibitors interfere w/ CA cell growth

• Which vitamins are also isoprenoids?
• Phytanic acid is also an isoprenoid in what disease does it accumulate (hint we've seen it before in FA catabolism)

• the fat-soluble vitamins are isporenoids
• Phytanic acid accumulates in pts w/ Refsum's disease (due to deficiency of α-hydroxylase)

*Activation → Hydroxylation (Refsum's) → Decarboxylation → β-oxidation (normal)

• Vit D₃ (cholecalciferol) arises by synthesis from __ (intermediate in cholesterol biosynthesis)
• Vit D₃ is a __

• Vit D₃ (cholecalciferol) arises by synthesis from 7-dehydrocholesterol (intermediate in cholesterol biosynthesis)
• Vit D₃ is a pro-hormone

• steroid nucleus can't be degraded in the human body, sol'n?

• the sol'n to steroid nucleus not being able to be degraded in the human body is to dispose of CHL via the biliary system

Bile acid synthesis (24C')

• Where does feedback regulation take place?
• At what gene(s)?
• Bile acids are formed from  CHL in the __
• Dietary CHL induces __ & inhibits __

• Feedback regulation takes place at the 1st step
• 1st step: 7-α-hydroxylase gene & upstream @ HMG-CoA reductase gene
• Bile acids are formed from  CHL in the liver 
• Dietary CHL induces 7α-hydroxylase (Thyr Hor does also) & inhibits HMG-CoA reducatase

• Bile acids are __ at ph 7.0
• 1° bile acids include: __ & chenodeoxycholic acid
• Bile acids → bile salts after conjugation w/ __ & __

• Bile acids are 90% deprotonated at ph 7.0 (100% when become salts)
• 1° bile acids include: cholic acid & chenodeoxycholic acid (cholic acid is more abundant, must 1st be activated)
• Bile acids → bile salts after conjugation w/ glycine & taurine (-SO₄; S from Met via Cys sulfinate decarboxylase)

Note: Ratio of glycine:taurine ≈3:1

Ascorbate deficiency impairs formation of bile salts

CoASH released upon gly or tau esterification

Bile salt features

1. hydrocarbon chain shortened by __, terminal C is oxidized to a carboxy group
2. __ groups added to original one
3. all -OH groups in the __ configuration
4. fusion btw A & B rings is in __-configuration
   1. result: bend btw rings A & B

1. hydrocarbon chain shortened by 3-C, terminal C is oxidized to a carboxy group
2. 1-2 more -OH groups added to original one
3. all -OH groups in the α- configuration
4. fusion btw A & B rings is in cis-configuration
   1. result: bend btw rings A & B

• What happens to the bile salts when they get to the small intestine? What does it?
• What gets removed to make "neutral sterols"

• The amide bond btw Tau/Gly & cholic acid gets deconjugated in the small intestine by bacteria (bile salts → bile acids)
• 7a-hydroxyl group gets removed to make "neutral sterols" → 2° bile acids

*1° & 2° bile acids/salts return to liver via entrohepatic circulation; ≈ 98% absorbed via Na-co-transport mech. in ileum

• Most common cause of gallstones?
• What's happening to pts w/ biliary duct obstruction?
• ↑ in CHL or ↓ in bile salts → supersaturation → __ → jaundice

• CHL precipates from mixed micelles
• biliary duct obstruction: serum levels of bile acids rise sharply - no neg feedback on 7α-hydroxylase
• ↑ in CHL or ↓ in bile salts → supersaturation → cholestasis (suppression of bile flow) → jaundice

Bile acid summary

• Rate limiting step: ?
• __ impairs formation of bile salts & collagen
• Bile salts returning through the portal system __ 7α-hydroxylase
• __ hormone ↑es synthesis of 7α-hydroxylase
• ↑CHL = ↑7α-hydroxylase = __

• Rate limiting step: 7α-hydroxylase (Cyt-P450)
• Ascorbate deficiency impairs formation of bile salts & collagen
• Bile salts returning through the portal system inhibit 7α-hydroxylase
• Thyroid hormone ↑es synthesis of 7α-hydroxylase
• ↑CHL = ↑7α-hydroxylase = ↓HMG-CoA reductase

Name the chemical signal route

• this describes the transport of hormones through blood & causes a response in a distant target tissue
• this is transport NOT through blood, instead diffuses to neighboring cells
• released messenger acts upon the synthesizing cell itself
• these paths are used by eicosanoids

• endocrine: the transport of hormones through blood & causes a response in a distant target tissue
• paracrine: transport NOT through blood, instead diffuses to neighboring cells
• autocrine: released messenger acts upon the synthesizing cell itself
• para- & autocrine are used by eicosanoids

Steroid Hormones

• What is their basis?

• Steroid hormones come from cholesterol

*most CHL comes from endogenous synthesis (liver)

Steroid Hormones

• What ctrls steroid hormone synthesis via tropic hormones?

• the anterior pituitary ctrls steroid hormone synthesis via tropic hormones

*tropic hormones also stimulate CHL esterase & LDL receptors

Steroid Hormones

• What's a unique property of the steroid nucleus?
• What are the possible modifications?

• The steroid nucleus is indestructible

1. creation of -OH groups
2. oxid/red of side groups
3. for estrogens - ring A aromatized (aromatase)

Biosynthetic Pathway Steroids

• side chain at C-17 cleaved to 2 carbons (__) or lost (__)
• All steroid hormones inherite __ @ C-3
• All mineralocorticoids - __ @ C-18
• Estrogens - aromatic __

• side chain at C-17 cleaved to 2 carbons (progestins, corticosteroids) or lost (androgens, estrogens)
• All steroid hormones inherit -OH group @ C-3
• All mineralocorticoids - aldehyde group @ C-18 (eg. aldosterone)
  
• Estrogens - aromatic ring A (aromatase) [testosterone is aromatized (aromatase) to produce the estrogens)

Hydroxylation of steroids

• What type of enzyme(s) are used in this rxn?
• What's req'd for this rxn?
• Write the equation

• mono-oxygenase enzymes - membrane bound: seens as cytosolic
• Cytochrome P-₄₅₀ is req'd for this heme containing rxn
• Steroid-H + O₂ +NADPH + H⁺ → Steroid-OH + NADP⁺ + H₂O

• What is the 1st step in steroid hormone synthesis?
• What is the starting material for all the steroids?
• What are the 2 paths for the mlc in the previous question?

• Side-chain cleavage rxn by desmolase (Cyp11a) [via NADPH requiring mono-oxygenase]
• Pregnenolone is the starting material for all of the steroids
• Pregnenolone can go to progesterone or 17-α-hydoxypregnenolone

• __ is the precursor to both estradiol & dihydrotestosterone (DHT)
• __ is mainly formed in granulosa (theca) cells of ovarian follicle (during pregnancy placenta is main source)

• testosterone is the precursor to both estradiol & dihydrotestosterone (DHT)
• estrogen is mainly formed in granulosa (theca) cells of ovarian follicle (during pregnancy placenta is main source)

*in menopausal women estrogen is produced as estrone from adrenal androgen androstenedione

Name the steroid

• synthesized in the testis (1°), adrenal cortex & the theca cells @ edge of ovarian follicle
• major androgen from Leydig Cells (LH) precursor to DHT
• major androgen from adrenal cortex

• Androgens: synthesized in the testis (1°), adrenal cortex & the theca cells @ edge of ovarian follicle
• Testosterone: major androgen from Leydig Cells (LH) precursor to DHT (via 5-α-reductase)
• Androstenedione: major androgen from adrenal cortex (zona reticularis)

• __ produces DHT from testosterone
• Defiency of this enzyme lead to what problem?

• 5-α-reductase produces DHT from testosterone
• DHT is req'd for prenatal development of ext male genitalia (& "drop" of testes) so w/ enzyme deficiency predominantly female ext appearance @ birth, puberty become male

*Testosterone production = Muellerian degeneration & Wolffian duct formation

Congenital Adrenal Hyperplasias (CAHs)

• What is the only hormone known to ctrl adrenal steroid synthesis?
• Adrenal cortex
  • zona __ = aldosterone synthesis
  • zona __ = glucocorticoids
  • zona __ = glucocorticoids (steroids)
• Adrenal medulla - __

• ACTH is the only hormone known to ctrl adrenal steroid synthesis
• Adrenal cortex
  • zona glomerulosa = aldosterone synthesis
  • zona fasicularis = glucocorticoids
  • zona reticularis = glucocorticoids (steroids)
• Adrenal medulla - catecholamines

Androgenital Syndrome

• What deficiency causes this and why?

• A deficiency in either 21-α-hydroxylase or 11-β hydroxylase can cause this b/c both are needed for glucocorticoid synthesis

Androgenital Syndrome

• lack of glucocorticoids → loss of __ of ACTH release → excess ACTH → __ → further stimulation of side chain rxn (desmolase)
• as corticosteroids blocked → initially formed progestins are diverted to __
• complete deficiency of either 11-β or 21-α-hydroxylase → __

• lack of glucocorticoids → loss of neg feedback ctrl of ACTH release → excess ACTH → adrenal hyperplasia → further stimulation of side chain rxn (desmolase)
• as corticosteroids blocked → initially formed progestins are diverted to androgen synthesis (1° cause of the CAHs)
• complete deficiency of either 11-β or 21-α-hydroxylase → hyponatremia (low Na⁺) or hyperkalemia (high K⁺)

*virilization & electrolyte imbalances can be cured w/ corticosteroid txs

Non-CAH egs of low corticosteroid

1. hypersecretion of ACTH, overproduction of corticosteroids (cortisol), often from a pituitary adenoma
2. from adrenocortical insufficiency (i.e. insufficient production of mineralo-/glucocoritcoids) think lesion/destruction of adrenal gland (↑ ACTH)

1. Cushing's syndrome (not disease as seen in FA oxid file)
2. Addison's disease

CAH mech'm

1. Precursor backs up → __
2. loss of __
3. __ stimulation continues
4. tissue grows in attempt to find __

1. Precursor backs up → another route
2. loss of neg feedback by product
3. ACTH stimulation continues
4. tissue grows in attempt to find the "right" level of the hormone that can't be produced (vicious cycle)

• major activator of gluconeogenesis
• Which gluconeogenic enzymes are most likel to be affected?

• cortisol: major activator of gluconeogenesis
• The main gluconeogenic enzymes to be affected are: G-6-Pase, PEPCK, Pyr decarboxlase

• Aldosterone formation from pregnenolone involves what?

• production of aldosterone from pregnenolone involves 3  hydroxylations, followed by 1 NAD⁺ requiring dehydrogenation (oxidation)

*Hydroxylations require NADPH; oxidations require NAD⁺

Aldosterone

• Why is aldosterone produced
• What is the mech'm?

• Aldosterone is produced in response to angiotensin & lowered Na⁺/K⁺ ratio
• Aldosterone raises Na⁺ absorption (↓Na⁺ loss, ↑K⁺ loss) → ↑ H₂O retention → ↑blood vol & pressure

Anabolic steroid side effects

• → elevated CHL
• male: __
• female: __
• Adolescence:__

• → elevated CHL (↑LDL, ↓HDL)
• male: testicular atrophy, gynecomastia, aggression
• female: hirsutism, deep voice
• Adolescence: cessation of long bone growth