1440 week 11-12

Steps of SGLT transport

1."Na+ Binding pocket" binds Na+ at high concentration in Sm. Intestine fluid 2.Na+ binding induces a shape change to form a "Glucose binding pocket" 3.Glc binds to high affinity pocket 4.SGLT "flips" to intracellular side 5.Low Na+ levels in cytosol, so Na+ unbinds, thus SGLT changes back to not having "Glc binding pocket", so Glc leaves and enters cytosol 6.SGLT "flips" empty Na+ binding site back to extracellular side, same as Step •Na+ removed from cytosol by Na+/K+ ATPase pump to maintain gradient (not shown)

NADPH acts as an enzyme cofactor to reduce various molecules in a variety of body functions.

1.NADPH for anabolic (build more complex molecules) reactions in adipocytes (fatty acid & cholesterol synthesis) and liver (CYP reductase). •In liver, 30% of Glucose-6P can diverted to PPP Ox-branch 2.NADPH is important in regenerating Glutathione. GSH (reduced thio) from GSSG (oxidized). GSH protects against reactive oxygen species (ROS) •Deficiency in G6PDH leads to ROS lysing RBCs when high oxidants/ROS (favism from fava bean). FYI: Maybe, a protection against malaria. 3.NADPH is an important cofactor for the macrophage enzyme that reduces O2 to form ROS radicals to destroy bacteria and possibly plaque formation. 4.NADPH is an important cofactor for Nitric Oxide Synthase (NOS). Nitric Oxide (NO) is important signaling molecule, including signaling vasodilation.

When Fe is abundant, liver makes and secretes a protein called hepcidin. Hepcidin binds to Ferroportin, causing its endocytosis. The transport of Fe into blood is down-regulated, thereby reducing Fe absorption. Any absorbed Fe is trapped inside the cells and ultimately is eliminated in stool when dead enterocytes slough off. Hepcidin is also secreted in response to inflammation- (bacteria need Fe to grow). Chronic inflammation can lead to anemia of inflammation.

A substantial amount of Tf-bound Fe arises from recycling. Macrophages in the spleen degrade old RBCs (~1%/day). The Fe is captured and released into the blood using the same ferroportin system described above. Hepcidin can also block the release of Fe from macrophages in the spleen.

Glucose- six H-C-OH carbon covalently linked

C1 is a aldehyde in linear glucose. C1 forms with C5 a hemiacetal in cyclic glucose. C6 is not part of the circle in cyclic glucose.

Glucose Polysaccharide "Starches":

Cyclic monosaccharides linked together by O-Glycosidic bonds to C1 in the hemiacetal form.

Lactate Fermentation for Anaerobic respiration: Recycle NADH + H+-> NAD+ by Hydrogenating Pyruvate-> Lactate

Cytosolic: Pyruvate + NADH/H+ -> Lactate/Lactic Acid + NAD+ Lactate DeHydrogenase (LDH) •Lactate/Lactic Acid can exit cell •May make cytosol acidic, Acid inhibits PFK-1, so indirect negative feedback •Lactate can be used by the liver in gluconeogenesis

Digestive Enzymes "Break" polysaccharide C1-O-C4 glycosidic bonds by:

Digestive Enzymes "Break" polysaccharide C1-O-C4 glycosidic bonds by: 1) bonding O-C4-end glucose to H+ and not C1-glucose of starch. 2) replacing C1-lost oxygen with OH-. Called hydrolysis, but H+ and OH- are not from the same water

Antioxidants: Glutathione (GSH):

Glutathione (GSH): Intracellular levels of this compound are ~ 5mM. This tripeptide is critical for managing oxidative stress. For example, overdose of acetaminophen depletes cellular stores of reduced glutathione and the subsequent oxidative overload causes hepatotoxicity. GSH is g-Glu-Cys-Gly. The isopeptide bond with Cys is made with the side chain carboxylate of Glu, not the normal alpha carboxy group. The Cys side chain sulfur is the redox active atom in GSH. (similar to peroxiredoxin above)

Glycolysis Definition/Purpose:

Glycolysis Definition/Purpose: Enzyme catalyzed phosphoryl-intermediates and bond 'shifts' to Transfer Energy from: Glucose-6-P (use ATP initially break glucose) with: 2PÒ, 2ADP & 2NAD+ to form: 2ATP, 2NADH, 2H2O & 2 Pyruvate

step 1 of glycolysis Reversible Isomerase: Flips Double bond of Glc-6-P C1-aldehyde to C2-ketone to form Fructose-6-P

Notice: C-6-PÒ is not altered in the isomerization http://clfs690.alivetek.org/CLFS690/glycolglucojmol/g6pisomerasejsmol.htm

Dietary Absorption of Fe

Only a fraction of dietary Fe is actually absorbed, typically 1-2 mg/d. Absorption of Fe can be either inorganic Fe (non-heme Fe) or heme in meat. Inorganic Fe in foods, especially plant-derived foods, shows low bioavailability. This is because the Fe is tightly bound to plant proteins, polyphenols and phytic acid. Taking ascorbic acid with the meal can enhance bioavailability of non-heme Fe. The Fe in heme is more bioavailable. Sufficient Fe levels in the body are maintained by regulating intake, since there is no active mechanism for elimination.

The decomposition of H202 is catalyzed by catalase (in peroxisomes) and peroxiredoxins: 2 H202 => 02 + 2H20

Put this reaction together with the one catalyzed by SOD (above) and we see that catalase and peroxiredoxin are required (indirectly) for the detoxification of superoxide

As we shall see below, the reaction of Ó or superoxide with organic molecules can produce organic peroxides:

RH + Ó => R-O-O-H (Don't confuse R-O-O-H with a carboxylic acid).

Catalase is specific for hydrogen peroxide and will not detoxify organic peroxides. Glutathione peroxidase is one enzyme responsible for eliminating organic peroxides. Glutathione (GSH) is a critical endogenous antioxidant [Slide 3]. Glutathione peroxidase has Se at the active site- hence Se is considered to be antioxidant.

ROOH + 2GSH => ROH + GSSG + H20

Peroxiredoxins are also involved in detoxification of organic peroxides. The enzymes contain one or two Cys residues at the active site that are oxidized by the peroxide. The oxidized enzyme is regenerated to the reduced form by another enzyme (e.g., disulfide reductase), which may use GSH for reducing equivalents.

The importance of peroxiredoxins is underscored by the fact that they are the third most abundant protein in RBCs and are present in high amounts in other cells. (R-SOH = sulfenic acid, Slide 4). Collectively, superoxide dismutase, catalase, glutathione peroxidase and peroxiredoxins are our body's first line of enzymatic defense against oxidative damage.

Toxic Arsenate

Toxic Arsenate (AsO43-) mimics PO43- in G3PDH enzyme; forming 1- AsO3-Glycerate-3-P instead of 1,3-BPG. So ATP cannot be formed by PG kinase •(Note: glycolysis is not the only metabolic process affected by arsenate) •One can build up tolerance to Arsenate (not yet understood, possibly involving liver)

Sodium-glucose transporter (SGLT)

Use Na+ gradient to move Glucose against its gradient from intestine fluid (chime) into intestinal epithelia cytosol SGLT1- Intestinal epithelia cell microvilli membrane SGLT2- In Kidney for reabsorption of Glucose (Glc) Other SGLT exist

Excess Fe that is not needed for proteins is stored as a complex with ferritin. Ferritin is a large multisubunit (24) protein with a central cavity that can store up to 4500 atoms of Fe. This Fe store is labile, meaning that the Fe can be easily released for use by cytochromes or enzymes as needed.

When Fe is high, it is stored as hemosiderin. Hemosiderin is an insoluble, modified form of ferritin. The Fe in hemosiderin is not as labile as in ferritin. Iron overload in tissues is characterized by the presence of hemosiderin in the tissues (can be detected histologically). • The importance of Tf for Fe delivery is illustrated by a naturally occurring mutation in mice that causes hypotransferrinemia (low blood transferrin). These mice show profound anemia, thus demonstrating the importance of Tf for providing the Fe needed for erythropoiesis.

Antioxidants: b-carotene:

b-carotene: The pigment in carrots. A lipid-soluble molecule, precursor to retinol that acts similarly to vitamin E.

glycolysis is like shooting a bow and arrow

glycolysis is like shooting a bow and arrow •Remove arrow from quiver---Glc to G-6-P to F-6-P; ATP->ADP •Notch the arrow. F-1,6-biP; ATP->ADP •Draw the bow. Split F-1,6-biP to GAP, DHAP •Aim, Release the string & hit target. Form 2ATP & 2NADH •Retrieve arrow. PEP to Pyruvate 2ADP->2ATP •Replace back in quiver. Pyruvate and NADH to lactate + NAD+ Alternative: to acetyl-CoA + NADH shuttle

Glucose Uniport Transporters (GLUT)- Transport Glucose by diffusion down its concentration gradient

only glut 2 not high affinity

Cellulose & Chitin have alternate Glucose orientation in the polysaccharide chain,

starch & glycogen (animal starch) do not. Thus, humans can only digest starch & glycogen, their enzymes can not attack the bonds

Antioxidants: Bilirubin:

• Bilirubin: A lipid-soluble metabolite of heme.

Antioxidants: Urate:

• Urate: A breakdown product of purines that the body puts to good use before eliminating it. In general, lipid soluble antioxidants cover membrane damage, and water soluble antioxidants deal with DNA and protein damage.

Mitochondria Shuttles (1 & 2) of NADH

•Cytosolic NADH->NAD+ by intermediate red/ox molecules that shuttle into mitochondria and either •Shuttle Malate/Asp/OAA: NAD+->NADH for ETS (in matrix) •Shuttle DHAP/Glycerol-3-P: FAD->FADH2 for ETS (in intramembrane space)

Transketolases (via Vitamin B1) and Transadolases use nucleophilic attack of the C=O bond of sugars to cleave & add CHO to sugars

•Vit B1 (Thiamin) is required for Transketolase •Schiff base is used for Transaldolase

anemia

A condition in which the blood is deficient in red blood cells, in hemoglobin, or in total volume.

Anaplerotic reactions Don't forget about OAA. To start the cycle we need both acetylCoA and OAA. The activity of the TCA cycle can be affected by the availability of OAA. In most cases, OAA levels in the mitochondria are subsaturating for citrate synthase. Thus, an increase in OAA will lead to increased TCA cycle activity.

As mentioned in the beginning, the TCA cycle also supplies precursors for anabolic reactions. Thus, some of the intermediates like citrate, aKG, succinate, OAA can be siphoned off for biosynthetic reactions. This will deplete the OAA levels. Acetyl-CoA cannot restore OAA because its two Carbons are lost as CÓ. (No net gain of carbon). Reactions that restore net levels of OAA are termed "anaplerotic reactions".

CELLULAR USE OF GLUCOSE-6-PHOSPHATE

CELLULAR USE OF GLUCOSE-6-PHOSPHATE Glucose-6-Phosphate is the initial substrate for Glycolysis, Pentose Phosphate Pathway and other Pathways.

Cellular Uptake. Cells get their Fe by endocytosis of Tf. [also see Slide 7] (But there are additional minor pathways) • Nearly every cell expresses a Transferrin Receptor (TfR). The amount of TfR is regulated by the cell's need for Fe. • TfR binds ferricTf and the complex is carried to late endosomes by the process of receptor-mediated endocytosis. The luminal pH of late endosomes is ~5.5. The low pH causes Fe(III) to dissociate from Tf. The free Fe is then transported into the cytoplasm by DMT1. (After reduction to Fe(II) by a ferrireductase) • The now apoTf is recycled back to the plasma membrane. Unlike ferriTf, apoTf cannot bind TfR at neutral pH and therefore is released into the extracellular fluid to search out more Fe.

Fe in the cytoplasm can now bind to proteins as needed. As usual, we have to be careful with free Fe, even in the cytoplasm. Thus, the distribution of Fe to cellular proteins is managed by Fe chaperones, which help deliver and often insert the Fe into the proteins that need it (Slide 8)

Regulation of proteins involved in iron metabolism is a specific example of translational regulation. Three key proteins involved in iron metabolism are:

Ferritin: stores excess iron in cells Transferrin: binds iron in plasma Transferrin receptor: a membrane protein, binds transferrin/Fe for uptake into cells. When iron levels are low, transferrin receptor (protein) synthesis increases and ferritin synthesis decreases (in line with the function of the proteins). When iron levels are high, the reverse happens. So, transferrin receptor and ferritin synthesis are reciprocally regulated. This regulation occurs at the translational level, as described below.

How does the body handle Fe?

For most biological organisms, Fe is a limiting nutrient. So, too for humans. Worldwide, the major cause of anemia is Fe deficiency. The body tries hard to hold its Fe. The body stores excess Fe that is not bound to protein and enzymes. Further, there is no active mechanism for eliminating excess Fe. Fe loss occurs through cell shedding or bleeding. The RDA for Fe is 18 mg/d for adult women, 27 mg/d during pregnancy; 8 mg/d for adult males and post-menopausal women. Approximately, two-thirds of the Fe in the body is present in hemoglobin and myoglobin. The remainder is present in cytochromes, enzymes and a variety of binding proteins, primarily ferritin.

Oxidized glutathione can be regenerated by glutathione reductase

GSSG + NADPH => 2 GSH + NADP+

PhosphoPentosePathway Oxidative Branch: Glucose-6-P oxidized & NADP+ reduced by DH enzymes form 2 NADPH & ribulose & CO2

Glucose-6P + 2NADP+ > > Ribulose-5P + 2NADPH + CO2

Glycolysis occurs, because CELLS NEED ATP

Glycolysis occurs, because CELLS NEED ATP •Glycolysis occurs in every cell's cytoplasm to quickly make ATP from inorganic HPO42- & ADP from glucose •it does not use O2. •End products are: 2ATP, 2NADH, 2H2O & 2 Pyruvate.

• Fe2+ (but not Fe3+) is transported by the Divalent Metal Transporter-1 (DMT1) into enterocytes in the duodenum. • DMT-1 also transports Co2+, Mn2+, and the toxic metal Cd2+. DMT-1 does not transport Fe3+, Zn2+, Ni2+, Cu, Cr or Hg.

Heme is transported by an unknown protein, also in the duodenum. The Fe(II) is released from heme in the cells by heme oxygenase. * On the basolateral side, Fe is transported out of enterocytes by Ferroportin. Fe(II) is then oxidized to Fe(III) by ferroxidases, Hephaestin and Ceruloplasmin (Cp the major ferroxidase). Ironically, ceruloplasmin is a copper-containing protein and is the major copper binding protein in plasma. [A Cu deficiency will affect Fe levels.] Finally, two molecules of Fe(III), bind to Transferrin, which carries the Fe in blood. Transferrin (Tf) is an abundant plasma protein (~300 mg/dL). In a normal healthy adult, about 1/3 of Tf is bound to Fe(III) (33% saturation). Transferrin does not bind Fe(II). It's better that iron is Fe(III) in the bloodstream (high O2). Want to avoid the risk of ROS formation.

Transferrin receptor mRNA has multiple IREs at the mRNA 3'end.

IRP also binds here. For the transferrin receptor mRNA, IRP binding serves to stabilize the mRNA. When iron levels rise, IRP binds iron and releases from the transferrin receptor mRNA. The exposed hairpins are targets for nucleases that degrade the mRNA. So, increased Fe levels lead to degradation of the transferrin receptor mRNA and the receptor is not translated. And no more Fe is taken up by the cell.

IRP1

IRP1 is cytoplasmic aconitase [the enzyme that converts citrate to isocitrate in the TCA cycle, there are cytoplasmic and mitochondria isozymes]. Aconitase is an Fe4S4 protein- the Fe4S4 cofactor is required for enzymatic activity. When Fe levels are low the Fe4S4 cofactor is disassembled and aconitase adopts a new conformation. The Fe-free protein version binds to the IRE and regulates Fe metabolism. Neat (and efficient) form of regulation! [Slide 12] When Fe levels rise, IRP1 has aconitase enzyme activity and no longer binds IRE. This leaves the IRE empty and the ferritin mRNA is translated, and the resulting ferritin protein sequesters excess Fe. TfR mRNA is degraded since cells don't need Fe.

IRP2

IRP2 regulates ferritin and TfR translation similar to IRP1. However, IRP2 is not an Fe4S4 containing protein. IRP2 abundance is regulated by ubiquitination in an Fe-dependent manner. When Fe is abundant, a specific E3 ubiquitin ligase (FBXL5) binds Fe, and polyubiquitinates IRP2- which is destroyed by the proteasome. In low Fe, the E3 ligase is inactive and IRP2 binds IREs and regulates translation. [Slide 13] Two IRPs mean we have redundant means to regulate cellular Fe.

Regulation of TCA

In simple terms, the TCA cycle is regulated by the need for ATP. When ATP levels (energy charge) are low, the activity of the TCA cycle will increase and vice versa. The availability of NAD+ and ADP signal the need for increased TCA activity. There is little activity under anaerobic conditions (NADH is high). The enzymes that are subject to regulation are: • pyruvate dehydrogenase (not strictly part of TCA) • citrate synthase • isocitrate dehydrogenase • aKG dehydrogenase It's no coincidence that these are also the irreversible steps in the TCA cycle.

Too much Fe

In this era of overnutrition, Fe can accumulate to unhealthy levels. The condition is called hemochromatosis. Tissues affected include liver, pancreas and heart, among others. This is where excess Fe is stored (as hemosiderin). The excess Fe increases oxidative stress, damaging the tissues. It affects primarily older men, in whom Fe accumulates over the decades since there's no easy way to eliminate it (don't take your wife's vitamins- with Fe). In most people, hemochromatosis is a genetic disease. Mutations have been demonstrated in the genes for HFE (which regulates hepcidin production), for hepcidin, for TfR and for ferroportin. The disease is treated by repeated blood draws (phlebotomy).

The hydroxyl radical will rip DNA to shreds. Radiation poisoning is explained in part by the production of the hydroxyl radical upon radiolysis of water from the products of radioactive decay. Hydroxyl radical also damages membranes. By reacting with the fatty acid moieties of phospholipids, the radical can crosslink the fatty acids or make them hydrophilic. Thought question: Why does this damage membranes? What stabilizes the structure of the lipid bilayer? The reactions of free radical oxidation of membrane lipids:

LH + HO• => H20 + L• L• + Ó => LOO• LOO• + LH => LOOH (peroxide) + L• L• + L• => L-L Notice the second and third reactions regenerate the lipid radical. Thus, the reaction is self-propagating and will continue unless something quenches it. Hydroxyl radical is so reactive that there is no enzyme to detoxify it. Instead the body makes use of antioxidants.

Explain NADPH major functions in the body and Phospho-Pentose Pathway-oxidative branch's use of Glucose-6-P to recycle NADPH and form ribulose.

NADP+ has an extra (3') PO4 than NAD + thus, used by different enzymes. Both structures have Niacin (Vitamin B3)

These reactive oxygen species (ROS) are produced as a consequence of metabolism (leakage of electron from CoQ in the electron transport chain is one source). They may also be produced intentionally as in the case of neutrophils/macrophages producing ROS (superoxide and/or nitric oxide) to kill phagocytosed bacteria. For example:

NADPH + 2Ó => NADP+ + 2 Ó•- (NADPH oxidase). Arginine + Ó + NADPH => NO• + Citrulline + NADP+ (Nitric Oxide Synthase- NOS)

Compare and contrast the biochemical mechanisms to recycle (redox) NADH + H+ <-> NAD+ + 2H

Nicotinamide (Niacin; Vit B3) is part of NAD+ (Nicotinamide adenine dinucleotide), cofactor of G3P DeHydrogenase enzyme

Pyruvate dehydrogenase regulation PDH is product inhibited by NADH and acetylCoA. They bind to the active site and prevent binding of the substrates NAD+ and CoASH. High levels of NADH and acetylCoA indicate that the cycle is not needed.

PDH is also subject to reversible covalent modification- namely phosphorylation. When PDH is phosphorylated it is inhibited. The PDH complex contains a PDH kinase subunit in addition to E1, E2 and E3. NADH and acetylCoA are allosteric activators of PDH kinase. A PDH phosphatase hydrolyzes Ser-P and activates PDH. Insulin stimulates the activity of PDH phosphatase, which activate the enzyme and allows utilization of glucose. In muscle, Ca++ activates PDH phosphatase. High levels of Ca++ in muscle indicate ongoing muscle contraction and the need for ATP. The figure illustrates regulation of the three main regulated enzymes of the TCA cycle. The theme is that if enough ATP and NADH are present, the enzymes will be inhibited. ATP and NADH inhibit all three enzymes (CS, IDH, aKDH) either by product inhibition or allosteric regulation. Ca++ stimulates IDH and aKDH. ADP stimulates IDH, while AMP stimulates aKDH. All three activators signal the need to make more ATP.

Phospho-Pentose Pathway- non oxidative branch Transforms monosaccharides between 3 to 7 carbons in length, (so other than hexoses) with Reversible Enzymes

PPP non-ox is crucial for Rapidly dividing cells, which need ribose-5P for DNA synthesis. Other carbon lengths of sugars are also needed by cells. •Isomerase Forms ribose-5P aldehyde isomer of ketone ribulose-5P product of PPP-ox, like fructose-6P <> glucose-6P, but ribulose-5P <> ribose-5P •Transformers: Transaldolase (evens) & transketolase (odds) reversible enzymes that change the sugar carbon length to another length. •Sugars can be 3 to 7 carbons in length •Transketolases uses phosphorylated Vitamin B1 (Thiamin) for adding/removing carbons bound to oxygen from sugar chain. (FYI: transaldolases uses Zinc ions)

step2. of glycolysis Phosphofructokinase-1 (PFK-1) adds phosphate to other end of Frc-6-P to form Frc-1,6-bisphosphate Committed, irreversible & Rate-Limiting step of glycolysis

Phosphofructokinase-1 (PFK-1) adds phosphate to other end of Frc-6-P to form Frc-1,6-bisphosphate Committed, irreversible & Rate-Limiting step of glycolysis PO3 to C-1 of Fructose-6-P-> P-6-Fructose-1-P (F-1,6-bisP) (bis- ; not di-)

Nicotinamide (Niacin; Vit B3) is cofactor in G3P DeHydrogenase enzyme that oxidizes it to allow for PO4 binding NAD+ (Nicotinamide adenine dinucleotide) + 2H <-> NADH + H+

R-C-O-H H Reduced (Hydrogenated) target R-C=O Oxidized (DeHydrogen) target NAD+->NADH/H+ removes 2e- & 2H+ (1e- in H-C bond, the other cancels N+ sort of)...

Reacting to ROS

Reacting to ROS In the event of oxidative stress (the condition of excess ROS causing damage in a cell), cells respond by inducing the expression of numerous antioxidant enzymes, including glutathione reductase, glutathione peroxidase, superoxide dismutase and peroxiredoxins, to list a few. Induction of gene expression is due to activation of an antioxidant transcription factor, named Nrf2. Nrf2 binds to antioxidant response elements (AREs), in the promoter of the genes. Under normal healthy conditions, Nrf2 is maintained at low levels by a ubiquitin ligase, KeapI. In response to oxidative stress, Cys residues in KeapI are oxidized, disrupting its binding to Nrf2. As a result, Nrf2 levels accumulate and transcription of the antioxidant genes is induced. [Slide 6 shows Nrf2 activation and lists some target genes.]

step 4. of glycolysis PAY DAY! Reason for glycolysis: HPO42- to ADPßà ATP by GA3P à BPG (with NAD+ à NADH) à 3PG

Reason for glycolysis: HPO42- to ADP <-> ATP by GA3P -> BPG (with NAD+ -> NADH) -> 3PG Step 1: G3PDH enzyme: De-Hydrogenates (oxidizes) with NAD+ to NADH and phosphorylates GA3P by HPO4 to form 1,3 bisPG (aka BPG). This is REVERSIBLE Step 2: PG Kinase: BPG forms ADP->ATP leaving an 3-PG acid. This kinase reaction is REVERSIBLE

Red Blood Cells

Red Blood Cells: ~20% of 1,3-BPG shifted to 2,3-BPG. 2,3-BPG does not form ATP, instead it skips to PEP. It decreases Hemoglobin affinity for O2

Ribose for dividing cells is a major purpose of PPP Non-Oxi branch. RibULose <> Ribose (by Isomerase).

Rib-you-lose Vs Rib-ose

step 3. of glycolysis Split Fructose-1,6-bisP in half to two 3-carbon sugars: Glyceraldehyde-3-P (GA-3-P) & DHAcetone-P (DHAP). DHAP can flip to GAP by an isomerase (like glucoseßàfructose).

Split Fructose-1,6-bisP in half to two 3-carbon sugars: Glyceraldehyde-3-P (GA-3-P) & DHAcetone-P (DHAP). DHAP can flip to GAP by an isomerase (like glucose <->fructose). (FYI) Adolase cleaves Uses Schiff base reaction.

Ferritin mRNA has a sequence called an Iron Response Element (IRE) near the mRNA 5'end.

The IRE adopts a stem-loop secondary structure, as shown in the figure [Slide 11]. A protein, Iron-Regulatory Protein (IRP, also called Iron Response Element Binding Protein- IRE-BP) binds to the IRE and blocks translation initiation. IRP only binds the stem loop when it lacks Fe. When Fe is bound to IRP, it cannot bind the stem loop. Thus, when Fe levels are high, it binds IRP, knocks it off the stem loop, which allows translation of ferritin mRNA. This allows storage of excess Fe.

What are the sources of the substrates for TCA? • Carbohydrate metabolism produces pyruvate, which is converted to acetyl-CoA by the pyruvate dehydrogenase complex. • Fat metabolism produces acetyl-CoA. • Protein metabolism produces amino acids, which feed into the TCA cycle at multiple points.

The Krebs cycle also has an anabolic function. It can serve as a source of precursors for the synthesis of more complex biological molecules, including carbohydrates, lipids, amino acids and amino acid derivatives. The reactions of the tca take place in mitochondria. This makes sense because the reducing equivalents can be fed directly into the electron transport chain.

The TCA cycle, also known as "Krebs cycle" or "citric acid cycle" is the final common pathway for the oxidation of fuels.

The TCA cycle produces more reducing equivalents and therefore more ATP than glycolysis, or beta-oxidation, or catabolism of amino acids. Because of this, genetic mutations or drugs that impair TCA cycle function will have their greatest effects on tissues/organs with the highest need for OxPhos and ATP. For example, muscle function and neuronal function are impaired and patients typically present with their first symptoms in these tissues.

Most Cells have high affinity transporter, GLUT1 (3 & 4 are similar)so, Glucose readily binds and is transported from the blood into cells even when Blood Glucose levels are relatively low.(Discuss other GLUT transporters later)

The diffusion is so, effective at Glc permeability that Glc is often not considered osmotically active in the blood.

Peroxides: Hydrogen peroxide is an oxidizing agent that can react with several different biological molecules such as DNA. H202 is a longer-lived molecule than superoxide and, because it is uncharged, it is able to diffuse through membranes. It can act as a signaling molecule, but more important it is toxic to infectious cells.

The enzymes, myeloperoxidase and eosinophilic peroxidase can catalyze the conversion of hydrogen peroxide to hypochlorous acid (bleach), which has oxidizing and antibiotic properties itself. H202 + Cl- + H+ => HOCl + H20

The overall stoichiometry for the TCA cycle is:

The overall stoichiometry for the TCA cycle is: (+OAA) + AcetylCoA + 3NAD+ + FAD + GDP + Pi => 2 CÓ + 3 NADH + FADH2 + GTP + CoASH +(OAA) GTP can be converted to ATP (GTP + ADP <-> GDP + ATP; DG ~ 0). NADH and FADH2 feed into OxPhos. Question: How many ATP's do we get per molecule of acetylCoA? 3?

Given the properties of the reactive oxygen species, it's no surprise that the body makes use of them during the inflammatory response. However, neutrophils/macrophages are playing with fire. One of the theories of aging is that aging reflects the accumulation of oxidative damage (reread Priestley's quote).

The oxidative damage would be due to ROS produced during inflammation, but also ROS that are produced during normal metabolic activities. It is estimated during normal oxidative phosphorylation that 1% of reduced oxygen species get loose from the electron transport chain before the oxygen is converted to water. Catching and eliminating these rogue ROS is one explanation for the presence of Mn-SOD in the mitochondrion.

In the event of hemolysis (lysis of RBCs), hemoglobin and free heme spill into the blood. Free heme can bind to membranes and carry out the Fenton reaction. This results in oxidative damage to cells and tissues.

The plasma contains two proteins, haptoglobin and hemopexin, to prevent such tissue damage. Haptoglobin binds to free hemoglobin in the blood. Hemopexin binds heme with high affinity (Kd < 1pM) and inhibits oxidative damage. Both complexes bind to specific receptors in the liver where they are endocytosed and destroyed. The free Fe is recycled to transferrin or ferritin.

Perhaps the most important anaplerotic reaction is that catalyzed by the enzyme, pyruvate carboxylase. Pyruvate + HCO3- + ATP => OAA + ADP + Pi The enzyme requires biotin as a cofactor. The endergonic conversion of pyruvate to OAA is made favorable by coupling the reaction to the hydrolysis of ATP. The reaction is a two-step mechanism. In the first step a covalent carboxybiotin intermediate is produced from bicarbonate and biotin. Hence, biotin functions as a "carrier of activated CO2". The standard free energy for hydrolysis of carboxybiotin is -4.7 kcal/mol and so the hydrolysis of ATP is needed to make carboxybiotin. In the second step, pyruvate reacts with the carboxybiotin yielding OAA. Biotin contains a 5C carboxylic acid chain that is covalently coupled to a Lys residue. So just like lipoamide in PDH, the biotin is attached to a flexible tether that can swing from one active site where biotin is carboxylated in an ATP-dependent fashion to a second active site containing pyruvate.

The pyruvate carboxylase reaction also serves as the first step in gluconeogenesis- the synthesis of glucose from pyruvate. Thus, the OAA produced by this enzyme has two fates, anaplerosis or gluconeogenesis, depending on what the cell needs. Acetyl-CoA is an allosteric activator of pyruvate carboxylase. A high level of acetyl-CoA signals the need for more OAA and therefore increased activity of the enzyme. Other anaplerotic reactions include the formation of aKG, fumarate and succinate from the catabolism of amino acids. These products can then be converted to OAA through the reactions of the CAC.

Hydroxyl radical: This is the most evil of the reactive oxygen species. Please note! This is NOT the same as hydroxide anion (OH-, i.e. base).

The reaction of hydroxyl radical with organic molecules is diffusion-limited, which means the reaction is limited by how fastOH can get to organics. The hydroxyl radical is produced by the reaction of hydrogen peroxide and superoxide, termed the Haber-Weiss reaction. This reaction is catalyzed by Fe(III) Ó.- + H202 => HO• + OH- + Ó

In addition to its role in oxidative phosphorylation, molecular oxygen is involved in many reactions in the cell. This poses a problem for the cell because oxygen is not all that reactive. Thus, oxygen has to be converted to more reactive species (ROS, reactive oxygen species) in order to oxidize molecules. But as Priestley points out, there can be too much of a good thing.

The reduction of oxygen to H2O is a 4-electron process:

The steps of the TCA cycle

The steps of the TCA cycle Reaction Enzyme Comments 1. AcetylCoA + OAA => Citrate + CoASH Citrate synthase Irreversible reaction 2. Citrate <-> [cis-aconitate] <-> isocitrate Aconitase Aconitate is an enzyme-bound intermediate 3. Isocitrate + NAD+ => a-ketoglutarate + NADH + CO2 Isocitrate dehydrogenase NADH! CO2! FIRST 3 IS IRREVERSIBLE 4. a-KG + NAD+ + CoASH => succinylCoA + NADH + CO2 a-ketoglutarate dehydrogenase NADH! CO2! Enzyme complex similar to PDH 5. succinylCoA +GDP + Pi -> succinate + GTP + CoASH succinylCoA synthetase GTP! substrate level phosphorylation Enzyme aka succinate thiokinase 6. succinate + FAD -> fumarate + FADH2 Succinate dehydrogenase FADH2! Part of ComplexII of ET chain 7. fumarate + H2O -> malate Fumarase 8. malate + NAD+ -> OAA + NADH Malate dehydrogenase NADH!

Superoxide breaks down spontaneously, termed dismutation. 2 Ó.- + 2H+ => H202 + Ó

This reaction is also catalyzed by superoxide dismutase (SOD) [Slide 2]. Human cells have two enzymes, Mn-SOD, located in the mitochondrion, and Cu,Zn-SOD in the cytosol. The importance of SOD is illustrated by: E. coli lacking SOD are much more sensitive to oxidative damage. (Higher rate of spontaneous mutations) Some cases of amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) are caused by mutations in Cu,Zn-SOD.

Pyruvate => AcetylCoA: Oxidative Decarboxylation AcetylCoA is the intermediate that is "fed" into the TCA cycle. As pyruvate is the endpoint molecule of CHO metabolism, it must be converted to acetyl-CoA before oxidation may be completed. The conversion of pyruvate to acetylCoA is an irreversible oxidative decarboxylation reaction and is catalyzed by a large multienzyme complex, pyruvate dehydrogenase (PDH). The reaction is: pyruvate + CoASH + NAD+ => acetylCoA + CO2 + NADH

This takes place in the mitochondrion and the NADH is fed into the ET chain. Pyruvate is generated in the cytoplasm (glycolysis) and gets into the mitochondrion using a transporter in the inner mito membrane. In acetylCoA, the acetyl group is joined to Coenzyme A through a high-energy thioester linkage. In this reaction, C2 of pyruvate is oxidized (by NAD+) with release of the carboxylic C as CO2. Some of the free energy of the decarboxylation is conserved in the thioester linkage. The reaction is irreversible. (PDH cannot make pyruvate from acetyl-CoA) Coenzyme A (CoASH) consists of an adenine nucleotide linked via pyrophosphate to pantothenic acid (vitamin B5), which is linked in turn via an amide linkage to 2-aminoethanethiol (decarboxylated Cysteine). The "business end" of CoASH is the thiol (-SH) group, which forms thioester linkages with a variety of acyl groups. In other words, CoASH is a "carrier of activated acyl groups".

Fe promotes ROS: Fe ions react readily with oxygen and are often an essential cofactor in enzymes that use oxygen. But free Fe reacts with oxygen non-enzymatically to produce ROS (Fenton reaction).

Thus, the cell must maintain tight control on free Fe levels. Fe(II) can react with oxygen to make superoxide; and with hydrogen peroxide to make hydroxyl radical: Ó + Fé+ => •Ó- + Fě+ H2Ó + Fé+ => HO• + OH- + Fě+ Don't forget the Haber-Weiss reaction catalyzed by Fe(III) mentioned above. Perhaps it's no coincidence that Fe-containing enzymes often "tie-up" the Fe in a heme group or FeS centers to prevent these reactions.

Fe deficiency

Too little Fe, whether due to inadequate intake or excess loss through bleeding, is first recognized as anemia. There are fewer RBCs and those present are smaller and less red due to poor Hb production. This manifests as weakness or malaise. Oxygen transport to tissues is compromised, as is oxidative metabolism in the cells. Given the stores of Fe in the body and the small amount lost each day (~ 1mg/d in men), an Fe-deficient diet can take a long time to develop anemia.

Translational Regulation of Fe Transport/Binding Proteins

Translational Regulation of Fe Transport/Binding Proteins The translation of specific mRNAs can be regulated independent of changes in global protein synthesis in order to regulate gene expression. This generally involves specific sequence elements in the mRNA itself, typically in the 5' or 3' untranslated regions (UTR)s. The sequence elements provide binding sites for specific proteins that then either inhibit translation, enhance translation or affect mRNA stability. [Slide 9,10]

In the TCA cycle, a two-carbon molecule (acetyl-CoA) is coupled to a 4-C molecule (oxaloacetate, OAA) to make a 6-C molecule (citric acid). In a series of steps, the 6C molecule is oxidized to a 5C then a 4C molecule, ultimately regenerating OAA. During this process the DeltaG released is captured in reducing equivalents (NADH, FADH2) and a molecule of GTP.

Two carbons enter the pathway, and 2 carbons leave the pathway as CÓ. Therefore, there is no net gain of C in the TCA cycle. In other words, following one turn of the TCA cycle, there is no net production of TCA cycle intermediates, ie, the cycle is catalytic. You begin with one molecule of OAA; you end with one molecule of OAA. As the intermediates are synthesized in one step, they are used in the next step.

Pyruvate Dehydrogenase Complex. (Irreversible) 2

Two other critical metabolic enzymes, a-ketoglutarate dehydrogenase (part of TCA, see below) and branched chain keto-acid dehydrogenase (amino acid catabolism) use a very similar reaction. In fact, the Ě subunit is the same in all three enzymes. Thiamine deficiency (beriberi) will interfere with the PDH reaction (it's needed for Ē). Given the central role of PDH and the TCA cycle in generating NADH for ATP production, it makes sense that thiamine deficiency is associated with symptoms of lethargy. Thiamine deficiency can lead to muscle and nervous system damage. Likewise, genetic mutations in PDH cause lactic acidosis, encephalopathy, motor dysfunction. Affected individuals usually die in childhood.

Antioxidants: Vitamin C (ascorbic acid):

Vitamin C (ascorbic acid): Linus Pauling maintained that megadoses (1-5g daily; RDA 45-60 mg/day) would reduce the frequency and severity of the common cold and prevent cancer. The concentrations of damaged DNA bases are decreased in individuals taking amounts of vitamin C in excess of the RDA.

Antioxidants: Vitamin E (a-tocopherol):

Vitamin E (a-tocopherol): This is a lipid-soluble antioxidant that has been demonstrated to be protective against lipid peroxidation caused by the hydroxyl radical.

What happens when small soluble molecules are not absorbable, such as disaccharides or similar molecules?

What happens when small soluble molecules are not absorbable, such as disaccharides or similar molecules? 1.They form osmotic gradient in the intestine that pulls water from blood and interstitial fluid ; and prevent water reabsorption... 2.Gut bacteria in large intestine digest the disaccharides releasing gasses 1.Incompletely digested complex carbohydrates also feed these bacteria, which leads to gasses 2.Bacteria in cattle, termites... can digest cellulose and chitin Results in DIARRHEA and FLATULENCE .... As people can attest that have Lactase deficiency or who took too much of a laxative

Superoxide anion: A short-lived species (~msecs at neutral pH). Superoxide anion is an example of a free radical (as is nitric oxide), meaning it contains a single unpaired electron in a molecular orbital. The unpaired electron "wants" to insert itself into a bond or find another electron with which to pair, thus oxidizing the substrate. Superoxide anion can also react spontaneously with nitric oxide to produce peroxynitrite. Peroxynitrite can react with and covalently modify Cys and Tyr side chains in proteins (nitrosylation), leading to (possible) impairment of function. [Recent experiments suggest that nitrosylation of proteins might be a normal physiological pathway as well]. The amount of protein nitrosylation is one measure of oxidative stress in cells. (pKa for superoxide is 4.8)

Ó.- + NO. => ONOO- (peroxynitrite)

Pyruvate Dehydrogenase Complex. (Irreversible) This is a large multienzyme complex with 5 distinct enzyme activities (labeled Ē, É and Ě) carried on different subunits. The complex contains multiple copies of each subunit in an ordered structure that facilitates the transfer of intermediates from Ē to É to Ě. The advantage of multienzyme complexes is that diffusion of substrates is minimized. That is, the product of the first active site is funneled into the second active site and so on. Thus, a multistep reaction occurs more efficiently

Ē- pyruvate dehydrogenase subunit: Carries out the decarboxylation of pyruvate and the transfer of the 2C fragment (hydroxyethyl) to thiamine pyrophosphate (TPP). É- dihydrolipoyl transacetylase: The 2C fragment is oxidized by reacting with a lipoamide prosthetic group releasing TPP. The acetyl group is then passed to CoASH in the active site of É. The lipoamide prosthetic group is an 8-C carboxylic acid with S at C6 and C8. The carboxylate end is covalently bound to a Lys side chain. This creates a 13 atom tether that can swing between the active sites in Ē, É and Ě. Ě- dihydrolipoyl dehydrogenase: The reduced lipoamide is reoxidized to the disulfide form. Electrons are passed through a FAD prosthetic group in Ě to NAD+ to produce the NADH product.

Important Points about the TCA reactions • The first step is irreversible. Citrate synthase will not make acetylCoA from citrate. The carbons are committed. • Fluoroacetate is used as rat poison and is toxic to humans. Citrate synthase converts fluoroacetate (CoA) to fluorocitrate and fluorocitrate is a potent inhibitor of aconitase. The TCA cycle comes to a grinding halt. • The release of CÓ by isocitrate dehydrogenase makes step 3 essentially irreversible. This step appears to be the most important for controlling the flux through the pathway. (i.e., rate-limiting)

• a-ketoglutarate dehydrogenase is a multienzyme complex like PDH. The Ē subunit- a-KG dehydrogenase, decarboxylates the substrate and transfers the 4C fragment to TPP. The É subunit- dihydrolipoyl transuccinase, oxidizes the 4C fragment to succinate and forms the CoA derivative. Ě is the same in both PDH and aKG dehydrogenase. Like PDH, a thiamine deficiency will reduce the activity of aKG dehydrogenase. Again, the release of CÓ along with a negative DG make this step irreversible. The three irreversible steps insure that the TCA cycle only goes in one direction. • The final three steps have the same chemistry as the main three steps of fatty acid ß-oxidation. 1. Oxidation of a C-C single bond to a double bond. 2. Hydration of the double bond. 3. Oxidation of the OH to a keto. • The CoA thioester of succinylCoA is a high energy bond (DG approx. -8kcal/mol), about the same as ATP. Succinyl CoA synthetase uses this free energy to drive GTP formation. This step is an example of substrate level phosphorylation. The GTP can then be converted to ATP. Some isozymes also accept ADP as substrate and make ATP directly.

Where happens to pyruvate?

•Anaerobic respiration: Skips Mitochondria; only uses glycolysis to get ATP from Glucose. •Pyruvate forms into Lactate (its reduced form) for regenerating NAD+ •Less efficient ATP/Glc but faster and mitochondria take up cell space •Examples: Fast-twitch muscles, red blood cells and ischemic cells • •Aerobic respiration: Cells with Mitochondria •Pyruvate forms into Acetyl-CoA in mitochondria for TCA •Much more efficient at ATP/Glc •NADH regeneration to NAD+ can also generate ATP •Shuttle Malate/Asp/OAA: NAD+->NADH for ETS (in matrix) ->3 or 2.5 ATP/Glc •Shuttle DHAP/Glycerol-3-P: FAD->FADH2 for ETS (in intramembrane space) ->2 or 1.5 ATP/Glc •

step5. Pay-Back ATP. Pyruvate kinase moves Phosphate from Phospho(Enol)Pyruvate to ATP leaving End product of glycolysis, Pyruvate

•Dehydration (& shift) forms phospho-enol-pyruvate (PEP) which can transfer PO4 to ADP forming ATP and pyruvate by Pyruvate kinase •Pyruvate can become Lactate (in cytosol) or Acetyl-CoA (in mitochondria)

GLUCOSE

•Glucose has many uses. Its main use is for the formation of ATP and thus fuel for the body •Glucose is obtained from the diet, but it can be synthesized by the liver. •Only "unchained, free" monosaccharide Glucose can be absorbed by into the blood from digestion •So, poly-, oligio- and di- saccharide chains from the diet have to be digested to monosaccharides

Glucose-6-P oxidation allows NADP+ -> NADPH and forms Ribulose via PhosphoPentosePathway (PPP) oxidative branch.

•Glucose-6-P & 2 NADP+ > > 2 NADPH & ribulose + CO2 •ATP and NADH are NOT involved in PPP •Glucose-6P dehydrogenase, G6PDH, is the rate limiting enzyme of NADP+ reduction to NADPH •NADPH (& ROS) feedback regulate G6PDH

HEXOKINASE (HK) traps and prepares the Hexose Glc as Glc-6-P in cell by: Glc & ATP -> Glc-6-P & ADP

•HK rapidly & irreversibly transfers Phosphate to Glc even at low cytosol Glc levels (low Km; ~0.1 mM). •GLUTs DO NOT transport Glc-6-P, thus HK traps Glc in cell as Glc-6-P and prepares it for uses. •This also keeps a low concentration of Glc in the cell to continually "pull" in more Glucose from the blood •HK feed-back inhibited by its product Glc-6-P. •high [Glc-6-P] inhibits HK •low [Glc-6-P] in cell increases HK activity

Determine the transformation/pathway of Fructose-6-Phosphate in a cell: 1. with reactive oxygen species; 2. undergoing cell division; or 3. requiring ATP.

•If cell needs NADPH for ROS or other needs • Fructose-6P > glucose-6P for G6PDH of PPPOx > ribULose-5P + 2 NADPH •If cell needs ribose-5P for DNA (replicating cell) 1.Frc-6P > G-6-P via isomerase > PPPOx > ribULose-5P (ketone) shifted by isomerase > ribose-5P (aldehyde) 2.Frc-6P transferred into ribose-5P via PPPNonOX (a bunch of intermediate molecules and paths that we are skipping) •If cell needs ATP then Fructose-6P> 2 Pyruvate + 2(3) ATP by glycolysis. •Notice Ribulose, Ribose-5P, Glucose-6P can all transform into Fructose-6P GA-3P can also be formed by PPPNonOx

NADPH and ROS feedback regulate G6PDH activity

•NADPH:NADP+ ratio held at ~70:1. This is by Low NADPH stimulation and High NADPH inhibition of G6PDH (product feedback) •Lack of NADP+ stops G6PDH (No NADP+ cofactor then the enzyme cannot function) •Reactive Oxygen Species upregulate G6PDH and Oxidized glutathione (GSSG) increases GPDH activity •NADPH needed to recycle glutathione to its reduced, antioxidant form. •Transcription factor from high reactive oxygen species increase G6PDH expression •Insulin also increases G6PDH transcription of adipose and liver cells because PPP removes glucose into ribulose •Cells undergoing cell division may increase PPPox to meet the demand for ribulose-5-P to become ribose-5-P. But they can also use PPP non-ox branch

(Disaccharide)ases "break" O-Glycosidic Bonds of Disaccharides to free absorbable Monosacchrides

••Glucose-1a -> 2b Fructose (=sucrose); hydrolysis by sucrase • •Galactose 1b -> 4a/b Glucose (=a/b lactose); hydrolysis by lactase • •Glucose 1a -> 4a Glucose (=maltose); hydrolysis by maltase