NADPH Maintains Glutathione in Reduced State

1. PENTOSE PHOSPHATE PATHWAY

- source of NADPH for reductive synthesis of fattyacids and cholesterol in liver, kidney, and adipose

- NADPH maintains glutathione in reduced state

- can also provide source of ribose-5-phosphate nucleotide synthesis nucleic acids

- provides for interconversion of pentoses with hexoses/trioses

Oxidative branch (irreversible)

- glucose-6-P  6-Phosphogluconate  Ribulose-5-Phosphate (Ribose-5-phosphate)

- key enzyme and rate-limiting step is glucose-6-P dehydrogenase; produces first of two NADPH, used NADP+as cofactor; glucose-6-PDHase has regulation  feed back inhibition by NADPH

- second molecule of NADPH produces in oxidative branch is product of 6-phosphogluconate dehydrogenase catalyzes decarboxylation of hexose to a pentose sugar (ribulose-5-P)

- end product is ribose-5-P precursor for synthesis of nucleic acids

Non-oxidative branch (reversible)

- includes sugar intermediates containing 4,5, or 7 carbons

- transketolase catalyzes 2/3 of reaction in branch; requires thiamine diphosphate (TDP)as a cofactor (prosthetic group) as well as Mg2+ ions(as does PDH and alpha-ketoglutarate dehydrogenase)

-thiamine (B1) deficiency affects both ribose-5-P production for nucleic acid synthesis and energy metabolism for carbohydrates; Wernicke-Korsakoff syndrome exhibits memoryloss due decreased synthesis of nucleic acids in nervous tissue

- high carbdiet thiamine deficiency  beriberi; tiring, weakness related to energy depletion; high carb diet  individuals rely on pyruvate and alpha-ketoglutarate DHase for aerobic production of ATP from glucose

- when non-oxidative branch begins with ribulose-5-P endproducts are glycolytic intermediates: glyceraldehyde-3-P and fructose-6-P; reversible reactions starting with these intermediates  ending with ribose-5-P

Cellular needs for ribose-5-P and NADPH

- cell requires NADPH but not ribose-5-P

- glucose-6-P from hexokinase in most tissues, but by glucokinase in liver/pancreatic beta-cells  oxidative branch to ribulose-5-P converted to fructose-6-P and glyceraldehyde-3-P in 2:1 ratio intermediates can be metabolized to pyruvate in glycolysis or returned to glucose-6-P via gluconeogenic pathway

- shunting around part of glycolysis (hexose monophosphate shunt)

- 2/3 of ribulose-5-P must be used to form xylulose-5-P with remaining 1/3 being converted to ribose-5-P

- both oxidative and non-oxidative branches working

Cell requires ribose-5-P for nucleic acid synthesis but does not require NADPH

- fructose-6-P and glyceraldehyde-3-P provided from glycolysis in 2:1 ratio for non-oxidative branch

- non-oxidative branch proceeds in opposite direction than figure 1

- ribose-5-P produced from glycolytic intermediates; oxidative pathway shutdown by NADPH feedback inhibition

Cell requires both NADPH and ribose-5-P

- cell operates only oxidative branch of pentose pathway

- all ribulose-5-P converted to ribose-5-P

2. REACTIVE OXYGEN SPECIES (ROS)

- toxic ROS generated during normal course of cellular metabolism; ROS inhibited through action of antioxidants

- look at table for ROS and antioxidants that reduce them (14-4)

Free radicals

- unpaired electron; extremely reactive against compounds that contain double bonds like unsaturated lipid (plasma membrane) and nucleic acids bases

- when damage occurs to membranes  oxidized molecules recycled

- antioxidants eliminate ROS before they cause damage

Singlet oxygen – generated from molecular oxygen in presence of UV light and heme iron; found in skin; not a radical; reacts with biological molecules and generates oxygen radicals (superoxide); contributes to oxidative damage in cells

Superoxide radical anion- superoxide; formed by transfer of a single electron to oxygen; accomplished via coenzyme Q in mitochondrial electron transport chain or by NADPH oxidase in WBCs

Hydrogen peroxyl radical – produced by protonation of superoxide; participate in same reactions as hydroxyl radicals (less reactive)

Hydrogen peroxide – central role in formation of ROS; produced from superoxide via superoxide dismutase (antioxidant) and Haber-Weiss reaction

Hydroxyl radical – most reactive ROS; produced from superoxide via Haber-Weiss reaction or via spontaneous degradation of hydrogen peroxide catalyzed by free metal ions such as iron; basis for antibiotic actions of hydrogen peroxide

Lipid peroxyl radical – depends on action of hydroxyl radicals; primary targets of hydroxylradicals are membrane phospholipids because most fatty acids in these structures are unsaturated (double bonds) reaction forms a lipid radical intermediate rapidly oxidized by molecular oxygen to form lipid peroxyl radical further oxidizes membrane lipids  regenerates more hydroxyl radicals

3. ANTIOXIDANT DEFENSE

Glutathione and selenium

Glutathione peroxidase: 2 GSH + H2O2 GSSG + 2 H2O

- glutathione, a tripeptide (glu-cys-gly), exists predominantly in reduced form (GSH) when cys has free SH group

- oxidized form (GSSG) consists of two tripeptides linked by a disulfide bond (-S-S-)

- GSH oxidized to GSSG via glutathione peroxidase (uses hydrogen peroxide)

- reduced glutathione (2 GSH) is a natural antioxidant

- selenium required for activity; cysteine residue is active site modified to selenocysteine

- GSH/GSSG = 100:1

Glutathione reductase: GSSG + NADPH + H+ 2 GSH + NADP+

- reductase contains bound FAD to transfer electrons from NADPH to GSSG

- NADPH derived from pentose pathway (niacin is precursor of NADPH)

Glutathione and pentose phosphate pathway

- defect in glucose-6-PDHase in RBC  defects in membrane stability

- event that demands RBC for NADPH increase defect in G6DPH prevents cell from maintaining adequate amount of GSH to reduce peroxides

- peroxides in blood can convert hemoglobin  methemoglobin and destroy RBC membrane through oxidation of phospholipids RBC destruction  rise in insoluble bilirubin in blood

- bilirubin is a breakdown product of heme that is insoluble until processed by liver into a soluble form

Catalase and superoxide dismutase

- catalase: 2 H2O2 H2O + O2

- hydrogen peroxide catalytically removed by catalase

- catalase contains fourheme groups; enzyme found in liver, kidney, blood, and mucous membranes

- often localized in peroxisomes of cell

- hydrogen peroxide produced by catabolism of superoxide by superoxide dismutase that catalyzes reaction of 2O2-. + 2H+ H2O2 + O2

- superoxide dismutase in cytoplasm contains two identical subunits requiring Cu2+and Zn2+ as cofactors; mitochondrial enzyme requires Mn2+ as a cofactor

- although ROS hydrogen peroxide is form  it is easily reduce to water by catalase

- mutants in Cu,Zn-superoxide dismutase gene occurs in patients with familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder

Antioxidant cascade

Glutathione – reducedglutathione (GSH) protects cell against peroxides (glutathione peroxidase) and then is regenerated ultimately by NADPH produced in pentose phosphate pathway

Vitamin C –

- GSH plays an additional antioxidant role through its ability to regenerate reduced vitamin C (ascorbic acid) protects cells against oxidant effects of both hydroxyl radicals and superoxide radical anion by reducing them

- reduced vitamin C restores vitamin E to its reduced state

- vitamin C maintains iron in reduced state (in prolyl and lysylhydroxylase) of collagen synthesis

- deficiency scurvy

- excess vitamin C intake  production of oxalates formation of kidney stones as calcium oxalate

Vitamin E

- antioxidant role of reduced Vitamin E is to rid cell of lipid peroxyl radical; it also acts on singlet oxygen and peroxyl free radical

- vitamin E is a fat-soluble vitamin; its major constituent is alpha-tocopherol

- vitamin E deficiency (rare) leads to increased peroxidation of membrane lipids, decreased mitochondrial activity, DNA mutations, and damage to cell transport processes; fragility of RBCs