biochem

PPP

- no ____ is directly consumed or produced in the cycle

PPP

1. Consists of

a. _________

- oxidation of glucose by the pentose phosphate pathway generates ______

- 1st stage of the pentose phosphate pathway

b. _________

- interconversion reactions can function in several different directions

- 2nd and 3rd stages of the pentose phosphate pathway

2. Carbon 1 of Glucose 6-Phosphate

 released as ______

3. Rate and Direction of the Reactions

- determined by

a. _________

b. _________

PPP

A. Functions

1. Source of _____

- produces __ _____ for each glucose 6-phosphate molecule entering the oxidative part of the

pathway

a. _____

- as a reducing equivalent for _____ / _____ (the premier antioxidant system in the cell)

- for _____ and _____ biosynthesis

2. Source of _____

- shunts G6P to form _____ for _____ synthesis

3. Route for Use of Ingested _____

- and for their conversion to _____ ( _____ ) and _____

( _____ )

PPP

B. Location

1. In ______

a. ______ - active in fatty acid synthesis

b. ______

- active in fatty acid synthesis

c. ______

- active in fatty acid synthesis

d. ______

- active in NADPH-dependent synthesis of steroids

e. ______

- require NADPH to keep glutathione reduced

2. Within ______

- ______

PPP

FIRST STAGE of the PENTOSE PHOSPHATE PATHWAY (IRREVERSIBLE OXIDATIVE REACTIONS)

1. Consists of 3 Reactions

-> formation of:

a. _________

b. _________

c. ______ molecules of _________ (for each glucose 6-phosphate oxidized)

2. Important in the

a. _________, _________

-> fatty acid biosynthesis

b. _________

-> NADPH-dependent steroids synthesis

c. _________ -> NADPH requirement to keep glutathione reduced -> membrane integrity

PPP

FIRST STAGE of the PENTOSE PHOSPHATE PATHWAY (IRREVERSIBLE OXIDATIVE REACTIONS)

A. Dehydrogenation of Glucose 6-Phosphate

- PPP is regulated primarily at this step

1. Enzyme

- glucose 6-phosphate dehydrogenase (G6PD)

2. Irreversible Reaction

- glucose 6-phosphate oxidation -> 6 phosphogluconolactone (6-phosphoglucono-δ-lactone)

3. Coenzyme

- NADP+ reduced to NADPH

4. NADPH

- potent competitive inhibitor of the enzyme

- high NADPH/NADP+ -> enzyme activity inhibition

a. Increased NADPH Demand

-> decreased NADPH/NADP+ -> increased glucose 6-phosphate dehydrogenase activity

-> increased flux through the cycle

5. Insulin

- enhances glucose 6-phosphate dehydrogenase gene expression -> flux through the pathway increases in well-fed state

PPP

FIRST STAGE of the PENTOSE PHOSPHATE PATHWAY (IRREVERSIBLE OXIDATIVE REACTIONS)

B. 6-Phosphogluconolactone Hydrolysis and Ribulose 5-Phosphate Formation

1. 6-Phosphogluconolactone Hydrolase/Lactonase

- hydrolyze the lactone (cyclic ester) of 6-phosphogluconolactone to 6-phosphogluconate

- irreversible reaction

- not rate-limiting

2. 6-Phosphogluconate Dehydrogenase

- catalyzes oxidative decarboxylation of 6-phosphogluconate by NADP+

- irreversible

a. Products

i. Pentose sugar-phosphate (ribulose 5-phosphate)

ii. CO2 (from carbon of glucose)

iii. 2nd molecule of NADPH

PPP

FIRST STAGE of the PENTOSE PHOSPHATE PATHWAY REVERSIBLE NONOXIDATIVE REACTIONS

1. Occurrence

- in all cell types synthesizing

- nucleotides

- nucleic acids

2. Interconversions

- of 3-, 4-, 5-, 6-, and 7-carbon sugars

3. Ribulose 5-Phosphate Conversion to

a. Ribose 5-Phosphate

b. Intermediates of Glycolysis

- fructose 6-phosphate

- glyceraldehyde 3-phosphate

4. Control

- primarily by the availability of intermediates

5. TPP - required in the transketolase reaction

a. Other TPP-Requiring Enzymes

i. Pyruvate Decarboxylase

- of the pyruvate dehydrogenase complex

ii. α-Ketoglutarate Dehydrogenase

- of the TCA cycle

iii. Branched-Chain α-Keto Acid Dehydrogenase

- of branched-chain amino acid metabolism

PPP

FIRST STAGE of the PENTOSE PHOSPHATE PATHWAY REVERSIBLE NONOXIDATIVE REACTIONS

A. Conversion of Pentose Phosphate to Intermediates of Glycolysis

- cells that carry out reductive biosynthetic reactions -> greater need for NADPH (than for ribose

5-phosphate) -> transketolase (transfers 2-carbon units), transaldolase (transfers 3-carbon units)

convert ribulose 5-phosphate to glyceraldehyde 3-phosphate and fructose 6-phosphate ->

glycolysis

B. Formation of Ribose 5-Phosphate from Intermediates of Glycolysis

- under conditions where demand for pentoses (for nucleotide and nucleic acid synthesis) > NADPH need

-> ribose 5-phosphate synthesis from glyceraldehyde 3-phosphate and fructose 6-phosphate

SECOND STAGE of the PENTOSE PHOSPHATE PATHWAY (REVERSIBLE OXIDATIVE

REACTIONS)

A. Ribulose 5-Phosphate to Ribose 5-Phosphate and Xylulose 5-Phosphate

- both products are formed by isomerization of ribulose 5-phosphate

1. Enzymes

a. Phosphopentose Isomerase (Ribulose-5-Phosphate Isomerase)

- converts ribulose 5-phosphate to ribose-5-phosphate

- ribose-5-phosphate can be used to produce nucleosides for RNA and DNA synthesis

b. Phosphopentose Epimerase (Ribulose-5-Phosphate Epimerase)

THIRD STAGE of the PENTOSE PHOSPHATE PATHWAY (REVERSIBLE OXIDATIVE REACTIONS)

- 3 five-carbon sugars are converted to 2 fructose-6-phosphate and 1 glyceraldehyde-3-phosphate

A. Ribose 5-Phosphate and Xylulose 5-Phosphate to Sedoheptulose 7-Phosphate and G3P

1. Reaction

- 2-carbon unit is transferred from a ketose (xylulose 5-phosphate) to an aldose (ribose 5-

phosphate) -> seven-carbon sugar sedoheptulose-7-phosphate and glyceraldehyde-3-

phosphate

2. Enzyme

- transketolase (requires TPP as cofactor)

3. Wernicke-Korsakoff Syndrome

a. Cause

- results from chronic thiamine deficiency -> reduced transketolase activity ->

clinical manifestations of this syndrome

b. Defective Transketolase Activity

- exhibiting reduced affinity to TPP (other TPP-dependent enzymes in these patients

appear to be normal)

c. Symptoms

- weakness or paralysis

- impaired mental function

THIRD STAGE of the PENTOSE PHOSPHATE PATHWAY (REVERSIBLE OXIDATIVE REACTIONS)

B. Sedoheptulose 7-Phosphate and G3P to Erythrose 4-Phosphate and F6P

1. Reaction

- 3-carbon unit is transferred from sedoheptulose 5-phosphate to G3P -> fructose-6-phosphate and

the four-carbon sugar erythrose-4-phosphate

2. Enzyme

- transaldolase (requires no cofactor)

C. Xylulose 5-Phosphate and Erythrose 4-Phosphate to F6P and G3P

1. Enzyme

- TPP-dependent transketolase

SUMMARY of REACTIONS (STOICHIOMETRY)

3G6P + 6NADP+

-> 2F6P + 3CO2 + G3P + 6NADPH + 6H+

REGULATION of the PATHWAY

A. Rate of the Glucose-6-Phosphate Dehydrogenase (G6PD) Reaction

- regulate the flux of glucose-6-phosphate

- controlled by the availability of its substrate NADP+

so that the pathway flux increases in response to

increasing levels of NADP+

(which indicates increased cellular demand for NADPH)

B. NADP+

- cellular concentration is the major factor

- availability regulates the rate-limiting G6PD reaction

USES of NADPH

A. Reductive Biosynthesis

- part of the energy of glucose 6-phosphate is conserved in NADPH

1. NADPH

- high-energy molecule (part of the energy of glucose 6-phosphate is conserved in NADPH)

- electrons are destined for use in reductive biosynthesis of macromolecules (of fatty acids and

steroids -> anabolism)

- cells keep the [NADP+

]/[NADPH] ratio near 0.01 (which favors reductive biosynthesis)

2. NADH

- used in oxidative metabolism (catabolism)

- cells keep the [NAD+

]/[NADH] ratio near 1000 (which favors metabolite oxidation)

USES of NADPH

B. Reduction of H2O2

1. H2O2 - one of a family of reactive oxygen species

- formed from partial reduction of molecular O2

- formed continuously

- as by-product of aerobic metabolism

- through reactions with drugs and environmental toxins

- diminished levels of antioxidants -> oxidative stress

highly reactive -> damage to

- DNA

- proteins

- unsaturated lipids -> cell death

- reactive oxygen intermediates are implicated in

- reperfusion injury

- cancer

- inflammatory disease

- aging

- protective mechanisms of the cell

USES of NADPH

B. Reduction of H2O2

2. Enzymes that Catalyze Antioxidant Reactions

a. Glutathione Peroxidase

- selenium-requiring

- converts reduced glutathione (tripeptide thiol, ->-glutamylcysteinylglycine) -> oxidized

glutathione -> glutathione reductase using NADPH as a source of reducing

electrons -> regeneration of reduced glutathione

- chemically detoxify H2O2

i. RBCs - totally dependent on HMP pathway for NADPH supply

- glucose 6-phosphate dehydrogenase defect -> decreased NADPH levels -> nonreduction of oxidized glutathione -> H2O2 accumulation -> membrane instability -> lysis

b. Superoxide Dismutase

c. Catalase

USES of NADPH

B. Reduction of H2O2

3. Antioxidant Chemicals

- detoxify oxygen intermediates

- correlated with

- reduced risk for certain types of cancers

- reduced frequency of other chronic health problems

(- clinical trials with antioxidants as dietary supplements have failed to show clear

beneficial effects)

a. Ascorbic Acid (Vitamin C)

b. Vitamin E

c. B-Carotene

- dietary supplementation -> rate of lung cancer in smokers increased rather thandecreased

C. Cytochrome P450 Monooxygenase System

1. Functions

- major pathway for the hydroxylation of aromatic and aliphatic compounds (steroids, alcohol,

drugs, other compounds)

- detoxify drugs and other compounds -> converting to soluble form -> renal excretion

2. Monooxygenases (Mixed Function Oxidases)

- incorporate one atom from molecular oxygen into a substrate (creating a hydroxyl group)

- the other atom reduced to water

a. NADPH

- provides the reducing equivalents

- a supply of NADPH is critical for the liver microsomal cytochrome P450

monooxygenase system

b. Overall Reaction Catalyzed by a Cytochrome P450 Enzyme

4. Mitochondrial Cytochrome P450 Monooxygenase System

- participate in hydroxylation of steroids -> more water soluble

a. Placenta, Ovaries, Testes, Adrenal Cortex

- hormone-producing tissues

- hydroxylate intermediates in the conversion of cholesterol to steroid hormones

b. Liver - uses the system in bile acid synthesis

c. Kidney

- uses the system to hydroxylate 25-hydroxycholecalciferol (vitamin D) -> biologically

active 1, 25- hydroxylated form

5. Microsomal Cytochrome P450 Monooxygenase System

- associated with the endoplasmic reticulum membranes particularly in the liver

a. Detoxification of Foreign Compounds (Xenobiotics)

i. Drugs

ii. Pollutants

- petroleum products

- carcinogens

- pesticides

b. Purposes of the Modification

i. Activate or inactivate a drug

ii. Make a toxic compound more soluble -> excretion in the urine or feces

c. New Hydroxyl Group

- serve as site for conjugation with a polar compound (glucuronic acid) -> increased

solubility

D. Phagocytosis by WBCs (Neutrophils, Macrophages, Monocytes)

1. Phagocytosis

- ingestion by receptor-mediated endocytosis of

- microorganisms

- foreign particles

- cellular debris

- by - neutrophils

- macrophages (monocytes)

2. Bacterial Killing Mechanisms

a. Oxygen-Dependent Mechanisms

i. Myeloperoxidase (MPO) System

- most potent of the bactericidal mechanisms

ii. Other Systems

- involving the generation of oxygen-derived free radicals

b. Oxygen-Independent Systems

- utilize pH changes in the phagolysosome and lysosomal enzymes  destruction of

pathogens

D. Phagocytosis by WBCs (Neutrophils, Macrophages, Monocytes)

3. Mechanism

- invading bacterium -> recognized by the immune system -> attacked by antibodies -> binding to

a receptor on a phagocytic cell -> phagocytosis -> internalization of the microorganism

-> NADPH oxidase (WBC cell membrane) -> conversion of molecular O2 to

superoxide (respiratory burst - rapid oxygen consumption accompanying superoxide

formation) -> converted to H2O2 by superoxide dismutase (SOD) -> addition of

chloride ions -> hypochlorous acid (HOCl) formation -> bacterial killing

a. Excess H2O2

- neutralized by

- catalase

- glutathione peroxidase

D. Phagocytosis by WBCs (Neutrophils, Macrophages, Monocytes)

4. NADPH Oxidase

- hormonally regulated complex enzyme

- subunits contain

- cytochrome

- flavin coenzymes

- electrons move from NADPH to O2 via FAD and heme -> generate O2

-

5. Genetic Deficiencies of NADPH Oxidase -> Chronic Granulomatosis

a. Chronic Granulomatosis

- severe, persistent chronic pyogenic infections

- formation of granulomas (nodular areas of inflammation) that sequester the bacteria

that were not destroyed

G6PD DEFICIENCY

- most common disease-producing abnormality in humans

- highest prevalence in the

- Middle East

- tropical Africa

- Asia

- parts of the Mediterranean

- inherited disease (X-linked) characterized by hemolytic anemia due to inability to detoxify oxidizing

agents

- family of deficiencies caused by >400 different mutations in the gene coding for G6PD

- some cause clinical symptoms

- shortened life span of patient due to complications of from chronic hemolysis

- increased resistance to falciparum malaria

A. Role of G6PD in RBCs

- decreased G6PD activity -> impaired NADPH formation increases the sensitivity of red blood cells to

oxidative stress -> failure to maintain adequate amount of reduced glutathione -> impaired

detoxification of free radicals and peroxides -> diminished stability -> hemolysis

1. Glutathione

- help maintain the reduced state of sulfhydryl groups of proteins (hemoglobin)

- oxidation of sulfhydryl groups -> protein denaturation -> form insoluble

masses (Heinz bodies) -> attach to RBC membranes

- additional membrane protein oxidation -> rigid and nondeformable RBCs ->

removed from the circulation by macrophages (liver, spleen)

- all cells are affected but most severe in RBCs

- PPP provides the only means of NADPH generation in RBCs

- RBCs are devoid of nucleus and ribosomes -> cannot renew supply of enzymes

- other tissues have alternative sources of NADPH (NADP+ dependent malate dehydrogenases)

B. Precipitating Factors in G6PD Deficiency

- most individuals who have inherited 1 of the many G6PD mutations do not show clinical manifestations

- some patients with G6PD deficiency develop hemolytic anemia if they

- are treated with oxidant drugs

- ingest fava beans

- contract a severe infection

1. Oxidant Drugs

a. Antibiotics

- sulfamethoxazole

- chloramphenicol

b. Antimalarials

- primaquine

c. Antipyretics

- acetanilid

- aspirin

d. Nitrofurans

2. Favism

- G6PD deficiency exacerbated (hemolytic effect) by consumption of fava beans usually within

24-48 hours after consumption

- not observed in all patients with G6PD deficiency

- all patients with favism have G6PD deficiency

- Mediterranean variant of G6PD deficiency is particularly susceptible

a. Fava Bean

- dietary staple in Mediterranean region

B. Precipitating Factors in G6PD Deficiency

3. Infection

- most common precipitating factor of hemolysis in G6PD deficiency

- infection -> inflammatory response -> generation of free radicals in macrophages -> diffuse

into RBCs -> oxidative damage

4. Neonatal Jaundice

- from impaired hepatic catabolism or increased bilirubin production

- 1-4 days after birth

- may be severe

C. Properties of the Variant Enzyme

1. Some Mutations

- do not disrupt the structure of the enzyme’s active site -> no effect on enzymatic activity

2. Mutant Enzymes

- show altered kinetic properties

a. Decreased catalytic activity

b. Decreased stability

c. Alteration of binding affinity for NADP+

, NADPH, or glucose 6-phosphate

3. Severity of the Disease

- correlates with the amount of residual enzyme activity in the RBCs

C. Properties of the Variant Enzyme

Severity of the Disease Table

C. Properties of the Variant Enzyme

4. G6PD A-

- prototype of the moderate (class III) form of the disease

- RBCs contain unstable but kinetically normal G6PD with most of the enzyme activity present in

the reticulocytes and younger RBCs (oldest RBCs contain the lowest level of enzyme

activity -> preferentially removed in hemolytic episode)

5. G6PD Mediterranean

- prototype of the more severe (class II) deficiency

- enzyme shows normal stability but scarcely detectable activity

6. Class I Mutations

- often associated with chronic nonspherocytic anemia (occurs even in the absence of oxidative

stress)

D. Molecular Biology of G6PD

1. Mutations

- cloning and G6PD gene and sequencing of its complimentary DNA -> identification of

mutations (most are missense point mutations in the coding region of the gene) that

cause G6PD deficiency

a. G6PD A-

, G6PD Mediterranean

- have mutant enzymes that differ from the normal variant by a single amino acid

b. Locations

i. Clustered Near the Carboxyl Ends of the Enzyme

- cause nonspherocytic hemolytic anemia

ii. Amino End of the Enzyme

- cause milder forms