Biochemistry Exam 2 LGT Heinz Schwarzkopf

hs0215@live.unthsc.edu

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Bioenergetics and Metabolism Glycolysis

PDH & TCA Cycle Electron Transport System Oxidative Phosphorylation

Anabolic pathway: Requires energy gluconeogenesis.

Metabolic pathways are divided into two categories:

Catabolic pathway: glycolysis

What type of metabolic pathway is the TCA cycle?

An

ab

olic

An

ab

olic

Cata

bo

lic

Cata

bo

lic

Pathways in Carbohydrate Metabolism

What stops these two pathways from occurring at the same time?

RELEVANT CELLULAR COMPARTMENTS

Cytosol / cytoplasm

Glycolysis, portions of gluconeogenesis, glycogen metabolism, pentose phosphate pathway.

Mitochondria

TCA, electron transport, >90% of ATP synthesis,

fatty acid oxidation.

Compartmentalized. Driven by the net free energy change of

the sum of individual reactions.

Regulated.

Metabolic Pathways are:

Regulation Allosteric effectors (e.g., ATP) VERY FAST!!

Some metabolic enzymes are regulated by allosteric effectors. An allosteric effector may be a substrate, intermediate or product of the pathway.

Short-term (at the protein level): some enzymes are modified covalently by phosphorylation. This regulatory mechanism requires a protein kinase (phosphorylating enzyme) and protein phosphatase (dephosphorylating enzyme).

Covalent modification (e.g. phosphorylation) FAST!....

Long-term (at the DNA level): the amount of the enzyme/protein is adjusted by a change in its synthesis or degradation.

Change enzyme amounts - SLOW. Metabolic enzymes have life spans ranging from 1 hour to several days; therefore this is a long-term regulation.

Many reaction products are used as the reaction feedback inhibitors ATP is used as feedback inhibitor in many catabolic processes

Feedback Inhibition and Feedforward Stimulation

Substrate A B C D Product E

In feedforward stimulation, a substrate stimulates the pathway by which it is utilized.

What class of enzyme is a kinase?

When an enzyme is missing as a result of a genetic defect, the immediate effects are always the accumulation of the substrate and the lack of the product.

If the intracellular degradation of a macromolecule (lipid, polysaccharide, etc.) by hydrolytic enzymes is blocked, the undegraded molecule accumulates mostly within the cells. The result is a "storage disease.

Inherited Enzyme Deficiencies cause

Metabolic Diseases

Oxidation-reduction: reactions involve the loss or gain of electrons (or protons) one reactant gains electrons and is reduced while the other loses electrons and is oxidized. Oxidation reactions generally release energy and are important in catabolism. (beta Oxidation is an oxidation or reduction taking place?) What is the fate of glucose? 6CO2 + 6H2O

When you see Dehydrogenase that is a clue that you are doing a redox reaction. Then remember that an NADH/NADPH/FADH2 is either used or produced.

NAD+ and FAD

1. In some enzymes bound covalently, called prosthetic group, e.g. succinate dehydrogenase.

2. NAD+ or NADP+ are a soluble co-factor free to diffuse from one enzyme to another. Both accept two electrons and one proton. Derived from vitamin B3 niacin

3. FAD or FMN are coenzyme derived from the vitamin riboflavin, B2, often tightly bound to specific enzymes called flavoproteins.

4. Great majority of NAD+/NADH is located in the mitochondria.

5. Most of NADP+/NADPH is in the cytosol.

Electron Carriers

Both coenzymes (NAD+ and NADP+) undergo reversible reduction of the nicotinamide ring. What can niacin deficiency lead to? a) Rickets b) Diarrhea c) Sterility d) Dermal sensitivity

Pellagra: Remember Niacin and the four Ds

Most dehydrogenases that use NAD or NADP bind the cofactor in a conserved protein domain, called Rossmann

fold.

Rossman fold What it for?

FAD/FADH2

AdenineAdenine

Flavin AAdenine denine DDinucleotideinucleotide

NN--glycosidicglycosidic bondbond

Flavoproteins are enzymes that catalyze oxidation-reduction reactions using either FMN or FAD as coenzyme. FAD or FMN are coenzyme derived from the vitamin B2, riboflavin.

NAD+ NADH

NADP+ NADPH

In a Healthy Cell

What is NADPH used for?

Metabolism

All major nutrients (carbohydrates, fat, protein, etc.) are degraded to acetyl coenzyme A (acetyl-CoA)

Think of acetyl-CoA as the center molecule of metabolism

In the mitochondria, the two carbons of the acetyl group become oxidized to CO2 in the TCA cycle, but

may be used to produce all your other molecules.

Do you remember what vitamins B2 and 3 are for?

Which of the following ways can the body not obtain glucose?

A. Monosaccharides

B. From the breakdown of glycogen

C. Glucose synthesis via amino acids

D. Glucose synthesis via acetyl CoA

E. Glucose can be obtained from all of these sources

Sources and Fates of Acetyl-CoA

Glucose occupies a central position in the metabolism of plants, animals and microorganisms. Glucose is the principal transported carbohydrate in humans and most abundant monosaccharide in dietary carbohydrates. Glucose can be transported by the blood, but it cannot be stored in the cells. For storage, it has to be converted to the polysaccharide glycogen.

Glucose: Sources, Uptake, Transport, Storage and Oxidation

What are the organs that store glycogen?

Which of the following glucose transporters is insulin dependent?

A. Glut1

B. Glut2

C. Glut3

D. Glut4

E. Glut5

What about the kidneys?

If a person is on a salt diet and has not ingested salt in 3 days can there intestines still absorb glucose?

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Why are certain organs such as the brain and retinas affected by high blood glucose levels? a) Because they run out of glucose transporters

b) Because they dont have a sweet tooth

c) Because glucose freely diffuses into the cells and is

toxic above certain levels

d) Because they cant use insulin

During the well-fed state: decreased levels of glucagon and elevated levels of insulin

During starvation: elevated levels of glucagon and low levels of insulin

Glucose metabolism by the liver depends on the insulin/glucagon but insulin is NOT required for the entry of glucose into the liver cell. Why? What about entry into pancreatic islet cells?

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Why doesnt the muscular glycogen store contribute to gluconeogenesis? a) Muscles are stingy

b) Muscles use up all the glucose before it can leave

c) Because it cant convert glucose-1-phosphate to glucose-6-

phosphate

d) Because it lacks glucose-6-phosphatase

Which of the following is only depend on glucose for energy?

A. Brain

B. Red Blood Cells

C. Liver

D. Adipose Tissue

E. Skeletal Muscle

Remember blood glucose maintained at 5 mmol/liter

Which of the following enzymes is involved in an irreversible step in glycolysis? A. Hexokinase B. Phosphoglycerate kinase C. Glyceraldehyde-3-phosphate DH D. Enolase E. Phosphoglycerate mutase

Where does glycolysis take place?

What does the enzyme do? How many irreversible steps?

6 - Carbon sugar

3 - Carbon sugar 3 - Carbon sugar

first priming reaction

second priming reaction

Glycolysis: the reactions of glycolysis, the major catabolic pathway for glucose. It is active in the cytoplasm of all cells in the human body. Breakdown of the six-carbon glucose into two molecules of three-carbon pyruvate, occurs in 10 steps and 2 phases:

Step 1

Step 2

Step 3

Step 4

Step 5

1. energy investment phase:

(steps 1 5)

Phosphorylation of glucose

and its conversion to

glyceraldehyde 3-phosphate

22

2 2

22

22

22

22

22

22

22

22

22

22

Oxidation and phosphorylationOxidation and phosphorylation

first ATP forming reactionfirst ATP forming reaction

Second ATP forming reactionSecond ATP forming reaction

Step 6

Step 7

Step 8

Step 9

Step 10

2. energy generation phase: (steps 6 10) Oxidative conversion of glyceraldehyde 3- phosphate to pyruvate

No oxygen needed

Be able to walk yourself Through the steps.

Which of the following enzymes is only found in the liver and pancreas? A. Hexokinase B. Phosphoglycerate kinase C. Glucokinase D. Aldolase E. Pyruvate kinase

Remember that a kinase catalyzes and irreversible reaction. Phosphorylation of glucose traps it in the cell (neg charge).

In the liver, the enzyme that converts glucose to glucose-6-phosphate is: A. Inhibited by ATP B. Inhibited by ADP C. Inhibited by Glucose 6-phosphate D. Inhibited by Insulin E. Inhibited by Fructose-6-phosphate

E. In peripheral tissue, hexokinase is inhibited by its product, Glucose-6-P, but glucose-6-P has no effect on glucokinase (which is found in the liver and pancreas)

Glucokinase (Liver & Pancreas)

- Highly specific for glucose.

- High Km for glucose (~10 mM).

- Not saturated at physiological blood glucose concentrations (4 - 5 mM).

- Inhibited by fructose 6-P, but NOT glucose 6-phosphate.

- Induced by insulin (transcriptionally).

The liver acts like a glucose sponge

Hexokinase (Peripheral Tissues)

- Low specificity - phosphorylates most relevant hexoses (glucose, fructose, mannose).

- Low Km (0.1 mM); saturated at all plasma glucose concentrations.

- Inhibited by glucose 6-phosphate.

- Activated by fructose 1-phosphate and glucose.

- Insulin has no effect on expression.

Km of the two isozymes of hexokinase

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Cytosolic NADH+

- It must be re-oxidized:

1. anaerobic conditions: by conversion of pyruvate to lactate by lactate dehydrogenase, and NADH is re-converted to NAD+, without oxygen participation.

What are the products of gylcolysis?

An increase in intracellular ATP in working muscle would cause? A. an decrease in glycolysis B. an decrease in glycogen synthesis C. An decrease in gluconeogenesis. D. Activation of pyruvate kinase E. Activation of PFK-1

Regulation of Glycolysis - Phosphofructokinase-1 (PFK-1), is the most important regulated

enzyme of glycolysis. PFK is:

- stimulated by insulin and inhibited by glucagon (in the liver).

- inhibited by citrate as well as by low pH.

- inhibited by ATP and stimulated by AMP and ADP.

- Hexokinase / Glucokinase: also regulated

- Pyruvate Kinase: also regulated. Inhibited by ATP.

- In RBCs, rates of glycolysis are regulated by ATP / (AMP+ADP) only.

Remember 1, 3 and 10 those are the irreversible steps of glycolysis.

Phosphofructokinase-1(PFK-1)

- Is the most important regulated enzyme of glycolysis.

- Commits glucose to pyruvate in glycolysis.

- Activated by:

- AMP

- Fructose 2,6-bisphosphate synthesized by PFK-2

- levels determined by insulin/glucagon

- levels determined by fructose 6-phosphate in muscle

- Inhibited by:

- ATP

- Citrate

- Glucagon

2,3-bisphosphoglycerate is an important allosteric effector of hemoglobins affinity for oxygen.

It shifts the oxygen binding curve to right = DECREASED affinity, resulting in more efficient release of oxygen at tissues.

2,3-bisphosphoglycerate in Red blood cells.

Pyruvate Kinase

In glycolysis, step 10, phosphoenolpyruvate is converted to

pyruvate by pyruvate kinase. Pyruvate kinase is more

active in the fed state than in the fasting state.

- inhibited by:

- ATP, acetyl-CoA, alanine

- activated by:

- Fructose 1,6-bisphosphate

Fates of Pyruvate Pyruvate formed by glycolysis, is only the first stage in the complete degradation of glucose. First route: pyruvate is oxidized, with loss of its carboxyl group as CO2, to yield acetyl group of acetyl-CoA by PDH. Acetyl group is then oxidized to CO2 by TCA cycle.

Second route: pyruvate is reduced to lactate by lactate dehydrogenase via lactic acid fermentation. Third route: pyruvate catabolism leads to alcohol and CO2, under hypoxic conditions, a process called alcohol fermentation.

What is the fate of glucose: a) Will be used up faster under anaerobic

condition than aerobic conditions b) Will be used up faster under aerobic conditions

than anaerobic conditions c) Will produce 36-38 ATP in all cells d) Will be used to produce 2,3-BPG and this is

know as the Warburg effect

What is this phenomenon called and where is it not seen? What is the Warburg effect?

[1] Lactate Is Produced under Anaerobic Conditions

Fructose: a) is highly regulated b) primarily metabolized by muscle c) Uses GLUT5 transporter d) is essential in fructosuria patients

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FRUCTOSE ENTRY INTO LIVER METABOLISM

glucokinase

Disorders of Fructose Metabolism

1 Essential fructosuria: a deficiency of fructokinase.

-asymptomatic

2 Hereditary fructose intolerance: a deficiency of fructose

1-phosphate.

-accumulation in your cells

Pryuvate is formed in the ______ and oxidized in the ______. A. cytoplasm Cytoplasm B. mitochondrial matrix cytoplasm C. cytoplasm mitochondrial membrane D. mitochondrial inner space mitochondrial matrix E. cytoplasm mitochondrial matrix

Which of the following is a cofactor for pyruvate dehydrogenase complex (PDC)? A. NADP+ B. ATP C. Vitamin B1 D. FADH2

What is Thiamin used for?

Glycolysis PDH TCA ETC

Pyruvate Dehydrogenase Complex (PDC)

- Pyruvate, derived from glucose and other sugars by glycolysis, is

oxidized to acetyl-CoA and CO2 by PDH complex.

The three enzymes that make up the complex:

E1 pyruvate dehydrogenase; It is inhibited by its products NADH

and acetyl-CoA and stimulated by AMP. It is inactivated by the

phosphorylation and reactivated by dephosphorylation.

E2 dihydrolipoyl transacetylase, catalyzes the transfer of the acetyl group to coenzyme A, forming acetyl-CoA.

E3 dihydrolipoyl dehydrogenase, catalyzes the regeneration of the disulfide (oxidized) form of lipoate.

regulatory enzymes: phosphorylate / dephosphorylate E1 (a kinase and phosphatase). The kinase is activated by ATP, thus ATP inhibits E1.

The overall reaction is irreversible; fat cannot be converted into carbohydrate.

Why cant fat be converted into glucose? a) As a means to punish people who cant cook b) Because anaperotic reactions are inhibited during

the fasting state c) Because the PDH reaction is irreversible d) Because of an accumulation of oxaloacetate

Key for the future: Phosphorylation of gluco-metabolic enzymes tends to correlate with getting energy out. Dephosphorylation of gluco-metabolic enzymes tends to correlate with producing ATP.

Mechanism of PDH Action

Five consecutive reactions:

TPP lipoic acid CoA FAD NAD

Decarboxylated then transferred to CoA

Requires 5 different cofactors:

Thiamine pyrophosphate, TPP (B1) - E1

Lipoic acid (lipoate) - E2

Flavin adenine dinucleotide, FAD (Riboflavin) - E3

Nicotinamide adenine dinucleotide, NAD+ (Niacin)

Coenzyme A (CoA) (synthesized from pantothenic acid (B5)

Pyruvate Dehydrogenase Complex (PDH)

Make sure youve read through the details on the PP.

Thiamine Pyrophosphate (TPP)

Thiamine deficiency leads to inability to oxidize pyruvate and thus

has a major neurological impact (Beriberi).

- removal of carboxyl (-COOH) groups from organic acids

- is a vitamin B1 derivative which is produced by the enzyme thiamine

Regulation of PDH

Allosteric

- Inhibition by ATP, acetyl-CoA and NADH.

- Activation by AMP, CoA and NAD+.

Covalent

- Activation by dephosphorylation

- Inhibition by phosphorylation of E1 (pyruvate dehydrogenase). Kinase activated by ATP.

Pyruvate is a potent inhibitor of pyruvate dehydrogenase kinase.

TCA Cycle Degradation of acetyl-CoA derived from carbohydrates, fatty acids

and amino acids.

- produces most of the CO2 generated in tissues (2).

- is the major source of NADH (3).

- allows excess energy to be used for fatty acid biosynthesis.

- provides precursors for many metabolites.

Most TCA cycle enzymes in the mitochondrial matrix; some in the

inner mitochondrial membrane.

How many molecules of NADH are formed from the breakdown of 1 molecule of pyruvate during one complete turn of the TCA cycle? A. 1 B. 2 C. 3 D. 4 E. 5

Remember: 2 in glycolysis, and one more than that in TCA

TCA Strategy - Activated 2C fragment (acetyl-CoA) reacts with a 4C oxaloacetic acid, to

yield 6C citrate.

- In a series of seven reactions two carbons are released as CO2 (oxidative decarboxylations), regenerating oxaloacetate.

- 2 carbons in, 2 carbons out = no net gain.

- Oxaloacetate must be present to "prime" the cycle.

- Four pairs of electrons are transferred during one turn of the cycle: three pairs of electrons reducing three NAD+ to NADH, and one pair reducing FAD to FADH2.

- One complete turn of the TCA cycle generates only one ATP (GTP) (in the conversion of succinyl-CoA to succinate), three molecules of NADH and one molecule of FADH2. Oxidation of one NADH leads to formation of 3 ATP, whereas oxidation of FADH2 yields 2 ATP.

- Total 12 ATP generated by one round of TCA cycle from one acetyl residue. From two acetyl residues, total 24 ATP generated.

TCA Cycle Acetyl-CoA

1

citrate

synthase

aconitase

2

3

isocitrate

dehydrogenase

oxidative decarboxylation 4

-ketoglutarate

dehydrogenase

phosphorylation 5

succinyl-CoA

synthetase

succinate

dehydrogenase

6

fumarase

hydration 7

8

malate

dehydrogenase TCA Cycle

(also called oxoglutarate)

(ATP)

Know the 8 steps

Which of the following is an example of substrate level phosyhorylation: A. Fumarase. B. Alpha-Ketogluterate Dehydrogenase C. Hexokinase D. Succinyl-CoA Synthetase. E. PFK1

Synthetase vs. Synthase

What is the total ATP yield from glycolysis plus TCA? -but what about ETC?

Regulation of the

TCA Cycle

Regulation of metabolite flow

from the PDH complex through

the TCA cycle in mammals.

The PDH complex is allosterically

inhibited when [ATP]/[ADP],

[NADH]/[NAD+], and [acetyl-

CoA]/[CoA] ratios are high,

indicating an energy-sufficient

metabolic state. When these ratios

decrease, allosteric activation of

pyruvate oxidation results.

The overall rate of the TCA cycle is controlled

by the rate of conversion of pyruvate to acetyl-CoA.

Anaplerotic Pathways

TCA

cycle in

term

ed

iates

Pyruvate carboxylase, malic enzyme, PEP-carboxykinase. Four rxns

Pyruvate Carboxylase Defficiency

A rare recessively inherited condition (enzyme deficiencies are normally genetic recessive)

Pyruvate accumulates because pyruvate carboxylase is a major consumer of pyruvate

Accumulated pyruvate is converted to lactate and alanine

Hypoglycemia

Caused by the inability to convert pyruvate to glucose

Neurological deficits and mental retardation

Dysfunction of TCA cycle due to insufficient oxaloacetate

Energy dysfunction normally results with neurological problems

Antiporters Transport Metabolites Across the Inner Mitochondrial Membrane

Transport across the inner mitochondrial membrane usually requires specific carriers

ADP, ATP (but not the other nucleotides); Pyruvate; Phosphate; Alanine, Aspartate; Glutamate; Substrates, intermediates and products of the TCA cycle

except: acetyl-CoA, oxaloacetate, fumarate, NAD, NADH (3ATP)

Does NADH really not need a transporter?

UNDER AEROBIC CONDITIONS (1)

Regeneration of NAD

What about the outer membrane?

UNDER AEROBIC CONDITIONS (2) Skeletal and cardiac muscle

More efficient 1 to 1 transfer

- Reaction intermediates donate electrons to specific coenzymes, such

as NAD+ and FAD to form the energy-rich reduced coenzymes, NADH

and FADH2.

- These reduced coenzymes can donate a pair of electrons to a electron

carriers, collectively called electron transport chain, and they lose their

free energy, that can be stored by the production of ATP from ADP and Pi,

this process is called oxidative phosphorylation.

- The electron transport chain is present in the inner mitochondrial

membrane.

- Electron transport chain and oxidative phosphorylation (ATP synthesis): are

tightly coupled processes. Therefore, inhibition of the electron transport

chain also results in inhibition of ATP synthesis.

Electron transport chain (respiratory chain)

and oxidative phosphorylation

Oxidative phosphorylation involves the reduction of O2 to H2O with

electrons donated by NADH and FADH2.

Photo-phosphorylation involves the oxidation of H2O to O2, with

NADP+ as electron acceptor.

Oxidative phosphorylation and photo-phosphorylation, both are

mechanistically similar both processes involve the flow of electrons.

Oxidative phosphorylation begins with the entry of electrons into the

respiratory chain.

NADH (carries electrons from catabolic reactions) and NADPH (supplies

electrons to anabolic reactions), are water-soluble electron carriers that

associate reversibly with dehydrogenases.

Oxidative phosphorylation

None of the functional groups in proteins can transfer hydrogen or

electrons, the components of the respiratory chain have to employ

metal ions and coenzymes.

Respiratory chains contains: (e- carriers)

- Flavoproteins

- Iron-sulfur proteins

- Cytochromes

- Ubiquinone (UQ) or Coenzyme Q (CoQ)

- Protein-bound copper

Note: With the exception of coenzyme Q, all members of this chain

are proteins.

Major Mitochondrial Electron Carriers (or Electron-Transfer System or Respiratory Chains)

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Flavoproteins

- The FAD or flavin mononucleotide (FMN) in the flavoproteins can transfer either one- or two-electrons at a time.

- Electron transfer occurs because the flavoprotein has a higher

reduction potential than the compound oxidized.

- They accept hydrogen/electrons from NADH and donate it to the

cytochromes.

Prosthetic group (tightly bound) to complex 2

Iron - Sulfur Proteins

(e- carriers) - also known as non-heme iron proteins. What is a heme?

- anchored in proteins through cysteine.

- both the iron-sulfur proteins and the flavoproteins of the respiratory

chain are integral membrane proteins.

Iron-sulfur complexes in proteins: iron in these complexes can change its

oxidation state reversibly between the ferrous (Fe2+) and ferric (Fe3+) forms.

FeFe3+3+ + e+ e-- FeFe2+2+

Cytochromes Cytochromes: Heme proteins (is a complex of protoporphyrin IX and

ferrous iron) that functions as electron carriers in respiration, photosynthesis and other oxidation-reduction reactions.

- One-electron carriers (Fe3+ Fe2+).

- All but one (cytochrome c) are integral membrane proteins.

- Cytochrome c is a small and water-soluble protein.

What does it do?

Ubiquinone (UQ) or Coenzyme Q (CoQ)

Makes molecule hydrophobic

- Ubiquinone, a hydrophobic quinone, is a lipid-soluble benzo-quinone

with a long isoprenoid side chain.

Match the following with where they are found: A. Fe-S proteins B. Flavin proteins C. Cytochrome C D.Cytrochome b E. Coenzyme Q

(ubiquinone)

1. Integral membrane protein

2. Free to diffuse

Which of the following complexes contain a protein-bound copper? A.NADH dehydrogenase B.Succinate Dehydrogenase C.Cytochrome b-c Complex D.Cytochrome c Oxidase

What are each of the above mentioned structures?

Protein-bound copper

- Protein-bound copper participates in the last reaction of

the respiratory chain, the transfer of electrons to molecular

oxygen. It switches between the Cu+ and Cu2+ forms during

these electron transfers. Part of complex 4

Respiratory Chain

Four members of the respiratory chain are freely diffusible:

NADH, ubiquinone, cytochrome c, and molecular oxygen.

1 Complexes I and II catalyze electron transfer to ubiquinone from two

different electron donors: NADH (complex I) and succinate or

FADH2 (complex II).

2 Complex III carries electrons from reduced ubiquinone to cytochrome

c, and

3 Complex IV transferring electrons from cytochrome c to O2.

Which side of the mitochondrion is the most acidic after oxidative phosphorylation? A.The inner membrane space B.The matrix C.Outer membrane space D.pH is the same across

Cells with high rates of respiration (e.g. heart

muscle) have mitochondria with many

densely packed cristae. Liver cells have

much fewer cristae.

COMPONENTS OF THE

ELECTRON TRANSPORT CHAIN COMPLEX I COMPLEX III COMPLEX IV

What happened to the last 2H+ at complex 4?

Increasing reduction potential

Complex I

(NADH to Ubiquinone)

- Complex I, also called NADH:ubiquinone oxidoreductase or

NADH

dehydrogenase, transfers electrons from NADH to ubiquinone, is a

large enzyme composed of 42 different polypeptide chains, including

FMN-containing flavoprotein and at least six iron-sulfur centers.

-no FAD?

- Electrons are transferred from NADH -> FMN -> iron-

sulfur -> ubiquinone.

- Amytal (a barbiturate drug), rotenone (a plant product commonly

used as an insecticide), and piericidin A (an antibiotic), inhibit electron

flow from the Fe-S centers of Complex I to ubiquinone.

If you block complex 1 can oxidative phosphorylation take place?

Complex II

(Succinate to Ubiquinone)

Complex II, also called succinate dehydrogenase, is the only

membrane-bound enzyme in the TCA cycle.

- It contain three Fe-S centers, bound FAD

- Electrons from succinate pass through a flavoprotein and several Fe-S

centers to ubiquinone.

- Acyl-CoA dehydrogenase (the first enzyme of oxidation) transfers electrons to ETF (electron-transferring flavoprotein, FAD), from which they pass to ubiquinone.

Complex III (Ubiquinone to Cytochrome c)

Complex III, also called cytochrome c oxidoreductase or cytochrome

bc1 complex, couples the transfer of electrons from ubiquinol (QH2) to

cytochrome c.

- It contains cytochrome b, an Fe-S protein, and cytochrome c1.

- Cytochrome c is a soluble protein. After its single heme accepts an

electron from Complex III, cytochrome c moves to Complex IV to

donate electron to a binuclear copper center.

INHIBITED BY ANTIMYCIN A

Complex IV (Cytochrome c to O2)

This is the final step of the respiratory chain. Complex IV, also called

cytochrome oxidase, carries electrons from cytochrome c to molecular

oxygen, reducing it to H2O.

- Complex IV is a large enzyme (13 subunits; Mr 204,000) of the inner

mitochondrial membrane.

- Oxygen is tightly bound between heme a3 and copper, to be released

after its reduction to H2O by the transfer of four electrons.

- Cytochrome oxidase has a very high affinity for oxygen.

- INHIBITED BY: CYANIDE, CO, AZIDE

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ATP Synthase

FO

F1 O for Oligomycin

Proton Gradient Oxidative phosphorylation proceeds in two steps: 1. Protons are pumped out of the mitochondrion: Proton pumping is

driven by the redox reactions in the respiratory chain, and creates a

electrochemical gradient across the inner mitochondrial membrane.

The inner membrane must be impermeable to protons to maintain

gradient.

Protons are admitted back into the mitochondrion, down their

concentration gradient, via proton channel: This process drives

ATP synthesis.

- four protons are pumped by the NADH-ubiquinone reductase complex and another four by the ubiquinone-cytochrome c reductase complex.

- cytochrome oxidase removes four protons from the mitochondrial

matrix, translocating two to the inter-membranous space and other two

consuming in the reduction of O2, thus, four protons leads to the synthesis

of one ATP molecule.

- ATP is synthesized only when protons flow, and protons can flow

only when ATP is synthesized.

Respiratory Control

- There is no rate-limiting step in oxidative phosphorylation, but its rate depends on substrates availability (ADP, inorganic phosphate and O2), and an oxidizable metabolite, like NADH and/or FADH2.

- ATP synthesis is absolutely dependent on continuous electron flow (electron transport) and electron transport occurs only during ATP synthesis.

- Oxidative phosphorylation produces most of the ATP.

- In healthy cells, the level of ATP exceeds that of ADP by a factor of 4 - 10.

- High demand for ATP, generates ADP, which stimulates respiration.

- At rest, accumulation of ATP depletes ADP levels and suppresses respiration.

2,4-dinitrophenol: A. Inhibits the flow of electrons at complex 4 B. lowers the basal metabolic rate C. Inhibit the flow of electron at complex 2 D. decrease the H+ gradient across the inner mitochondrial membrane

Uncouplers do which of the following: A. Diminish the proton gradient B. Disrupts the electron transport chain C. Prevent Complex I from transferring electrons from NADH to ubiquinone D. Disrupts oxidative phosphorylation but allows ATP synthesis to occur

Uncouplers of Oxidative Phosphorylation

Uncoupler - Any substance that inhibits ATP synthesis but have no effect on electron transport.

(Uncouplers means the dissociation of oxidative phosphorylation (not reduction) from ATP synthesis. The most common uncoupler is 2,4-dinitrophenpl (DNP)).

specific chemical agents:

- 2,4-dinitrophenol (DNP) and pentachlorophenol

- Valinomycin: an antibiotic that makes the inner mitochondrial membrane permeable for potassium.

Oxygen utilization is maintained as electron transport (and H+ pumping) continue.

ATP synthesis is diminished or inhibited, as proton gradient (electron-flow) is weak or disrupt, or non-existent.

- Thyroxin: a hormone produced by the thyroid glands to regulate

metabolism by controlling the rate of oxidation in cells.

- Thermogenin: a protein of the inner mitochondrial membrane that

allows trans-membrane movement of protons, also called uncoupling

protein, a membrane-spanning protein that forms a natural

channel to allow proton return.

increases heat generation.

found in specialized tissues (usually hairless, hibernators or cold-adapted).

brown fat (particularly high content of cytochromes) localized in the neck and upper back.

physiological un-couplers:

Uncouplers of Oxidative Phosphorylation

Inhibitors of Electron Transport

Oxidative phosphorylation is inhibited by many poisons.

Electron flow through the respiratory chain, can be blocked by:

Rotenone - an insecticide, blocks flow of electrons from Fe-S complexes in the NADH-Q reductase complex to ubiquinone through Complex I, (Parkinsons?). Blocks transfer of electrons associated with NADH.

Amytal (a barbiturate), site of action same as rotenone, also inhibit electron-flow through the NADH-Q reductase complex (Complex I).

Antimycin A, an antibiotic, that blocks electron flow from cytochrome b to c1 - through cytochrome c oxido-reductase complex (Complex III).

Cyanide - binds to cytochrome oxidase (Complex IV) and prevents electron transfer to oxygen. Hydrogen sulfide, carbon monoxide and azide also act as cyanide.

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Mitochondrial DNA

Mutations in mitochondrial DNA can cause:

- Lebers hereditary optic neuropathy (LHON): blindness, caused by degeneration of the optic nerve.

- Myoclonic epilepsy and ragged-red fiber disease (MERRF).

- Mitochondrial DNA has a higher mutation rate than nuclear DNA.

Which of the following can be damaged by ROS? A. Hemoglobin B. Proteins C. DNA D. Cell Membranes E. All of the above

Reactive Oxygen Species (ROS) Are Formed

During Oxidative Metabolism

Superoxide: O2 + e O2

O2 HO2

H2O2

o Also: Reduced flavoproteins (FADH2) in locations other

than the respiratory chain can produce hydrogen

peroxide (H2O2)

Hydroxyl radical:

H2O2 + Fe2+ + H+ HO + Fe3+ + H2O

HO2 O2

H+

H+

Electrons may leak out of the respiratory chain:

Enzymes Evolved to Destroy ROS

Superoxide dismutase (SOD)

2O2 + 2H+ H2O2 + O2

Catalase (a heme-containing enzyme that destroys H2O2)

2H2O2 2H2O + O2

Peroxidases Glutathione peroxidase (2GSH + H2O2 GSSG + 2H2O)

SOD

Catalase

Many Metabolites, Vitamins, Hormones and

Phytochemicals Can Eliminate Dangerous Free

Radicals

These molecules form stable free radicals that are sufficiently reactive to react readily with free radicals

but not sufficiently reactive to damage normal

constituents of the cell.

Bilirubin

Uric acid

Ascorbate (Vitamin C)

Estrogens

Carbohydrate Metabolism Most important function of carbohydrate metabolism is: maintain blood

glucose level at all times.

- Brain alone consumes ~ 120 g of glucose / day. Therefore, a blood glucose level of 4.0 to 5.5 mmol/liter (70 to 100 mg/dL) must be maintained at all times.

- Only liver glycogen (NOT muscle glycogen), can be used to maintain the blood glucose level.

- Gluconeogenesis produces glucose from amino acids, lactic acid, and glycerol. It is the only source of glucose during prolonged fasting.

- Only the liver and kidneys have a complete gluconeogenic pathway.

- Liver produces 10 times more glucose than kidney, because the liver is so much larger than a kidney.

Which of the following cannot be utilized for the net synthesis of glucose? A. Leucine B. Glycerol C. fatty acids D. Aspartate E. Alanine F. Acetyl CoA Remember that the 2 L amino

acids are only ketogenic.

Gluconeogenesis new formation of sugar

- Occurs primarily in the cytosol, although some precursors are generated in the mitochondria.

- Glucose is a universal fuel and building block in humans, some tissues (brain, erythrocytes, testes) depend almost completely on glucose for their metabolic energy.

- Brain alone requires ~ 120 g of glucose each day this is more than half of all the glucose stored as glycogen in liver and muscle.

- Liver is the major gluconeogenic organ of the body.

- Muscle does NOT contribute to gluconeogenesis, because no glucose 6-phosphatase and no glucagon receptors

Glucose stored in the liver and muscle, in the form of glycogen. But this stored glucose

is not sufficient between meals and fasting. At that time, organisms synthesize glucose

from non-carbohydrate (such as pyruvate). This pathway called gluconeogenesis.

Gluconeogenesis

Gluconeogenesis: synthesis of carbohydrate (such as glucose)

from non-carbohydrates such as oxaloacetate or pyruvate

(Synthesis of one molecule of glucose from two molecules of pyruvate requires energy, equivalent to 6 molecules of ATP)

- An energy-requiring, biosynthetic pathway of generating

glucose, generally from 3- and 4- carbon compounds.

- Gluconeogenesis must get around the highly exergonic

glucokinase, phosphofructokinase-1 (PFK-1) and pyruvate

kinase reactions by a new set of exergonic reactions that

drive the process in the opposite direction. (1, 3 & 10)

Gluconeogenesis

Gluconeogenesis and glycolysis are NOT identical pathways, running in opposite directions. Reverse of glycolysis except the irreversible (kinase) reactions.

Three reactions of glycolysis are irreversible and cannot be used in gluconeogenesis:

1. Conversion of glucose to glucose 6-phosphate by hexokinase/glucokinase.

2. Phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by phosphofructokinase-1 (PFK-1).

3. Conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase.

Match the glycolytic enzymes with their gluconeogenic counterparts:

1. Hexokinase/ Glucokinase

2. PFK-1 3. Pyruvate Kinase

A. PEP carboxykinase B. Fructose -1,6-

bisphosphatase C. Pyruvate carboxylase D. Glucose-6-

phosphatase

Glycolysis and

Gluconeogenesis

Glycolysis Gluconeogenesis

[1]

2

3

4

5 6

7

[8] 9

[10]

Irreversible reactions # [1], [8],

and [10] Know the enzymes.

Notice the use of an ATP and a GTP

Which of the following liver enzymes would be active in the fasted state? A. pyruvate kinase B. PEP carboxykinase C. Fructose 1,6 bisphosphatase. D. Both A and B E.Both B and C

Which of the following metabolic reactions can pyruvate carboxylase be found? A. Glycolysis B. Krebs cycle C. Glycogen synthesis D. Gluconeogenesis E. Both B and D F. Both A and D

Conversion of Glucose 6-phosphate to Glucose

- The irreversible glucokinase reaction is reversed by the action of glucose-6 phosphatase (a simple hydrolysis). This

enzyme is located in the ER and is present only in

the liver. - Glucose can thus leave the liver and enter the blood.

Note: glucose-6 phosphatase is involved in both gluconeogenesis and

glycogenolysis.

Fructose-2, 6-bisphosphate: A. Stimulates glycolysis B. Is inhibited by PFK1 C. Is inhibited by PFK2 D. Favors gluconeogenesis E. Stimulates Fructose-1,6-bisphosphatase

Activates glucose breakdown

Overall Gluconeogenesis reaction

- Leading from pyruvate to free glucose:

2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4H2O

glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+

How do amino acids lead to free glucose?

Gluconeogenic Substrates

- Lactate (Pyruvate) Cori cycle. Lactate is released into the blood by

muscle, and by cells that lack mitochondria, such as RBCs and taken

to the liver to be used for gluconeogenesis

- Glucogenic amino acids (all, except leucine and lysine).

- Glycerol (from the breakdown of triglycerides in adipose tissue),

- All TCA intermediates (except acetyl-CoA) are substrates of gluconeogenesis.

Cori Cycle

After vigorous exercise, lactate produced by anaerobic glycolysis in muscle

returns to the liver and is converted to glucose, which moves back to muscle

and is converted to glycogen a circuit called the Cori cycle.

Cori Cycle: Shuttling of glucose and lactate between muscle and liver

during physical exercise.

Regulation of Gluconeogenesis Hormones are important for the regulation of gluconeogenesis:

Insulin: is a hormone, released from pancreatic -cells in response to hyperglycemia (increased blood glucose level). Insulin lowers the blood glucose level (stimulates glycogenesis and glycolysis in liver).

Glucagon: is a polypeptide hormone from the -cells of the

pancreas that stimulates the glucose-producing pathways of the liver (glycogenolysis & gluconeogenesis). It is released in response to hypoglycemia (decreased blood glucose level). Glucagon raises the blood glucose level.

Regulation of Gluconeogenesis

Glucagon and insulin secreted by pancreatic and cells,

respectively. The release of glucagon and insulin is inhibited

by somatostatin, which is secreted by the pancreatic

delta cells.

Epinephrine and norepinephrine: are stress hormones that

are released during physical exertion and cold exposure. In the

liver, they favor gluconeogenesis over glycolysis by inducing

cAMP. EPI->G-protein->adenylyl cyclase->cAMP->PKA

->blood glucose

Glucocorticoids: are also stress hormones that affect gene

transcription. Glucocorticoids stimulate gluconeogenesis by

inducing gluconeogenic enzymes.

Regulation of Gluconeogenesis (3)

Pyruvate kinase is the most important regulated enzyme in the PEP-pyruvate cycle. It is inhibited by ATP and activated by

fructose 1,6-bisphosphate.

Phosphofructokinase and fructose-1,6-bisphosphatase in the liver are oppositely regulated. ATP and citrate stimulate fructose-1,6-

bisphosphatase but inhibit phosphofructokinase (favor

gluconeogenesis).

Glucose-phosphorylating enzymes in the liver are hexokinase/glucokinase the glucose/glucose 6-phosphate reaction.

Just know that when one pathway is activated the other is inhibited.

-ATP and citrate stimulate fructose 1,6 bisphosphatase but inhibit phosphofructokinase, means favor

gluconeogenesis.

Ethanol increases both, cytosolic and mitochondrial NADH + H+ concentrations. Bad if it uses all the NAD+

- Increased NADH + H+ inhibits the conversion of lactate to pyruvate, thus inhibiting gluconeogenesis.

Ethanol and Liver Metabolism

Gluconeogenesis - Gluconeogenesis takes place almost exclusively in the liver, and the

liver receives large quantities of fatty acids from adipose tissue during fasting. Insulin inhibits lipolysis, thus in fed state the level of free fatty acids is low and in fasting state, when insulin is low, FFAs are high.

- Fatty acid oxidation is less controlled by feedback inhibition than is glucose oxidation. Therefore, the levels of ATP and acetyl-CoA in the liver are actually elevated during fasting.

- Fatty acid oxidation provides the necessary energy required by liver for gluconeogenesis. What is the substrate?

Fatty acid oxidation: The burning of stored fat.

What is the Livers Primary energy source?

A.Glycolysis B.Gluconeogenesis C.Fatty Acid Oxidation D.A & B

Why Glycogen?

- Fat cannot be as rapidly mobilized in muscle.

- Fat cannot be oxidized to produce energy under

anaerobic conditions.

- Requires energy input to initiate fat oxidation.

- Fat cannot be converted into glucose!

Glycogenesis vs. Glycogenolysis

Liver glycogen only to maintain blood glucose, liver doesnt use it.

Storage of Glucose and Glycogen

in a normal healthy adult

-Most people have more muscle than liver, the total amount of muscle

glycogen exceeds that in the liver.

There is more glycogen per gram of tissue weight in the liver (it is more

concentrated in the liver)

Glycogen Structure

1

2 3

4

5

6

1

2 3

4

5

6

1

2 3

4

5

6

Know alpha-1,4- linkages (like starch) -vs. beta which is cellulose With alpha-1,6-branching Add glucose molecules at the reducing end, take them off at non.

Glycogen Synthesis

Glycogen is readily synthesized from glucose.

(Glucose is a most abundant monosaccharide in dietary carbohydrate)

For glycogen synthesis:

- Primer required to initiate synthesis.

- The protein glycogenin (Mr 37 kDa) serves as the primer,

or that primes the synthesis of new glycogen chains.

- Glycogenin is self glycosylating; attaches glucose from

UDP-glucose to tyrosine residue (Tyr194) of glycogenin.

Glycogen Synthesis

Synthesis of uridine diphosphate (UDP)-glucose. UDP-glucose is the

activated form of glucose, or the precursor for glycogen synthesis.

2 high energy bonds

Which of the following favors glycogen synthesis? A. Binding of epinephrine to alpha2 receptors B. The activation of Protein Kinase A and its phosphorylation

cascade C. Rising levels of AMP in the muscle D. Activation of glycogen phosphorylase E. A high insulin/glucagon ratio

122

What does glycogen phosphorylase do?

Glycogen Synthesis

Glycogen synthase is the key regulatory enzyme for glycogen synthesis; creates the -1,4 glycosidic bonds in glycogen by transferring the glucose residue from UDP-glucose to non-reducing end of the glycogen molecule.

Glycogen synthase cannot form the -1,6 glycosidic bonds at the branch points of glycogen. Branching requires a glycogen-branching enzyme, called transglycosylase or glycosyl-transferase, that breaks an -1,4 bond and forms an -1,6 bond .

Glycosidic bonds: are covalent bonds, formed between two monosaccharide molecules by means of a dehydration reaction.

Which of the following enzymes are not found in muscle? A. Glycogen phosphorylase B. Hexokinase C. Glucose-6-phosphatase D. Phosphoglucomutase E. All of the above are found in muscle

In the muscle glycogenolysis is always coupled to glycolysis!!

Regulation of Glycogen Metabolism

Glycogen metabolism is regulated by hormones and metabolites.

- Glycogen synthesis and glycogen degradation should not be active

at the same time to avoid an ATP-consuming futile cycle, achieved

by the phosphorylation/dephosphorylation of the key enzymes

glycogen synthase and glycogen phosphorylase.

- Glycogen synthase is active in the de-phosphorylated state, whereas

glycogen phosphorylase is active in the phosphorylated state.

Phosphorylation state of the enzymes is regulated by:

- Insulin- Dephosphorylated state both in the liver and in muscle. - Glucagon- Phosphorylated state (only affects liver)

- Norepinephrine and epinephrine are powerful activators of

glycogen breakdown both in muscle and liver.

protein kinase phosphorylates both glycogen

phosphorylase and glycogen synthase &

protein phosphatase de-phosphorylates both glycogen

phosphorylase and glycogen synthase .

Regulation of glycogen synthase and glycogen

phosphorylase

C C

R R C C

R R

OH

OH OH

Phosphorylase b

OP OP

Phosphorylase a

Glycogen Glucose-1P

ATP cAMP

Inactive Protein Kinase A

Active Kinase A

ATP

Inactive phosphorylase kinase

Active

2 ATP

Adenylyl cyclase

O-P

+

Phosphoprotein

phosphatase

Phosphoprotein phosphatase

Phosphorylase kinase

Ca++ (muscle)

+

Glucose

-

HORMONE (GLUCAGON / EPINEPHRINE)

AMP(muscle)

128

Glycogen metabolism enzyme deficiencies -> storage diseases

Von-Gierkes disease (type I) Glycogen buildup in liver Pompe disease (type II) Glycogen buildup in muscle (heart) Cori disease Anderson disease McArdle disease Hers disease Tarui disease

The binding of epinephrine to receptor on liver cells would result in intracellular increase in: A. glucose-6-phosphate dehydrogenase. B. CAMP C. Glycogen synthetase activity. D. glycolysis E. All of the above.

What is the route of energy mobilization?

131

Fructose Metabolism in the Liver

Excess fructose is toxic

The liver metabolized fructose faster than it metabolizes glucose. Fructose-1-phosphate tends to accumulate because fructokinase activity exceeds aldolase B activity Fructose-1-phosphate directs dietary glucose into glycogen synthesis

Fructose phosphorylation ties up inorganic phosphate (Pi), which impairs oxidative phosphorylation

NADPH: A. Maintains an oxidative environment of the

cytosol B. Provides electrons for oxidative

phosphorylation C. Is a coenzyme for fatty acid synthesis D. Is mainly found in the mitochondrion

PPP two general products: NADPH for biosynthesis Ribose for nucleic acid synthesis

Primary Function of the

PPP

Oxidation of glucose-6-phosphate, makes two important products: NADPH and ribose-5-phosphate

- occurs in the cytosol of the cell: all the enzymes in the PPP are located in the cytosol.

- provides NADPH, a coenzyme, for redox reactions or reductive biosynthetic pathways (such as fatty acid and cholesterol synthesis) and maintenance of reductive environment of the cytosol -the defense against oxidative damage or stress.

- provides ribose-5-phosphate for nucleotide (purine and pyrimidine) and nucleic acid biosynthesis and recycles nucleic acid-derived pentoses for potential energy use.

Which Tissues?

- all tissues:

- Most active in tissues involved in fatty acid and steroid synthesis (adrenal gland, liver, adipose tissue and mammary gland).

- Red blood cells (RBCs): maintain their membrane integrity. Rapidly dividing cells, such as bone marrow, use ribose 5-phosphate to make RNA, DNA, coenzymes as ATP, NADH, FADH2 and coenzyme A.

.

2 phases

Oxidative Phase: oxidative branch of the PPP is concerned

with the synthesis of NADPH. Glucose-6-phosphate

dehydrogenase catalyzes the rate-limiting step. The reaction

is irreversible, maintains a high NADPH / NADP+ ratio.

Non-oxidative Phase: non-oxidative branch of the PPP links

ribulose 5-phosphate (product of the oxidative branch) to the

glycolytic and gluconeogenic pathways. This reaction is

reversible. The most important enzymes in this reaction, are

transketolase and transaldolase.

-Pentose can be recycled or used.

Pentose Phosphate Pathway (PPP)

Glucose 6-phosphate dehydrogenase (G6PDH) is inhibited

by a high NADPH / NADP+ ratio.

G6PDH is the first, and most important, enzyme of the

pentose phosphate pathway.

The amounts of glucose 6-phosphate dehydrogenase and

phosphogluconate dehydrogenase are increased in the well-

fed state, and this effect is mediated by insulin.

Why is this enzyme sometimes an issue?

Oxidative reactions of PPP

In some organs, the Pentose Phosphate Pathway ends at

this point, and its overall equation is:

Glucose 6-phosphate + 2 NADP+ + H2O

ribose 5-phosphate + 2 NADPH + 2 H+ + CO2

Non-oxidative reactions of PPP

- Pentose phosphates produced in the oxidative phase, are recycled into glucose 6-phosphate in non-oxidative phase.

- In a series of reactions or rearrangements of the carbon skeletons:

- six 5-carbon sugar phosphates are converted to five 6-carbon

sugar phosphates.

- produces glucose 6-phosphate and NADPH.

Two enzymes are unique in PPP in inter-conversions of sugars:

1. Transketolase: catalyzes the transfer of 2-carbon from a ketose donor to an

aldose acceptor, forming the 7-carbon product sedoheptulose 7-phosphate.

The remaining 3-carbon fragment is glyceraldehyde 3-phosphate.

2. Transaldolase: a 3-carbon fragment is removed from sedoheptulose

7-phosphate and condensed with glyceraldehyde 3-phosphate, forming

fructose 6-phosphate, and finally convert to glucose 6-phosphate by

phosphohexose isomerase.

Summary of the flow of 15 C atoms through Pentose Phosphate Pathway reactions by which 5-C sugars are converted to 3-C and 6-C sugars.

C5 + C5 C3 + C7 (Transketolase)

C3 + C7 C6 + C4 (Transaldolase)

C5 + C4 C6 + C3 (Transketolase)

3 C5 2 C6 + C3 (Overall)

Glucose 6-phosphate may be regenerated from either the 3-C glyceraldehyde 3-phosphate or the 6-C fructose 6-phosphate, via enzymes of gluconeogenesis.

Balance Sheet

140

If nucleic acid synthesis is required (e.g., in actively dividing cells), the product of the oxidative branch, ribose 5-phosphate (5C), is used

and will not enter the non-oxidative branch.

When the cell needs more NADPH than ribose-5-phosphate, both the oxidative and non-oxidative branches work in series to yield NADPH (+CO2) and form fructose-6-phosphate and glyceraldehyde-3-phosphate. These products are recycled to glucose-6-phosphate which goes back to feed the oxidative branch of PPP again, and again (i.e., all carbons of glucose-6-phosphate may convert to CO2).

If energy is also needed, 5C products are recycled to glucose-6-phosphate for glycolysis and repeat PPP reactions (e.g. RBCs).

Pentose Phosphate Pathway Scenarios

Role of Cytosolic NADPH - Maintain reductive environment in the cytosol of cell (GSH,

glutathione).

- Provide reducing equivalents for anabolic pathways (fat, cholesterol synthesis).

- Provide electrons for the generation of Reactive Oxygen Species (ROS). Making peroxides

- In red blood cells, NADPH produced by the pentose phosphate pathway, is very important in preventing oxidative damage

- Cytosolic environment must be maintained in a reduced form to maintain enzyme functionality.

Reducing environment: An environment without oxygen available for organisms.

Glutathione

- Glutathione (GSH, tripeptide; Glu-Cys-Gly), functions as a

reducing agent.

- Glutathione Peroxidase (GPx) catalyzes degradation of

hydroperoxides by reduction, as two glutathione molecules

(represented as GSH) are oxidized to a disulfide (GSSG).

Only the reduced form of glutathione (GSH) is an antioxidant. Therefore, the dimeric, oxidized form (GSSG) has to be reduced back

by the enzyme glutathione-reductase (GR). Can be faulty

(GR depends on NADPH from the pentose phosphate pathway)

2 GSH + ROOH GSSG + ROH + H2O

GSSG + NADPH + H+ 2 GSH + NADP+

GPx

GR

GS-SG 2 GSH

HSProteinHS Protein

S S

Active enzyme

Denatured enzyme

NADPH + H+ NADP+

Glucose 6-phosphate Dehydrogenase

(G6PDH) Deficiency

- Pentose phosphate pathway is the only source of NADPH in

RBCs.

- G6PDH is the first enzyme of the pentose phosphate pathway.

- G6PDH deficiency has a profound effect on the stability of red

cells, and cannot maintain antioxidant defenses.

- The red cells are unable to handle the additional oxidative stress,

after exposure to drugs (sulfonamides, acetanilid, nalidixic acid,

nitrofurantoin) or food items (fava beans).

- Over 400 genetic variants of G6PDH, mostly coding for less active/stable enzyme.

- The most common human enzyme deficiency; > 400 million people affected worldwide, with a shortened life span.

- Confers resistance to malaria. -Sickle cell anemia

Glucose 6-phosphate Dehydrogenase

(G6PDH) Deficiency

Major Organs of Metabolism Four major organs play a dominant role in fuel metabolism:

1.Liver

2.Adipose

3.Muscle, and

4.Brain.

Which of the following enzymes are favored by insulin? A. Glycogen synthetase B. Glycogen phosphorylase C. Protein Kinase A D. Cyclic AMP E. Glucokinase

Insulin: is a hormone, secreted by the cells of pancreas, to

lower blood sugar levels. Insulin is needed to convert sugar,

starches, and other food into glucose (blood sugar) or regulates

storage of glycogen in the liver and accelerates oxidation of sugar

in cells.

Insulin stimulates glycolysis and glycogenesis and

inhibits gluconeogenesis and glycogenolysis.

Insulin: Being a protein, insulin is not orally active because

it is destroyed by digestive enzymes, so mostly used as a

i.p. injection.

Its metabolic effects are anabolic, for example, synthesis of

glycogen, triacylglycerols (TAG), and protein.

The most important hormone for growth and development

Insulin

Insulin is a Satiety Hormone

- Insulin is the hormone of the well-fed state. Its synthesis and release

are stimulated by glucose, and this effect is further increased by amino

acids. Therefore, the plasma level of insulin is highest after a

carbohydrate-rich meal. During fasting, plasma level of insulin falls.

- Insulin stimulates the utilization of nutrients such as glucose, amino

acids, and triglycerides. It diverts excess nutrients into the synthesis

of glycogen, fat, and protein.

- Insulin regulates the metabolism of fat and protein as well as of

carbohydrate. It induces the conversion of excess carbohydrate to fat

by glycolysis and fatty acid synthesis.

Insulin decreases lipolysis Puts your energy stores away

Glucagon: is a polypeptide hormone secreted by

the -cells of the pancreas that stimulates the

glucose-producing pathways of the liver. It is

released in response to hypoglycemia (decreased

blood glucose Level, < 40 mg/dl). Hypoglycemia is

a medical emergency.

Glucagon raises the blood glucose level.

Glucagon promotes the release of glucose from the

liver. Glucagon stimulates glycogenolysis and gluconeogenesis

and inhibits glycolysis. in the liver

Glucagon

Glucagon Maintains the Blood

Glucose Level

- Glucagon secretion from the pancreatic cells is increased two to

three fold by hypoglycemia and reduced to half of the basal release by

hyperglycemia.

- Glucagon up-regulates the blood glucose level when dietary

carbohydrate is in short supply. Its actions on glucose metabolism

pathways, are opposite to those of insulin.

- Unlike insulin, glucagon acts exclusively on the liver; it has

negligible effects on adipose tissue, muscle, and other extra-hepatic

tissues.

For Glucagon think of releasing your energy stores like catecholamines

Which of the following tissues depends upon insulin for the transport of glucose? A. brain B. red blood cells C. liver D. Muscle E. All of the above

What transporter does it use?

What is a normal blood glucose level? a) 55mmol/liter b) 90mg/dL c) 5mg/dL d) 0.5mmol/liter

Unchecked rise in blood glucose following a meal would result in severe dehydration, and lead to hyperosmolar coma.

Fasted State (Liver)

Inhibition of:

PFK-2

Pyruvate kinase

Glycogen synthase

Activation of:

F-2,6 bisphosphatase

(loss of fructose-2,6-bisphosphate)

Phosphorylase kinase

Glycogen phosphorylase

Hormone-sensitive lipase

- The net result is the inhibition of glycolysis and glycogenesis and

- Activation of gluconeogenesis and glycogenolysis.

In the fasting state, Glucagon levels are high.

Fasted State (Liver) - Four hours after a meal, the liver becomes a net producer of glucose.

Glucose is initially formed by glycogen breakdown, but liver glycogen

lasts for < 1 day.

- During more extended fasting, humans depend entirely on gluconeo-

genesis to produce ~160 g of glucose per day. At least one half

of this is consumed by the brain.

- Liver supplies glucose and ketone bodies (such as acetoacetate and

-hydroxybutyrate) during fasting.

- Liver first uses glycogen degradation and then gluconeogenesis to

maintain blood glucose levels.

- Under fasting conditions, glucagon is elevated and stimulates

gluconeogenesis.

Prolonged Fast / Starvation

- Major adjustment in tissue glucose utilization occurs:

- Brain and muscle adapt to use ketone bodies and fatty acids thus decreasing the demand for hepatic gluconeogenesis.

- This spares protein; energy requirement is satisfied by fatty acid oxidation.

- Red cells remain the major glucose utilizers.

Re-feeding The levels of glycolytic enzymes in the liver are very low, and

patients severely starved show profound carbohydrate intolerance.

Therefore, re-feeding should be started slowly.

Ethanol and Liver Metabolism

- Alcohol is metabolized in the liver by two oxidation reactions:

1 Ethanol is first converted to acetaldehyde by alcohol dehydrogenase

in the cytosol, and produces NADH (& uses up cytosolic NAD+).

(The capacity to metabolize ethanol, is thus dependent upon the ability

to shuttle NADH into mitochondria (to regenerate NAD+).

2. Acetaldehyde enters mitochondria and is subsequently oxidized to

acetate by aldehyde dehydrogenase and generates NADH. (Note:

This enzyme is inhibited by disulfiram (antabuse), a drug that has

found use in patients desiring to stop alcohol ingestion.

- Increased NADH inhibits gluconeogenesis and fatty acid oxidation.

Reversal of Glucagon action by Insulin

(Re-feeding)

Activation of:

- PFK2

- Pyruvate kinase

- Glycogen synthase

Inhibition of:

- F-2,6 bisphosphatase

- Phosphorylase kinase

- Glycogen phosphorylase

- Hormone-sensitive lipase

Diabetes is caused by Insulin Deficiency or

Insulin Resistance

Type 1 diabetes (insulin-dependent): usually starts in childhood. It is an autoimmune disease that leads to the destruction of pancreatic

cells. Without endogenous insulin production, the patients depend on

insulin injections for life. Type 1 diabetes afflicts 1 per 400 individuals.

Type 2 diabetes (insulin-resistant): was a disease of middle-aged and older individuals. It is more common than type 1, is less severe. The

pancreatic cells are intact, and the plasma level of insulin may be

normal, reduced, or elevated. The problem may be either reduced insulin

secretion or insulin-resistance of the target tissues, or a combination of

both.

Diabetes is caused by Insulin

Deficiency or Insulin Resistance The complete absence of insulin in type 1 diabetes leads to:

- Diabetic ketoacidosis, with severe ketonemia, acidosis, and

blood glucose levels as high as 1000 mg/dL, in which the blood

becomes acidic due to a buildup of ketones.

- Large amounts of glucose and ketone bodies are lost in the

urine, and osmotic diuresis causes dehydration and

electrolyte imbalances. The coma is caused by dehydration,

electrolyte disturbances, and acidosis.

Major ketone bodies: acetoacetate, b-hydroxy butyrate, acetone

Know what ketonuria is

Diabetes is caused by Insulin

Deficiency or Insulin Resistance

- Patients with type 2 diabetes are not afflicted by ketoacidosis b/c they

have insulin to inhibit the breakdown of Fat to acetyl CoA, Acetyl-CoA

doesnt accumulate, so it doesnt get converted to ketones. It is

characterized by excessive glucosuria (glucose in the urine) with

osmotic diuresis (high glucose levels in the urine leads to concentrated

urine, which pulls water into the urine to dilute it) . If the patient

forgets to drink, the resulting dehydration can become sufficiently

severe to affect the central nervous system.

- The over-treatment of diabetes with insulin or oral anti-diabetic drugs

leads to hypoglycemic shock.

Diabetes leads to late complications Urinalysis is a quick screening test for diabetes

Eighty percent of Type 2 diabetics are obese. Obesity is the most common cause of insulin resistance.

What are Xenobiotics?

What is polydipsia?

Monooxygenase reaction catalyzed by cytochrome P-450 (CYP) to metabolize foreign chemicals.