3 April 2008 Sugars, concluded; Electron Transport and Oxidative Phosphorylation Andy Howard...

3 April 2008

Sugars, concluded; Electron Transport

and Oxidative Phosphorylation

Andy HowardIntroductory Biochemistry

3 April 2008

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Electron Transport

This shows how we can really make ATP from all those reducing equivalents that we amassed during glycolysis and the TCA cycle…but first we have some unfinished carbohydrate business to complete!

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What we’ll discuss Remaining

carbohydrate issues Entner-Doudoroff

Pathway Pentose Phosphate

Pathway Glyoxylate Pathway TCA cycle evolution

ETS and Oxidative Phosphorylation Generalizations about

oxidation-reduction reactions

Electron Transport: Complexes I-IV

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Entner-Doudoroff Pathway Alternative catabolic pathway from glucose-6-

phosphate to smaller molecules Found in some bacteria as alternative to normal

glycolytic pathway Other bacteria that do have glycolytic pathway

possess these enzymes as a side-path We’ve already discussed this: this is a review

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Entner-Doudoroff reaction 1:G6PDH Oxidizes glucose-6-phosphate to 6-

gluconolactone We’ll meet this enzyme in the PPP

shortly

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Entner-Doudoroff Pathway 2:Gluconolactonase

Dehydratase: Converts 6-P-gluconolactone to 6-P-gluconate

An example of a phosphorylated sugar acid

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Entner-Doudoroff Pathway 3:6-P-gluconate dehydratase Converts 6-phosphogluconolactonate to

2-keto-3-deoxy-6-phosphogluconate with release of water

First step differentiating this pathway from the pentose phosphate pathway

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Entner-Doudoroff Pathway 4:KDPG Aldolase As usual, breaking C-C bonds is somewhat

special Energetics fairly near isoergic, though Cleaves KDPG to pyruvate and glyceraldehyde-

3-phosphate Analogous to ordinary aldolase but secondary

product is more oxidized Thus only one ATP produced per molecule of

glucose degraded here

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KDPG aldolase

EC 4.1.2.14 Class I aldolase TIM barrel protein Strong similarities to other

aldolases, including fructose 1,6-bisphosphate aldolase

PDB 2C0A71 kDa trimerE. coli

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Significance of this pathway

Primary pathway for glucose degradation in some organisms

Secondary pathway in some organisms that do have standard glycolysis:

Provides degradative pathway for gluconate and related compounds

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Pentose Phosphate Pathway Pathway for converting 6-carbon sugar

phosphates to 5-C sugar phosphates Provides ribose-5-phosphate Provides reducing equivalents in the form

of NADPH that can be used in anabolic reactions

Catabolic Can be regarded as a cycle

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Pentose Phosphate Pathway:Oxidative Phase

Begins with G6PDH and gluconolactonase, just like Entner-Doudoroff pathway

Proceeds to ribulose-5-phosphate via a second oxidative step

Remember that NADPH generally is used in anabolism, and it has to come from somewhere PPP NADP+ + NADH NADPH + NAD+

NAD kinase

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Glucose-6-Phosphate Dehydrogenase

Catalyzes oxidation of G-6-P to6-phosphogluconolactone

Some isozymes will oxidize other hexoses

Others are specific to glucose

PDB 1DPG107 kDa dimerLeuconostoc mesenteroidies

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Isozymes: G6PDH & H6PDH

G6PDH is specific to glucose-6-P: Found almost exclusively in erythrocytes Coded for on X chromosome

(1 copy/cell: Male has 1, female’s second is inactive)

H6PDH: runs several hexose phosphates Found in many other tissues Coded for on Chromosome 22

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iClicker quiz Why does it matter that the G6PDH gene

is located on the X chromosome?(a) males don’t possess the gene(b) females don’t possess the gene(c) only one copy available per cell(d) no DNA-repair mechanisms available

for X-Chromosome genes

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Medical issues with G6PDH

Numerous identified mutations found in human erythrocytes

All involve partial interference with first reaction

Total absence of G6PDH is fatal Survival of defective G6PDH genes:

individuals with these erythrocytes have increased resistance to malaria

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Malaria: critical influence on human evolution

G6PDH Sickle-cell anemia (Hb E6V)

Similar natural history: Heterozygotes for sickle-cell have

increased resistance to parasite Behavior (post-WWII)

DDT, eradication of Anopheles mosquitoes, thin eggshells in birds

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Gluconolactonase

Converts gluoconolactone to 6-phosphogluconate

Remember this is a hydratase, not an oxidoreductase

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6-phosphogluconate dehydrogenase

Catalyzes oxidative decarboxylation of 6-phosphogluconate to ribulose-5-phosphate

NADP is electron acceptor Same superfamily of enzymes

as glycerol-3-P dehydrogenase

PDB 2PGD106 kDa dimerSheep

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Non-oxidative phase

Once we’ve made ribulose-5-phosphate, we can go a couple of directions

Two transketolase reactions:Kn + Am An-2 + Km+2

One transaldolase: Kn + Am An-3 + Km+3

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Non-oxidative steps Epimerases

, isomerases, trans-ketolases, trans-aldolases

Chart courtesyMichael King,Indiana State

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Ribulose-5-phosphate 3-epimerase

Converts RuBP to xylulose-5-P

TIM-barrel protein Co-regulated with

RuP isomerasePDB 2FLI290 kDa dodecamerStreptococcus pyrogenes

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RuP Isomerase

1.25Å structure available from Midwest Structural Genomics Project

Illustrates utility of high-resolution structures Finding hydrogens Identifying secondary

conformations of sidechains

PDB 1O8B24 kDa monomerE.coli

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Transketolases Transfer 2-C fragment

from ketose to aldose TPP-dependent enzyme

(characteristic of two-carbon transfers)

P. Asztalos et al (2007) Biochemistry 46: 12037

PDB 2R8O147 kDa dimerE.coli

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Transaldolases Transfer 3-carbon unit—

effectively moves adihydroxyacetone groupfrom ketose to aldose

Reaction:sedoheptulose-7-phosphate +glyceraldehyde-3-phosphate D-erythrose-4-phosphate + D-fructose-6-phosphate

Schiff-base intermediate Structurally related to TIM-

barrel aldolases

PDB 3CLM39 kDa monomerNeisseria gonorrhoeae

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Significance of the PPP

Generates NADPH where it’s needed Source of Ribose-5-phosphate Several medical conditions associated

with deficiencies in these enzymes G6PDH problems already mentioned Deficiencies in transaldolase lead to liver

problems (Verhoeven et al (2001) Am J Hum Genet. 68: 1086)

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Glyoxylate pathway

Alternative fate for isocitrate Absent in animals; fundamental in

bacteria, protists, fungi, plants Especially prevalent in oily seed plants,

where seed oils are converted to carbohydrates during germination

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Glyoxylate pathway reactions

Isocitrate lyase:isocitrate glyoxylate + succinate

Malate synthase:glyoxylate + acetyl CoA + H2O L-malate + CoASH + H+

This pathway skips two decarboxylations,so it produces less NADH but doesn’t lose as much carbon

Net reaction enables creation of oxaloacetate that can go into gluconeogenesis

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TCA Cycle and Evolution The entire pathway didn’t evolve together Some reactions much older than others Some ran backward in early

implementations Several enzymes adapted from amino

acid degradation Youngest enzyme:

-ketoglutarate dehydrogenase

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Aerobes and anaerobes

Because of close coupling between TCA cycle and oxidative phosphorylation, the complete TCA cycle is an aerobic phenomenon

Anaerobes do have most of these enzymes, but the sequence of reactions is different

Oxygen is actually toxic to many anaerobes

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Overall role of electron transport Last 3 lectures: we discussed

carbohydrate metabolism and the Krebs cycle, each of which produced some high-energy phosphate energy directly.

In both of those systems much of the energy generated took the form of reduced cofactors--NADH in both systems, and FADH2 (or QH) in the Krebs cycle.

Now we’ll see what happens to those!

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Reduced cofactors to ATP We will discuss how the energy latent

in these reduced cofactors can be turned into energy in the form of high-energy phosphate bonds in nucleoside triphosphates--the standard currency of energy.

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What the ETS does The electron transport system (ETS) is

responsible for these transformations. Like the Krebs cycle or glycolysis, the

electron transport chain is a series of chemical transformations facilitated by proteins.

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Roles of these systems Some of these proteins are enzymes in

the conventional sense others are not--they're electron transport

proteins only: so they can only be regarded as enzymes

if we allow that the entire ETS is a large, multi-polypeptide transformation system--a multi-component enzyme

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The overall reactions NADH + H+ + (1/2)O2 + 2.5 ADP

+ 2.5 Pi NAD + H2O + 2.5 ATP ETS also catalyzes transformations of

the flavin coenzyme FAD: FADH2 + (1/2)O2 + 1.5 ADP + 1.5 Pi

FAD + H2O + 1.5 ATP These are mediated through other

cofactors: Q, cytochromes, Fe-S proteins, etc.

Proton translocation is crucial

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Chemiosmotic theory:What it says Protons are translocated from outside

of mitochondrial inner membrane into its interior

That passage actually generates both chemical and electrical energy.

This is because they are moving down a concentration and electrical-potential gradient.

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How it works

This energy is used to drive the synthesis of ATP from ADP and Pi within an enzyme called ATP synthase, which is (big surprise!) anchored on the inside of the inner mitochondrial membrane.

The structure of two components of this enzyme system were determined in 1999 by Andrew Leslie and others.

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Oxidation state and energy

We typically measure oxidation states in volts.

We can relate the energy associated with an oxidation-reduction reaction--the so-called change in redox potential--with the change in the oxidation state of the molecules involved in the reaction.

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What is a volt? A volt is actually a measure of energy

per unit charge; in fact, a volt is one joule per coulomb.

When we say that a double-A battery has a voltage of 1.5 V, we mean that it can (under optimal conditions) deliver 1.5 joules of energy( = 0.359 cal, or 3.59*10-4 kcal) per coulomb of charge.

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Charge and energy One electron carries a charge of

1.602 * 10 -19 coulomb If change in redox potential in a reaction is

0.32 V and all of that change is delivered to a single electron:then energy imparted to that electron is

eΔE =(1.602 * 10-19 coulomb / e-) *(0.32 J/coulomb)= 0.513*10-19J / e- = 1.23* 10 -23 kcal / e-

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… in biochemical units … That doesn't sound like much, but if we

look at that on a per mole basis it's (1.23 * 10-23 kcal/e-) *6.022 * 1023 e -/mole= 30.87 kJ/mol = 7.38 kcal/mol

which is a reasonable amount of energy on the scale we're accustomed to examining.

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So what can we get? There is enough energy bound up in the

reduced state of NAD relative to the oxidized state to drive the net creation of 2.5 molecules of ATP from ADP and phosphate, as indicated in the equations shown above.

Since there are NADH molecules created in several steps in glycolysis and the Krebs cycle, there numerous net ATP molecules that arise from the overall process.

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Results from Krebs cycle 3 NADH produce 7.5 ATP 1 FADH2 produces 1.5 ATP 1 substrate-level phosphorylation Total: 10 ATP per round, if we don’t

get interrupted!

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ETS: The big picture

5 membrane-associated, multi-enzyme complexes in mitochondrial inner membrane

Complexes I-IV associated with electron transport and proton translocation

Complex V uses proton gradient to produces ATP from ADP and Pi

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Complexes I-IV There are several multi-enzyme complexes

involved in converting the reductive energy in NADH to its final products.# NameI NADH-Ubiquinone oxidoreductaseII Succinate-ubiquinone oxidoreductaseIII Ubiquinol-cytochrome c oxidoreductaseIV Cytochrome c oxidase

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Overview of Oxidative Steps

Chart courtesyMichael King,Indiana State

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Complex I

NADH:Ubiquinone oxidoreductase Embedded in inner mitochondrial

membrane Passes electrons from NADH to

ubiquinone