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3 April 2008
Sugars, concluded; Electron Transport
and Oxidative Phosphorylation
Andy HowardIntroductory Biochemistry
3 April 2008
3 April 2008Sugars, concl’d; Electron Transport p. 2 of 47
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!
3 April 2008Sugars, concl’d; Electron Transport p. 3 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 4 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 5 of 47
Entner-Doudoroff reaction 1:G6PDH Oxidizes glucose-6-phosphate to 6-
gluconolactone We’ll meet this enzyme in the PPP
shortly
3 April 2008Sugars, concl’d; Electron Transport p. 6 of 47
Entner-Doudoroff Pathway 2:Gluconolactonase
Dehydratase: Converts 6-P-gluconolactone to 6-P-gluconate
An example of a phosphorylated sugar acid
3 April 2008Sugars, concl’d; Electron Transport p. 7 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 8 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 9 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 10 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 11 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 12 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 13 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 14 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 15 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 16 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 17 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 18 of 47
Gluconolactonase
Converts gluoconolactone to 6-phosphogluconate
Remember this is a hydratase, not an oxidoreductase
3 April 2008Sugars, concl’d; Electron Transport p. 19 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 20 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 21 of 47
Non-oxidative steps Epimerases
, isomerases, trans-ketolases, trans-aldolases
Chart courtesyMichael King,Indiana State
3 April 2008Sugars, concl’d; Electron Transport p. 22 of 47
Ribulose-5-phosphate 3-epimerase
Converts RuBP to xylulose-5-P
TIM-barrel protein Co-regulated with
RuP isomerasePDB 2FLI290 kDa dodecamerStreptococcus pyrogenes
3 April 2008Sugars, concl’d; Electron Transport p. 23 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 24 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 25 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 26 of 47
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)
3 April 2008Sugars, concl’d; Electron Transport p. 27 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 28 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 29 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 30 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 31 of 47
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!
3 April 2008Sugars, concl’d; Electron Transport p. 32 of 47
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.
3 April 2008Sugars, concl’d; Electron Transport p. 33 of 47
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.
3 April 2008Sugars, concl’d; Electron Transport p. 34 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 35 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 36 of 47
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.
3 April 2008Sugars, concl’d; Electron Transport p. 37 of 47
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.
3 April 2008Sugars, concl’d; Electron Transport p. 38 of 47
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.
3 April 2008Sugars, concl’d; Electron Transport p. 39 of 47
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.
3 April 2008Sugars, concl’d; Electron Transport p. 40 of 47
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-
3 April 2008Sugars, concl’d; Electron Transport p. 41 of 47
… 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.
3 April 2008Sugars, concl’d; Electron Transport p. 42 of 47
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.
3 April 2008Sugars, concl’d; Electron Transport p. 43 of 47
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!
3 April 2008Sugars, concl’d; Electron Transport p. 44 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 45 of 47
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
3 April 2008Sugars, concl’d; Electron Transport p. 46 of 47
Overview of Oxidative Steps
Chart courtesyMichael King,Indiana State
3 April 2008Sugars, concl’d; Electron Transport p. 47 of 47
Complex I
NADH:Ubiquinone oxidoreductase Embedded in inner mitochondrial
membrane Passes electrons from NADH to
ubiquinone