1 slides or areas with a blue background are subjects that were skipped in lecture. they are...

1

Slides or areas with a blue background are subjects that were skipped in lecture. They are included here just for your interest and will not be included in any exam.

Note for lecture 9, 2010

Links to movies: Rotation of actin filament arm

Animation 1 Animation 2

Animation 3

http://www.columbia.edu/cu/biology/courses/c2005/movies/fof1_rot_2700nm.mpg

http://www.columbia.edu/cu/biology/courses/c2005/movies/F1catalise%20-Wang.mpeg

http://www.columbia.edu/cu/biology/courses/c2005/movies/F1_top_2.mpeg

http://www.columbia.edu/cu/biology/courses/c2005/movies/ATPase%20rotation%20-Wang.mov

2

-2 ATP+ 4 ATP= + 2 ATP

Handout 7-2“glycolysis” ends here

ATP debt paid in full

3

1 glucose + 2 ADP + 2 Pi + 2 NAD 2 pyruvate + 2 ATP + 2 NADH2

Δ Go = -18 kcal/mole

So overall reaction goes essentially completely to the right.

4

(Handout 7-3)

Handout 7-4b

5

Handout 7-4b

pull

pull

•Δ G = Δ Go + RTln([products] [reactants])

6The second way the cell gets a reaction to go in the desired

direction:

1) First way was: a coupled reaction (i.e., a different reaction) .One of two ways the cell solves the problem of getting a reaction to go

in the desired direction

Glucose + ATP glucose-6-P04 + ADP, Δ Go = -3.4 kcal/mole

2) The second way:• Removal of the product of an energetically unfavorable

reaction• Uses a favorable downstream reaction• “Pulls” the unfavorable reaction• Operates on the second term of the Δ G equation.

• Δ G = Δ Go + RTln([products]/[reactants])

7• So glucose pyruvic acid

• ADP ATP, as long as we have plenty of glucose• Are we all set?

• No…. What about the NAD?.. We left it burdened with those electrons.

• Soon all of the NAD will be in the form of NADH2

• Very soon

• Glycolysis will screech to a halt !!• Need an oxidizing agent in plentiful supply to keep taking

those electron off the NADH2, to regenerate NAD so we can continue to run glucose through the glycolytic pathway.

8

Oxidizing agents around for NAD:

1) Oxygen

Defer (and not always present, actually)

2) Pyruvate, our end-product of glycolysis

In E. coli, humans:

Pyruvate lactate, NADH2 NAD, coupled

In Yeast:

Pyruvate ethanol + CO2

10

Glucose NADH2NAD

Lactate Pyruvate

GAL-3-P 1,3-Di-PGA

Biosynthetic pathway to NAD

Handout 7-1b

excreted

Glucose

ATPATP

11

yeastE.

coli

humansHC=O |CH3

Acetaldehyde detail

12

Fermentation: anaerobiosis (no oxygen)

Lactate fermentation

Ethanolic fermentation

Mutually exclusive, depends on organism

Other types, less common fermentations, exist– (e.g., propionic acid fermentation, going on in Swiss cheese)

13

The efficiency of fermentation

glucose--> 2 lactates,

without considering the couplings for the formation of

ATP's (no energy harnessing):

Δ Go = -45 kcal/moleSo 45 kcal/mole to work with.

Out of this comes 2 ATPs, worth 14 kcal/mol.

So the efficiency is about 14/45 = ~30%

Where did the other 31/45 kcal/mole go?

Wasted as HEAT.

14Fermentation goes all the way to the right

Since 2 ATPs ARE produced, taking them into account, for the reaction:

Glucose + 2 ADP + 2 Pi 2 lactate + 2 ATP

ΔGo = -31 kcal/mole (45-14)

Very favorable.All the way to the right. Keep bringing in glucose, keep spewing out lactate,Make all the ATP you want.

glucose--> 2 lactates, without considering the couplings for the formation of ATP's (no energy harnessing): Δ Go = -45 kcal/mole kcal/moleOut of this comes 2 ATPs, worth 14 kcal/mol. So the efficiency is about 14/45 = ~30%

That’s fermentation.

15

glycerolphosphate

glycerol

ATP +NAD

DHAP(dihydroxy acetone phosphate)

+ NADH2

glycolysis+O2

CO2 + H2O

- O2

?

Gl;ycerol as an alternative sole carbon and energy source for E. coli

and ADP + Pi ATP

16Glycerol + ATP → glycerol phosphate → DHAP

NAD → NADH2

17

glycerolphosphate

glycerol

ATP +NAD

DHAP(dihydroxy acetone phosphate)

+ NADH2

glycolysis+O2

CO2 + H2O

- O2

Glycerol cannot be fermented.E. coli CANNOT grow on glycerol in the absence of airThese pathways are real, and they set the rules. Stoichiometry of chemical reactions must be obeyed. No magic is involved

18Energy yield

Complete oxidation of glucose,

Much more ATP

But nature’s solution is a bit complicated.

The fate of pyruvate is now different

But all this spewing of lactate turns out to be wasteful.Using oxygen as an oxidizing agent glucose could be completely oxidized, to: … CO2That is, burned.

How much energy released then?

Glucose + 6 O2 6 CO2 + 6 H2O

ΔGo = -686 kcal/mole !Compared to -45 to lactate (both w/o ATP production considered)

19

2 NADH2 NADH

2 ATP

Acetyl-CoA

2 CO2

Score:Per glucose

Handout 8-1

20Acetyl-CoA

O

||

CH3 - C –OH + Co-enzyme A Acetyl ~CoA

Acetic acid, acetate

Acetate group

Pantothenic acid (vitamin B5)

21

2 ATP

Acetyl-CoA

2 CO2

2 CO2

2 CO2

2 oxaloacetatePer glucose

2 NADH2 NADH2 NADH2 NADH

6 CO2

B

22

GTP is energetically equivalent to ATP

GTP + ADP GDP + ATP

ΔGo = ~0

G= guanine (instead of adenine in ATP)

23

2 ATP

Acetyl-CoA

2 CO2

2 CO2

2 CO2

2 oxaloacetatePer glucose

2 NADH2 NADH2 NADH2 NADH

6 CO2

B

24

FAD = flavin adenine dinucleotide

FAD + 2H. FADH2

adenine

ribose

ribose

Business end(flavin)

25

2 NADH2 NADH2 NADH2 NADH2 FADH22 NADH

2 ATP

2 ATP

Acetyl-CoA

2 CO2

2 CO2

2 CO2

Succinic dehydrogenase

oxaloacetatePer glucoseD

Note label is in OA after one turn of cycle,half the time on top, half on bottom.So no CO2 from Ac-CoA after just one turn.(CO2 in first turn from OA).

26

2 NADH2 NADH2 NADH2 NADH2 FADH22 NADH

2 ATP

2 ATP

2 CO2

2 CO2

2 CO2

Glucose + 6 O2 6 CO2 + 6 H2O :

By glycolysis plus one turn of the Krebs Cycle:

1 glucose (6C) 2 pyruvate (3C) 6 CO2

2 X 5 NADH2 and 2 X 1 FADH2 produced per glucose

4 ATPs per glucose

NADH2 and FADH2 still must be reoxidized ….

No oxygen yet to be consumed

No water produced yet

Paltry increase in ATP so far

Per glucose

E

27Oxidation of NAD by O2

NADH2 + 1/2 O2 --> NAD + H2O

ΔGo = -53 kcal/mole

If coupled directly to ADP ATP (7 kcal cost),46 kcal/mole waste, and heat

So the electrons on NADH (and FADH2) are not passed directly to oxygen, but to intermediate carriers,

Each transfer step involves a smaller packet of free negative energy change (release)

28Handout 8-3

NADH2

Ubiquinone, or Coenzyme Q

Iron-sulfur protein

heme

Cytochromes are proteins

~ F

ree

en

erg

y

10

Up to 50 C’s long

29

Handout 8-4

Oxidativephosphorylation

30

Nelson and Cox, Principles of Biochemistry

(outside)

I II III IV

H+ ions (protons) are pumped out as the electrons are transferred

31

Schematic idea of H+ being pumped out

Handout 8-4

Conformationalchange

Relaxation back

32

FoF1 Complex:Oxidative

phosphorylation (ATP formation)

Handout 8-4

Pro

ton

flow

bac

k in

via

mas

s ac

tion

33

H+H+

H+H+H+H+

H+H+

H+H+

H+H+

H+

H+H+

H+

H+

H+

H+

H+H+

H+

H+

H+

H+

H+H+

H+

H+

H+

H+

H+H+

H+

H+

H+

ETC Complex I’s

NADH

H+

H+H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

NADH

Artificial phospholipid membrane

pH drops pH rises

Slides with a blue background are subjects that were skipped in lecture. They are included here just for your interest and will not be included in any exam.

34

H+

H+

H+

H+

H+

H+

H+

H+ H+

H+

H+

H+

H+

H+

H+H+

H+

H+

H+

H+

H+

H+

H+

H+H+

H+H+

H+

H+

H+

H+

H+

H+

H+

H+H+

H+

H+

H+H+

ADP + Pi

ATP

Artificially produced mitochondrial membrane vesiclewith ADP and Pi trapped inside

ATP is formed from ADP + Pi

ADP + Pi


35Dinitrophenol (DNP): an uncoupler of oxidative phosphorylation

DNP’s -OH is weakly acidic in this environment

DNP can easily permeate the mitochondrial inner membrane

Outside the mitochondrion, where the H+ concentration is high, DNP picks up a proton

After diffusing inside, where the H+ concentration low, it gives up the proton.

So it ferries protons from regions of high concentration to regions of low concentration, thus destroying the proton gradient.

Electron transport chain goes merrily on and on, but no gradient is formed and no ATP is produced.

- + H+


36Chemiosmotic theory

Proton motive force (pmf)Chemical gradientElectrical gradient Electrochemical gradient

Peter Mitchell 1961 (without knowing mechanism)

Water-pump-dam analogy

Some evidence:

Nobel Prize 1978

37

++

+ +

+

+ADP

ATP

++

crista

the outside

the inside

What about E. coli? Cell membrane houses all components

38

ATP synthetase

outside

inside

Gamma subunit: cam

The mechanism of ATP formation: The ATP synthetase (or ATP synthase)The F0F1 complex:

the outside

the inside

Gamma subunit is inserted inside

39

outside

inside

ATP synthetase

Flow of protons turns the C-subnunit wheel. C-subunits turn the gamma cam

40Outside

Outside

Inside

Inside

Mitochondria Chloroplasts

41

Norbert Dencher and Andreas Engel

View of the c-subunits making up the F0 subunit using atomic force microscopy

Animation of the Fo rotation driven by the influx of H+ ions (“wheels within wheels”).M.E. Girvin

42

Alpha+beta

Gamma

Handout 8-5

Three conformational states of the a-b subunit: L, T, and O

ADP Pi

(top view)

43

Movie link

http://www.columbia.edu/cu/biology/courses/c2005/movies/F1catalise%20-Wang.mpeg

45

Detach the C-subunits

Motor experiment

(MW 42K)

Attach the big arm

46

Testing the ATP synthetase motor model by running it in reverse (no H+ gradient, add ATP)

Actin labeledby tagging it with fluorescent molecules

Actin is a muscle protein polymer

Hiroyuki Noji, Ryohei Yasuda, Masasuke Yoshida & Kazuhiko Kinosita Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature, 386, 299 - 302.

Attached to the gamma subunit

47

1234 5

Run reaction in reverse: add ATP, drive counter-clockwise rotation of cam

ATP hydrolysis

ATP

x

Here the cam has no driving motor (c) attached any more

Start here

counter-clockwise

48

Movie link

http://www.columbia.edu/cu/biology/courses/c2005/movies/fof1_rot_2700nm.mpg

49

ATP accounting

• Each of the 3 ETC complex (I, III, IV) pumps enough H+ ions to allow the formation of 1 ATP.

• So 3 ATPs per pair of electrons passing through the full ETC.

• So 3 ATPs per 1/2 O2

• So 3 ATPs per NADH2

• But only 2 ATPs per FADH2 (skips complex 1)

50

Handout 8-1

51

ATP

ATP

ATPATP generated by the ATP synthetase is called is oxidative phosphorylation, or oxphos

FumarateX

X

More favorable ∆GO

with FAD than with NAD

H2

Succinate

Fre

e en

ergy

cha

nge

Areas with a blue background are subjects that were skipped in lecture. They are included here just for your interest and will not be included in any exam.

52

Nelson and Cox, Principles of Biochemistry


53

Substrate level phosphorylation (SLP): 2 ATP

1ATP from Glycolysis

1 ATP (GTP) from Krebs

OXPHOS:

1 NADH from glycolysis

1 NADH from Krebs entry

3NADH from Krebs

1 FADH2 from Krebs

Total: 17 ATP

5 NADH = 15 ATP 1 FADH2 = 2 ATP

Grand total (E. coli):17 + 2 = 19 per ½ glucoseor 38 per 1 glucose

Handout 8-6

Handout labeled 8-6

54

Cellular location (eukaryotes):

CYTOPLASM

MITOCHONDRIA

Handout labeled 8-6

55

ATP accounting

• 38 ATP/ glucose in E. coli

• 36 ATP/glucose in eukaryotes– Cost of bringing in the electrons from NADH from glycolysis into the

mitochondrion = 1 ATP per electron pair

– So costs 2 ATPs per glucose, subtract from 38 to get 36 net.

56

Efficiency

• 36 ATP/ glucose, worth 7 X 36 = 252 kcal/mole of glucose

• ΔGo for the overall reaction glucose + 6 O2→ 6CO2 + 6 H2O:-686 kcal/ mole

• Efficiency = 252/686 = 37%

• Once again, better than most gasoline engines.

• and Energy yield:36 ATP/ glucose vs. 2 ATP/glucose in fermentation(yet fermentation works)

• So with or without oxygen, get energy from glucose

57

Handout 9-2

glucose

pyruvate

acetyl-CoA

O.A.KREBS

STARCH

FATS

PROTEINS

Catabolism

E.T.C.

AMINO ACIDS ATP

ATP

You are what you eat

NADH2

O2 H2O

GLYCOLYSIS

NAD

Anabolism

FATSATP

GLYCOGEN

ATP

ATP

FATSATP

NAD

Anabolism

FATSATP

FATSATP

NAD PROTEINS

AMINO ACIDSATP-K.G.

PROTEINS

AMINO ACIDSATP-K.G.-K.G.

PROTEINS

AMINO ACIDSATP

PROTEINS

AMINO ACIDSATP

GLYCOGEN

ATP

ATP

Anabolism

FATSATP

PROTEINS

AMINO ACIDSATP

58

Metabolic map

Serine biosynthetic path in red

60Handout 9-2

Deamination and transamination of amino acids

Glutamic acidalpha-keto-glutaric acidAlanine Pyruvate

Transamination

Glutamic acid alpha-keto-glutaric acid

HOH

Oxidative de-amination

NAD NADH2


61

Biosynthesis of proteins

e.g., an enzyme like hexokinase: met-val-his-leu-gly …..

If this done like lipids and polysaccharides, we need an enzyme for each linkage

First an enzyme that will condense val to met to make met-val.

Then an enzyme with a different substrate specificity, which adds his to met-val to make met-val-his.

Since there are 500 AAs in hexokinase, we need 500 enzymes to do the job.

If there are 3000 proteins in E. coli, then we need ~500 X 3000 = 1.5 million enzymes to make all the different primary structure of all the proteins.

But even then, it won’t work, as each of these million enzymes is also a protein that needs to be synthesized.

We need a better plan to polymerize the amino acids in the right order.

62

glucose

monomers

MacromoleculesPolysaccharides LipidsNucleic AcidsProteins

biosynth

etic p

athw

ay

intermediates

Flow of glucose in E. coli

Each arrow = a specific chemical reaction

Problem 1: Getting specific reaction rates to go in real time:

Enzymes

Problem 2: Getting the reactions to go in the desired direction: Coupled reactions + favorable metabolic paths

(all mediated by enzymes) Problem 3: Getting the information to make the specific

3-dimensional enzymes:Just need to specify the primary structure ….. How?

63

Nucleic acids

Prof. Mowshowitz will continue with this next chapter in the story,

leading to the biosynthesis of the all-important proteins.

64

Lecture ended here


65Alternative sources of carbon and energy

Shake = milk:milk sugar = lactose = disaccharide= glucose – galactose

beta-galactosidase+HOH → glucose + galactose

glucose → glycolysis, etc. galactose (3 enzymatic steps) glucose Slides with a blue background are

subjects that were skipped in lecture. They are included here just for your interest and will not be included in any exam.


Bun = starch = poly-alpha-glucose G-1-P → G-6-P

glycolysis


Lettuce = cellulose = polysaccharidePoly-beta glucose

→| (stays as the polysaccharide)

We have no enzyme for catabolizing cellulose



French fries = fat (oil) =triglyceride

69

(Triglyceride)Lipases(hydrolysis)

Glycolysis (at DHAP)Slides with a blue background are subjects that were skipped in lecture. They are included here just for your interest and will not be included in any exam.

70

CH3-(CH2)n-CH2-CH2-C-OH

CH3-(CH2)n-CH2-CH2-C-CoA + HOH

CH3-(CH2)n-CH=CH-C-CoA

CH3-(CH2)n-CH-CH2-C-CoA

CH3-(CH2)n-C-CH2-C-CoA

CH3-(CH2)n-C-CoA + CH3-C-CoA

||

||

||

||

||

||

||

||

O

O

O

O

O

O

O

O

|OH

KrebsCycle

ATP + Coenzyme A-SH

FAD

NAD

+HOH

+ CoA

Fatty acid (oxidation)catabolism

Acetyl-CoAFatty acid -2o

NADH2

FADH2 FAD

etc.

FADH2

Handout 9-1 left



Hamburger = proteinProteases (e.g., trypsin) → → 20 AAsStomach acid (pH1) also helps by denaturing protein making it accessable to proteolytic attack

Each of the 20 AA’s has its own catabolic pathway, and ends up in the glycolytic or Krebs cycle pathways

But first, the N must be removed:


72E.g., degradation of phenylalanine (6 steps)

transaminase

Products = Fumaric acid → Krebs and Acetoacetate → 2 Acetyl-CoA → Krebs

PKU (phenylketonuria)

Phe builds up and gets metabolized to an injurious product (phenyl pyruvate)


73

Handout 9-2

glucose

pyruvate

acetyl-CoA

O.A.KREBS

STARCH

FATS

PROTEINS

Catabolism

E.T.C.

AMINO ACIDS ATP

ATP

You are what you eat

NADH2

O2 H2O

GLYCOLYSIS

NAD

Anabolism

FATSATP

GLYCOGEN

ATP

ATP

FATSATP

NAD

Anabolism

FATSATP

FATSATP

NAD PROTEINS

AMINO ACIDSATP-K.G.

PROTEINS

AMINO ACIDSATP-K.G.-K.G.

PROTEINS

AMINO ACIDSATP

PROTEINS

AMINO ACIDSATP

GLYCOGEN

ATP

ATP

Anabolism

FATSATP

PROTEINS

AMINO ACIDSATP


74Biosynthesis of monomers

E.g., • Fatty acids (acetyl CoA from Krebs cycle)

• Amino acids (Serine: 3-phospho-glyceric acid from glycolysis)


75

CH3-(CH2)n-CH2-CH2-C-OH

CH3-CH-CH-C-CoA2 2

CH3-CH CH-C-CoA=

CH3-C-CH2-C-CoA

CH3-C-CH2-C-CoA

CH3-C-CoA + CH3-C-CoA||

||

||

||O

||O

||O

||O

||O

O

O

O

|OH

KrebsCycle

NADP H2 NAD

-HOH

+ CoA

Fatty acid biosynthesis

Repeated addition of 2-carbon units (acetyl-CoA molecules via this same set of reactions)

Acetyl-CoA Acetyl-CoA

H

NADP H2 NADP

P

Handout 9-1

Start at bottom

Handout 9-1 right


76Handout 9-3

(Glycolytic intermediate)

Phosphoester group

hydrolysis

Glutamate is the amino donor

Handout 9-3


77

Biosynthesis of macromolecules

1) Lipids

2) Polysaccharides

3) Proteins


78

DHAP(dihydroxy-acetonephosphate)

+ Fatty acid 1

+ Fatty acid 2

+ Fatty acid 3

O || -C -(CH2)x -CH3

O || -C -(CH2)x -CH3

O || -C -(CH2)z-CH3

O || -C -(CH2)x -CH3

O || -C -(CH2)y -CH3

O || -C -(CH2)y -CH3

Triglyceride (fat) biosynthesis

Glycerol phosphate

Phosphatidic acid

Triglyceride

NADPH

Phospholipid

Handout 9-3


79


80Polypaccharide: Hyaluronic acid

n


81Polysaccharide synthesisHyaluronic acid (joint lubricant)

COO-

Glucuronic acidN-acetyl-glucosamine

Enz.1+

Enz. 2 Enz.1

Enz.1Enz. 2

Enz.1Enz. 2

Enz.1Enz. 2

Enz.1Enz. 2

Hyaluronic acid (polysaccharide) via ~2 enzymes


1 slides or areas with a blue background are subjects that were skipped in lecture. they are...

Documents

b slide

atp g o

b excreted glucose atp

kcalmole kcalmole

atp handout

atp debt

glucose pyruvic acid

swiss cheese slide