Lec. 9.   Biol C2005/F2401   2006   L.Chasin     10/5/06

Copyright 2006  Lawrence Chasin and Deborah Mowshowitz   Department of Biological Sciences   Columbia University   New York, NY

[ATP-synthetase 3-D animation (.mov)]

ATP synthetase
oxidative phosphorylation
substrate level phosphorylation
alternative energy sources:
other sugars
polysaccharides
fat
protein
transamination, deamination
biosynthetic pathways to monomers
polysaccharide and lipid biosynthesis
protein synthesis ......


Mechanism of ATP synthesis in mitochondria

(ATP synthetase)
The flow-back is through lollipop-like structures that populate the inner surface of the inner membrane. See handout on ATP-synthetase for the structure of this multi-subunit protein complex.  Each lollipop is a complex of proteins; the stem is called Fo and forms a channel through the membrane.  The sphere is called F1 and contains the ATP SYNTHETASE activity; that is, it is in the spheres that the generation of ATP from ADP + Pi takes place. It is the flow-back of H+'s through the F1 spheres that generates the phosphorylation of ADP by Pi to form ATP.  An analogy would be to use one source of energy to pump water up to a high level behind a dam (the pumping of protons tied to the free energy released in the oxidations of the electron transport chain components) and then letting the water drive turbines to generate electricity as it falls from the high level behind the dam (the generation of ATP)

. This is one place where reading of both texts can help in the understanding of this very indirect mechanism. Indeed, this theory was doubted for many years after its proposal by Mitchell (it is also known as the Mitchell Hypothesis; Mitchell was known to do experiments in the basement of his mansion, like in the movies). 

The mechanism of this reaction, the ATP synthetase, has only become clearer in the last few years.  The Fo channel includes  10 c-subunits surrounding a central stalk, the gamma subunit.  A proton outside the mitochondrial membrane flows back by entering the Fo stem channel where it bind to an amino acids on one of 10 c subunits comprising the cylindrical channel.  This binding produces a shift in the cylinder by 1/10 turn (see right).  As the c-cylinder turn so does its attached gamma stalk. The top the gamma subunit reaches into the center of the F1 head structure, and its shape there is in the form of an asymmetric cam. The F1 head is organized like a pie divided into 3 identical wedges [Purves picture].  The wedges can be configured in space  to provide a binding site for ADP and Pi or for ATP.  As the cam rotates amidst the alpha-beta subunits of the F1 head structure, it distorts their structure.  Three distinct distortions are produced and each has an effect on the ability of the alpha-beta subunits to bind ATP and ADP and Pi on their face that is exposed to the inside of the mitochondrion.  See the ATP synthetase handout to follow this process.

One distortion forces the ADP and Pi together in one wedge, while the ATP that just had been formed on another wedge is distorted in the opposite way to release the ATP. The sequence of these 3 events is thus 1) the binding of ADP and Pi (L), 2) a kind of mechanical force pushing them together (T), followed by 3) a quick release of the ATP (O).  The formation of these 3 conformations is driven by protons binding to specific amino acids in the Fo channel. Thus as the protons flow back into the mitochondrion, the Fo shaft with its cam  is spinning.  See a pretty  animation of ATP synthesis from the Website of W. Junge.

To review the action of ATP synthetase see problem 5-13.

(oxidative phosphorylation and substrate level phosphorylation)

From glucose, at least ... But we do not live from cake alone .

How about a carbon and energy source OTHER THAN GLUCOSE?

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Not in live lecture - not responsible for italicized text:

Where do we get this glucose? Ultimately from plants, who are able to synthesize it using an energy source obviously other than glucose, solar energy. I want to now rather briefly turn to photosynthesis, to summarize what is involved. Just sit back and listen, because photosynthesis will not be on the exam.

Photo - synthesis, the word and the problem consists of two parts.

1. Synthesis of glucose from simple CO2 in the air.

2. Photo: the source of the energy, in the form of ATP, from sunlight, to carry out this synthesis.

First, the synthesis. Overall:

6 CO2 + 6 H2O --> C6H12O6 + 6 O2

CO2 is highly oxidized carbon. Need to add H's from a reducing agent. The ultimate source of these hydrogen atoms is going to be water, but water is a poor reducing agent (O2 holds its electrons tightly). So for photosynthesis, we need a source of reducing power as well as a way to make ATP from ADP.

Overall with ATP:

6 CO2 + 12 NADPH2 + 18 ATP  -->  18 ADP + 12 NADP + 1 glucose.

So we need NADPH2 and ATP. Get them both from the light reactions.

Another piece of cellular machinery: the chloroplasts, like mitochondria, a membrane delimited cytoplasmic organelle. But they also contain light-absorbing pigments, including the chlorophylls. When light energy (one photon) is absorbed by these pigments (each at a characteristic wavelength), the energy is used to excite a water electron to a higher energy state.

The excited e- goes down a (different) E.T.C. ending up at NADP, where a pair of e-'s are accepted to produce NADPH2. This is the NADPH2 we needed to make glucose.

12 H2O + 12 NADP + 18 ADP + 18 Pi + 60 photons -->

6 O2 + 12 NADPH2 + 18 ATP + 18 H2O (from ADP+Pi)

The ATP is made via proton pumping to establish a hydrogen ion gradient as the electron travels down an electron transport chain, as in mitochondria.

Efficiency 27%, once e-'s have been excited. But much less from light overall, as many e- fall back from excited state.

In plants these reaction occur only in chlorophyll-containing tissues  (leaves, mainly); other plant tissues use glucose via respiration. And so do the leaves at night. Glucose is stored, transported, and used for energy.

So plants: CO2 --> glucose + O2

and we (and plants): glucose + O2 --> CO2

The carbon gets shuttles back and forth. In the planet as a whole, the CO2 is also in equilibrium with vast stores of inorganic CO2 in the oceans and in the form of carbonates (CaCO2), limestone.

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We discussed glucose. But one does not live by refined sugar alone. Most parts of a big Mac dinner will provide us with energy. These pathways of degradation of compounds is called CATABOLISM.

The roll:

1. POLYSACCHARIDES:

STARCH = Gluc-gluc-gluc-gluc-....Hydrolyzed to glucose-1-P, then PhosphoGlucoMutase catalyzes its isomerization to G6P = a glycolytic intermediate . Glycolysis takes it from there.

See problem 4-13 (4-11 in 16th ed.) Ask yourself, "What steps and enzymes are needed to carry out the reaction shown in the problem: Glucose-P + ATP --> 2 PGAlde?"

Milk shake:

More complex sugars: LACTOSE = glucose-galactose --> glucose + galactose; galactose then --> --> glucose.

Lettuce:

CELLULOSE --> nowhere (excreted). We can't handle it (no catabolic enzyme, no cellulase).

The French fry:

2. LIPIDS: fats = triglycerides (structures on board, refer to handout) = more interesting:

Triglyceride hydrolysis --> glycerol + 3 free Fatty Acids

For the main Fatty Acid (FA) part of the triglyceride (see handout):

FA + CoA (ATP [--> AMP] ) to get started --> FA-CoA --> via FAD (2,3-ene C=C double bond), then HOH addition (OH added distally), --> via NAD --> C=O

(3-keto, or beta-keto acid, so called "beta-oxidation", in mitochondria)

+ CoA again (no ATP needed here) --> FA-CoA (smaller by 2 carbons now), + CH3-CO-CoA = acetyl-CoA = the KC entry compound --> continues around the Krebs Cycle.

etc. etc. in blocks of 2 carbons. See problem 5-14.

About twice the energy as from glucose, pound for pound.

How about the glycerol part?

See glycolysis handout. On PowerPoint graphic,

glycerol  + ATP --> glycerol-1-P

glycerol-1-P + NAD --> DHAP (= dihydroxyacetone phosphate) + NADH2

DHAP --> continues in glycolysis (rxn 5). See recitation problems #3, problem 2.

Under aerobic conditions, no problem, the NADH2's produced will get re-oxidized in the E.T. chain.

Under ANAEROBIC conditions cannot be fermented (see last lecture).

The burger,

3. = PROTEIN:

Common step for all proteins: hydrolysis of the peptide bonds by several enzymes (pepsin, trypsin), stomach acid (pH1) denatures the protein, making it accessible to proteolytic attack  --> Amino Acids.
Subsequent metabolism depends on which of the 20 aa's we're talking about.

A central reaction is:

Glutamate (R= -CH2-CH2-COOH ) + NAD + H2O --> alpha-ketoglutarate (--> Krebs Cycle continuation) + NH3
This enzyme is glutamic dehydrogenase (GDH).

Also:

Aspartate: (R= -CH2-COOH) --> -NH3, --> HOOC-CH=CH-COOH (that's fumarate = a Krebs Cycle intermediate)
Aspartate deaminase (E. coli but not humans)

The rest of the amino acids cannot lose their amino group so directly. 
So a general problem for aa's: to get rid of the NH2 group: General solution: Transamination:

aspartate + alpha-KG --> oxaloacetate (OA) + glutamate (=transamination)

Pass the NH2 to alpha-KG to form glu, then use GDH to make alpha-KG + NH3 from glu.

Asp becomes OA, another old friend.

Another simple example: ala + a-KG --> glutamate + pyruvate ( --> KC)

What happens to this ammonia (NH3), sounds bad. High conc. of NH3 are toxic for many cells (OK for E. coli, excretes directly and quickly), so combine with CO2 to form urea for excretion (low urea conc. here, so OK, no denaturation of proteins).

Other catabolic paths are more complex, but the principle is the same, they get converted to intermediates of the Krebs Cycle or of glycolysis..

His --> --> --> --> glutamate in humans, but not in E. coli. So we can do something E. coli can't, for a change (my graduate work involved this pathway)

So we get energy from protein.  Fast for a day or two, and you start breaking down your own muscle protein to get energy to keep going. See Problems 5R-4 & 5R-5 for an example of amino acid metabolism.

4. Nucleic acids (in a burger, though not much)

Enough is enough:  again, --> KC + glycolytic pathway intermediates.

 

Now we are going to turn from the breakdown of chemicals to the BIOSYNTHESIS of new ones. The process of building up new compounds from simpler ones is called ANABOLISM.

1. POLYSACCHARIDES

Glucose: run glycolysis in reverse, but often with different enzymes for a particular step.

e.g., a phosphatase, uncoupled to ATP for F-1,6-diP to F6P. 

UTP (similar to ATP) + glucose-1-P --> UDP-gluc + PPi, + glycogen(n) --> UDP + glycogen(n+1) 

UDP + ATP --> ADP + UTP 

So 2 ATPs are used to put each glucose residue in glycogen (one to make the G-1-P plus one to regenerate the UTP)).

Also: glucose  --> --> gal --> +glucose --> lactose

All enzymatic, and 1 or a few enzymes for each polysaccharide, then a repeating structure, so the same few enzymes are used again and again. 

Hyaluronic acid = 2 modified sugars, repeating:

N-acetyl-glucosamine + glucuronic acid:   NAcGA-Glucu-NAcGA-Glucu-NAcGA-Glucu-NAcGA-Glucu - - - -

 (see handout) : glucose --> N-acetyl-glucosamine;  glucose --> glucuronic acid

N-acetylglucosamine + UTP --> UDP-N-acetyl-glucosamine; + growing chain --> growing chain+1 + UDP
glucuronic acid + UTP --> UDP-glucuronic acid; + growing chain --> growing chain+1 + UDP
Two alternating enzymes build the polymer from UDP-sugar monomers

2. LIPIDS, phospholipids: biosynthesis is like degradation, but reverse, and different enzymes (and not in mitochondria). See handout.

glucose --> DHAP,  (+NADH) --> glycerol-1-P,

+ 2 FAs --> phospholipid; + 3 FA = triglyceride

Biosynthesis of FATTY ACIDS (see handout)

1) Acetyl-CoA + NADPH2; 2) -HOH step ; 3) NADPH2 again --> repeat in blocks of 2, --> Fatty Acid

NADP is like NAD but with an additional phosphate group. NADPH is usually used as a reducing agent for biosynthetic reactions, while NAD is used as an oxidizing agent in catabolic reactions.

3. NUCLEIC ACIDS

Defer, but how about ATP, a NA monomer

glucose --> --> glyceraldehyde-3-P --> ser --> gly

G6P--> --> ribose-P

ser + gly + ribose-P + NH3 --> + about 10 steps -->ATP.

(Then ATP <--> ADP many times, for plenty of energy)

4. Proteins:

Monomers = aa's:

Serine from glycolysis: see handout

Krebs Cycle --> alpha-keto-glutarate, +NH3 + NADH2 --> glutamate,

Krebs Cycle --> OA --> transam (via glu) --> aspartate

Pyruvate, via transamination --> alanine

Isoleucine (ile):

Glucose --> Oxaloacetate --> asp --> asp-P --> --> --> thr --> --> --> --> --> ile
Added in this path: pyruvate, 2 NADPH2's, 1 NADH2, 2 ATP, transaminations

Made other aa's along the way: asp, thr

Typical biosynthetic pathway: branches, requires reducing power (NADH2, or NADPH2), ATP, subject to Feedback Inhibition.

Summary:

SEE OVERHEAD: YOU ARE WHAT YOU EAT (simplified). Yes and No. Same atoms, but different compounds. All paths funnel into (catabolism) or fan out from (anabolism) the central energy metabolism pathways of glycolysis and the Krebs cycle. Due to coupled reactions or an overall favorable free energy change, these pathways have Delta Go's that are large and negative.

So that finishes a long section that started with one important  function of proteins, being enzymes.  But we included a discussion of the directionality of the enzymatic reactions, the need for energy, the metabolic pathways that produce the needed energy, and then metabolic pathways leading to the needed small molecules in general. 

Now let's return to proteins as macromolecules, and finish off our consideration by seeing how they are synthesized, how the monomer amino acids are polymerized into polypeptides. 

We saw how polysaccharides are made by the action of a few biosynthetic enzymes, which had as substrates the growing end of a polymer chain and the free monomer that is being attached to it. So a handful of enzymes can take care of polysaccharide synthesis.  Similarly, a handful of enzymes can handle the biosynthesis of fatty acids and fats and phospholipids.

So now let's consider the biosynthesis of proteins in the same way.  Let's start with the synthesis of hexokinase, the first enzyme of glucose metabolism.  Suppose the first few amino acid were met-val-his-leu-gly. In this case, the growing end is different for each step, so we would need one enzyme to add val to met, and then another to add his to met-val, and a third to add leu to met-val-his, and so on. So if hexokinase has 500 amino acids, we would need about 500 enzymes to put it together.  If there are 3000 enzyme proteins that have to be made this way, then that's 3000 x 500 = 1,500,000 enzymes to do the job. That's a lot of enzymes. And wait a minute: each of these 1,500,000 enzymes is a unique protein itself, and has to be synthesized! So we quickly see the impossibility of making proteins by a series of simple enzymatic condensation reactions.  We need a new principle, a way of using some common synthetic apparatus that somehow knows what amino acid comes after what amino acid in every protein.  A mystery until the 1960's.  The discovery of how this information  (what a protein's primary sequence should be) is realized in a growing cell is one of the greatest achievements in the history of experimental biology.  The answer of course is written in the nucleic acids.

Copyright 2006  Lawrence Chasin and Deborah Mowshowitz   Department of Biological Sciences   Columbia University   New York, NY