Slide 1: the TCA Cycle

Fundamentals I

Dr. Miller 9/2/08

11:00-12:00 pm

The TCA Cycle

Slide 1: The TCA Cycle

Metabolism that’s perhaps why it has become the most prevalent form of metabolism for living beings.
Objective here is to oxidize two carbon fragments derived from metabolism of carbohydrates.
Problem is we have a three carbon fragment which is the product- mainly pyruvate.
In order to get into the TCA cycle, there is a big admission price to pay.
Ultimately uses carrier molecules like Oxaloacetate and the unique properties of thioester bonds and also the properties of oxo-acids that allow them to spontaneously decarboxylate in the TCA cycle.

Slide 2:

Just came from glycolysis and made 2 molecules of pyruvate.
Pyruvate will enter the TCA cycle as acetyl CoA;
Pyruvate is going to lose a carboxyl function because it has three carbons.
Will only take in the TCA cycle a 2 carbon acetyl CoA group.
Pyruvate enters TCA cycle;
TCA cycle is very productive of NADH and FADH2.
One substrate level phosphorylation will occur to convert GDP to GTP.
This cycle is mainly devoted to producing reduced electron carriers- NADH and FADH2.
These electron carriers will carry the oxidized products- the protons and electrons to the oxidative phosphorylation respiratory chain.
The respiratory chain is capable of using the energy brought by NADH and FADH2 to pump proton in the inner mitochondrial space; the protons will go passing down the F1 and F0 ATPase system (ATP synthase system) to make ATP.

Slide 3: The TCA cycle

Pyruvate is decarboxylated and the acetyl group is attached to coenzyme A.
The coenzyme A ushers the acetyl group into the TCA cycle.
The acetyl group combines with Oxaloacetate to make citric acid.
Citric acid is not useful for further operations so it is converted by aconitase enzyme to isocitric acid.
Isocitric acid is decarboxylate to give rise to CO2 and alpha-keto glutarate.
Alpha keto glutarate is also decarboxylated and gives succinyl CoA.
Succinyl CoA is high energy compound that when hydrolyzed gives rise to the formation of GTP to make succinate.
Succinate is oxidized to make fumerate.
Fumerate is hydrated to make malate.
Malate is oxidized to come back to Oxaloacetate.
This whole process creates CO2 and creates NADH and FADH2 winds up with the same material that provided one the reactants in the beginning.
The system is now ready to another acetyl group to enter the TCA cycle.

Slide 4: Preparation

Pyruvate goes into the mitochondria.
Pyruvate is decarboxylate to make Acetyl CoA.
Pyruvate dehydrogenase is a huge enzyme complex containing three enzymes and five coenzymes.
It contains five of the vitamins that we talked about on Friday- these are all used to get pyruvate admitted to the TCA cycle.

Slide 5

Pyruvate is decarboxylated and is done by thiamin pyrophosphate (TPP).
TPP for a while carries the acetyl group
TPP is the cofactor in the enzymes which does the decarboxylation.
TPP, vitamin B1, was talked about on Friday.
TPP gives up the acetyl group to lipoic acid.
Lipoic acid has a disulfide-bonded side chain; the disulfide bond is broken and reduced when TPP passes the acetyl group to the sulfur atom.
Lipoic acid is now carrying the acetyl group and is then passed onto Coenzyme A.
It is derivitized again by a thioester bond with Coenzyme A.
Lipoic acid has been reduced by the passing of the acetyl group; it has been reduced.
The Lipoic acid can be reoxidized to disulfide-bonded lipoic acid by giving first its hydrogen atoms to FAD to form FADH2
FADH2 gives its protons (H+ atoms) to NAD to give NADH & H+
Five coenzymes used: TPP (vitamin B1), lipoic acid (does not have B1 designation) and acts as an acetyl carrier, Coenzyme A (portion of this comes from panthenoic acid, vitamin B5), and the electrons are passed to FAD first (vitamin B2), and wind up with NADH (vitamin B3).
Keeps stressing 5 different vitamins were involved.

Slide 6

Coenzyme A will now usher two carbon fragments into the TCA cycle.
Enzyme can actually pluck a proton from the carbon atom.
Carbon atom is now available for reaction with carboxyl function with Oxaloacetate.
Oxaloacetate receives this and gives rise to citryl-CoA.
Hydrolyze CoA away and you get citrate (citric acid).
Called a ____condensation where a carbon atom condenses with another carbon atom.
This is the reversal of the guillotining of fructose 1, 6 bisphosphate and the third and fourth carbon atoms; the carbon-carbon bond was cut.
Here we created a carbon-carbon bond.
This is the acetyl group brought in and this is the carrier.
Goes into spiel about knowing about amino acids.
Look at Oxaloacetate- it is the oxidized form of aspartic acid; put an amino group on Oxaloacetate, you will have aspartic acid. Keto function has replaced the primary amine.

Slide 7

Now you wind up with citric acid…this is why it’s called the Tricarboxylic Acid Cycle.
Three carboxyl groups are involved to form one molecule.
This is the acid from lemons, grapefruit, plums- any citric fruit because they have lots of citric acid.
Fruits are valuable because you already have a product that is part of the TCA cycle right in the food.
Citrate is a poor substrate for oxidization; the hydroxyl group is a tertiary hydroxyl group and CANNOT be easily hydrated.
This molecule cannot lose water that well, so it must be changed to a secondary hydroxyl group.

Slide 8

It is changed to a secondary hydroxyl by aconitase enzyme.
Aconitase enzyme will remove a proton from this carbon atom and cause this hydroxyl group to leave and make an intermediate which is the dehydrated from of citric acid called cis-aconitate.
Cis-Aconitate has a double bond made from removing water.
When water is removed at that location, water can then be added back in a different direction so that the hydroxyl group is added a different carbon atom.
A hydrogen atom is also added to a different carbon.
You have just reversed the hydrogen and the hydroxyl groups to make a secondary alcohol in isocitrate.

Slide 9

This reaction requires a sulfur because the hydroxyl group does not leave easily.
The only reason this is shown is to show us the commonality which occurs in biochemistry.
Have to get rid of a group with an oxygen it in.
How do you handle oxygen when you want to do biochemical work? BRING IRON IN, just like how we did with hemoglobin.
The iron and sulfur boxes in the enzyme; take the proton away from the carbon atom, the hydroxyl group is aided in its leaving because of its affinity for the iron.
That’s how you get rid of a difficultly-removable group- the hydroxyl group.
The iron takes over and pulls it away.
The product is the aconitate, which has been dehydrated.

Slide 10

If we have this particular arrangement, isocitric acid can be oxidized and the hydrogen atoms can be taken way to form NADH.
Now we have oxalosuccinate.
Oxalosuccinate will oxidatively decarboxylate spontaneously because we now have a keto function of an oxo-function beta to this carbon atom.
This carboxyl group will be lost and alpha-keto gluterate is made.
Two things have been done by making isocitrate.
1. Set up a dehydrogenation system and dehydrogenated isocitric acid to make oxalosuccinate
2. Decarboxylated oxalosuccinate spontaneously (no enzyme needed) to make alpha-keto gluaterate.
A carboxyl group has been lost to form CO2.
Also gained an energy rich compound, NADH.
This is a key operation where isocitrate is essentially converted to alpha-keto glutarate.
Lose CO2 and make NADH.
The picture on the right is how the complex looks in a 3-D arrangement; don’t pay attention to it because we don’t have enough time.
We wind up with alpha-keto glutarate.

Slide 11

Alpha-keto glutarate looks like pyruvate.
Since they have similar structures, alpha-keto glutarate can also be decarboxylated just like pyruvate.
It can be decarboxylated with all the same cofactors used in decarboxylating pyruvate to acetyl CoA.
Since alpha keto glutarate lost a carboxyl group here, it is now complexed with succinyl CoA.
This carboxyl group is lost and succinyl CoA has been made by the addition of Coenzyme A (CoA).
It was just like how pyruvate was decarboxylated and then wound up as acetyl CoA.
Here, we decarboxylate alpha keto glutarate and it winds up as succinyl CoA.
The exact same reaction- the same cofactors- TPP, lipoic A, panthenoic acid, vitamin A, vitamin B2, and vitamin B3 are used in BOTH REACTIONS.
This is the same operation as converting pyruvate to succinyl CoA.
Lose carboxyl group, put Coenzyme A on there.
This is where our 2nd CO2 comes from….this carboxylation reaction.

Slide 12

Now succinyl-CoA, this thioester bond is a high energy bond; when it is hydrolyzed, the energy in that bond can be used to convert GDP to GTP and free CoA. That gives us succinic acid.

Slide 16

Succinic acid can be dehydrogenated to give fumarate, so you have a dehydrogenase. This particular reaction occurs in the membrane of mitochondria. Right in the respiratory chain itself; not in the matrix, but in the respiratory chain itself in complex #2, which we will talk about later.
The complex #2 is the location where this oxidation occurs and when it occurs, we reduce FAD to FADH2 and make fumarate. Fumaric acid, fumarate. Fumarate can by hydrated and this is a process where the FAD shows you the FAD; it is connected by a side chain of histadine.

Slide 14

The FAD is connected by a side chain of histadine to its enzyme. When the enzyme that does the oxidation will add the hydrogen atoms to this nitrogen and this nitrogen, it places a double bond here (refers to image.)
So this is FAD and this is the kind of cofactor that is carried along to receive the H atoms from the oxidation.
FAD, remember, is Vitamin B2. It is involved in this process and that occurs in the respiratory chain.

Slide 13

Now you hydrate fumarate, you get L-malonate. This is malonic acid; this name comes from the Latin name for apple. Now apple juice is very rich in malonic acid. That is why the name of the apple has been derived by the Latin name for apples.
So, this particular compound is made by simply adding water across a double bond so we have reduced this particular carbon atom to CH2 and this particular carbon atom to the hydroxyl group.

Slide 15

Now we can dehydrogenate malonate by taking hydrogen atom here, creating a double bond and reducing more NAD to NADH. Now we have oxaloacetate.
Now we come back to the substance that we started out with. The way we did this, the oxalanic acid or oxaloacetate was used a carrier to carry the 2 carbon fragment in there, get it oxidized and come back, and we have oxaloacetate again.
The 2 carbons that were lost in the first pass, came from the oxaloacetate. So what remains in oxaloacetate, in this part of the oxaloacetate, came in the form of the acetyl group in the very beginning. If you really go carefully through the whole process and you look at it with a critical eye, you will note that the 2 carboxyl groups that were lost were from the original oxaloacetate.
But, we wind up with another 4 carbon molecule, the oxaloacetate again, because we had 6 carbons in the citric acid and we lost 2 of them from oxaloacetate but we now incorporated the 2 acetyl carbons back into new oxaloacetate. That is essential how that system works.
Now note that the regeneration of oxaloacetate is not an energy producing reaction; it is energy utilizing. The only reason that this reaction occurs is because that oxaloacetate is a really sought after molecule.
It is used to make citric acid, more glucose or more carbohydrate incase the individual is carbohydrate deficient. You will take off oxaloacetate to make new glucose. You cannot go back directly from acetyl CoA; it’s irreversible. The formation of acetyl CoA is irreversible. It is an irreversible phenomenon.
If you are going to make new glucose, you go from oxaloacetate. And of course if you need protein, you can siphon off some oxaloacetate to make aspartic acid. (We will review some of these considerations again later.)
This is a very expensive reaction; it actually goes because the amount of oxaloacetate that is around in a normal individual is on the order of mircomolar amounts. Mircomolar; you don’t have at any given time much oxaloacetate available.

Q: Can’t hear the question – mic did not pick up.

A: ..Because there is some little of it, the precursors are in HUGE quantities. You can have any reaction go by adjusting the ratio of reactant to products even though it is energetically unfavorable, it will go because of the discrimination or differential between the reactants and products. So what is going to make oxaloacetate is generally quite plentiful.

Slide 17

Okay, now energy consequences of glycolysis and the TCA.
One glucose molecule in glycolysis – no CO2, 2 ATP, 2 NADH in the system.
In pyruvate dehydrogenation, we get 2 carboxyl groups off because each pyruvate molecule (there were 2 pyruvate) came from 1 glucose. The 2 pyruvates were decarboxylated in the pyruvate dehydrogenation reaction. We made 2 NADH molecules, all the way down from the 5th vitamin used – NADH. We got 2 NADH.
In the TCA cccyle, we have 4 CO2 molecules produced; remember 2 times of 2 CO2, made each time that molecule came through; there were 2 from 1 glucose. There were 4 CO2 made. 2 GTP made, 6 NADH made (there were 3 places that we made NADH) so that is a total of 6 for the whole glucose molecule. We had 2 FADH2 made. One place where we reduced FAD, and that was where succinate was oxidized to fumarate, and that occurs in the respiratory chain itself.
So the total, we have lost all our carbons now. As I told you, you have total loss of carbons in the form of CO2. We have made 4 ATPs by substrate level phosphorylation. That is, we made those ATPs by simply adding phosphate to ADPs. We had no oxidation going on, no respiratory chain, we made them by substrate level phosphorylation.
Then, we have a total of 10 NADHs made, and a total of 2 FADH2 made.
What this amounts to if we sum this up – We had substrate level glycolysis in the TCA (our 4 high energy phosphate bonds made) and in NADH in the electron transports, you get 3 ATP for every NADH molecule.
So we have 30 ATP coming from that, and since FADH comes into the respiratory chain from complex #2, we don’t get as much ATP per FADH. We only get about 2 electrons/2 ATP for every FADH molecule. We get a total of 4 there.
So the theoretical maximum high energy phosphate bond potential reduction from this whole business is 38. Remember that, but remember that is not the whole balance sheet.
The final balance sheet will be on the order of 30 to 32 because we have to make ATP useful; this is making ATP. For the ATP to be useful, it has to be transported out of the mitochondria and that takes energy, high energy phosphate bonds away from us in order to expend the energy to get the ATP out of where it is made.
ATP will accumulate, if the only idea was to make it and accumulate it in the mitochondria, that would kind of be ridiculous. It would be impossible to utilize muscle contraction in any other area.
So you can theoretically make 38 ATP from 1 glucose molecule, but that is an over production situation.

Slide 18

Now this is where we need to take a look at what the TCA does and how it can be utilized. There are many products that can be made from intermediates in the TCA.
Take a look at oxaloacetate, for instance. Now remember, I told you that it is simply deaminated aspartic acid but you can make aspartic acid and then make aspartic acid from all these other compounds, even pyridine nucleotides, which we will study later.
If you take a-K-G, it is essentially decarboxlyated glutamic acid – you make glutamate, proline, glutamine, (something else – can’t hear.) You can make all these compounds from a-K-G.
From succinyl CoA, you can make glycine, porphryins, you can do very many things with the intermediates in the TCA.
The TCA is a simple trading area where you can actually use aspartic acid to fill up oxaloacetate, or you can use a-K-G to make glutamate. Anyway, you can do a variety of things with that.
Acetyl CoA now, notice that there is no reverse sign here. Once you get to this point, you are either going to the TCA or you are going to use acetyl CoA to make fatty acids.
This is essentially the situation. If you have a high content (high metabolic status, high energy status) and isocitrate, the conversion of isocitrate to a-K-G is blocked, this is one of the major blocking regions in the whole TCA.
If there is a great deal of ATP around, then this particular reaction, the conversion of isocitrate to a-K-G is inhibited and then, essentially, the TCA is cut down.
The TCA is cut down and acetyl CoA comes pouring in here; basically it will not enter the TCA but will be siphoned off to make fatty acids.
This is where you get into high caloric restrictions, high calorie, well fed individual – more sweets are eaten.
Then you come down here and acetyl CoA cannot go into the TCA, the only thing to do is make fatty acids.
Other side of the coin, if you are starving, whether it being enforced starving because you are in a concentration camp or you are desiring to lose weight, what will essentially happen is that the body senses it is lacking carbohydrate.
All the bodies reagents will try to make glucose. The only way to make glucose here is through gluconeogensis and to go back to carbohydrate, you have to siphon off oxaloacetate.
Oxaloacetate is siphoned off to go make carbohydrate.
Pyruvate can be siphoned off to go back, but acetyl CoA cannot be going back to make pyruvate and then onto making carbohydrate.
Have to get new carbohydrate through sacrificing pyruvate which if you are starving, there is not much pyruvate. Don’t have much pyruvate.
If you then start to use oxaloacetate and oxaloacetate get it short supply- where do you get oxaloacetate? You break down muscle tissue or some protein to make oxaloacetate or glutamate and then go around here.
Anyway, oxaloacetate is a real central issue. It is siphoned off to make carbohydrate and when that occurs, the only thing that can happen with acetyl CoA is that it has to combine with itself to make acetoacetate.
Acetoacetate can be decarboxylated to make acetone.
A starving person’s breath smells like acetone because they are essentially existing by energy production from ketone bodies. (We don’t have time to go into ketone bodies – can read up on.)
Realize that this is a very important junction of health and metabolism, starvation and health and well being.
Acetyl CoA will combine with itself if there is NO oxaloacetate to make ketone bodies which can be decarboxlyated to make acetone. You can determine a person’s health status by that classification.

Slide 20