The Citric Acid Cycle

Hello there! It’s been a little while, hasn’t it? I certainly feel like it has been. I’ve returned to my study blog, which has proven to be a very effective study method (hint: YOU SHOULD TRY THIS TOO IT’S FUN), to talk about an important aspect of metabolism that will be on our third biochemistry test.

Now, I have written a post on the pentose phosphate pathway, but unfortunately, that one’s not quite finished. I’ll go back and polish it up, and then I’ll give it to you guys. After all, anyone who actually bothers to read this stupid blog deserves the best~


In my post on glycolysis, I mentioned that the anaerobic conversion of glucose to pyruvate was a catabolic pathway that united all of the organisms that got to call themselves “living.” It’s true, too. Glycolysis is super handy for any cell that can’t do aerobic respiration—that means for bacteria, red blood cells and yeast without oxygen, it’s pretty awesome.

However, when you throw some oxygen into the mix, you get a whole different ball game. Sure, with ten anaerobic reactions, we can make 2 ATP and 2 NADH by making pyruvate out of glucose. However, if we convert pyruvate to Acetyl-CoA and then push it through eight reactions of the citric acid cycle, we’ll get 3 NADH, an FADH2 (well, a CoQH2), and a GTP (basically an ATP). That doesn’t sound too impressive, but trust me: when we put those reduced suckers through the ETC and get some ATP out of them, glycolysis will look like small potatoes.

Before we can use oxygen as our final electron acceptor, though, we need some electrons to give it. We’ve got a perfectly good source of them already, thankfully—that pyruvate? Yep, there’s a ton of energy in that lil’ loser.

After pyruvate is made out of glucose in the cytosol of our cells, it enters our mitochondria by a transport protein. (Yes, Tumblr Side of Science, “The mitochondrion is the powerhouse of the cell.“) Once it gets there, it’s oxidized to Acetyl-CoA and carbon dioxide. Here’s the reaction:

Pyruvate + CoASH + NAD+ —> Acetyl-CoA + NADH + CO2

If you’ve already had an introductory bio sequence, you’re going, “Yes, Acetyl-Coenzyme A, my old friend.” Well, I’m about to show you a secret that will make you question your friendship.

This reaction is carried out not by a simple enzyme like triose phosphate isomerase, but by a complex called the “pyruvate dehydrogenase complex.” This complex is made up of three enzymes—pyruvate dehydrogenase, dihydolipoyltransacetylase, and dihydrolipoyldehydrogenase—that each require a different kind of cofactor (TPP, lipoamide, FAD/FADH2). If that sounds horrific to you, ‘grats! You are a mentally stable individual!

Perhaps even scarier than that is the amount of energy released by this reaction: in spite of the formation of a super energetic thioester bond, it releases a net 34 kJ/mol under standard conditions. That’s like busting up an ATP into ADP and Pi.

So, great, we’ve got Acetyl-CoA. Awesome! We’ve moved pyruvate into the mitochondrion (which is the powerhouse of the cell), and now we’re making a really high energy compound out of it. Now what?

Well, now we bring in our friend, the citric acid (or Krebs) cycle. Here’s what the net reaction looks like:

Acetyl-CoA + 3 NAD+ + FAD (CoQ) + GDP + Pi + 2 H2O —> 2 CO2 + 3 NADH + FADH2 (CoQH2) + GTP + CoASH

That’s a bit more complicated than the net reaction of glycolysis, so let’s break it down a little bit.

First off, we see that we’re starting with that really high energy compound and ending up making it into carbon dioxide. If you’re sitting there thinking, “Is this why we’re taught that we breathe in oxygen and breath out carbon dioxide?,” the answer would be, “Yes!” The carbons in the carbon dioxide that you breathe out ultimately come from what you eat. Just think about that for a minute.

Okay, okay, enough thinking. Now, while we’re doing that, we’re also transferring four hydrides (eight electrons) to reducing agents, making three NADH and one FADH2. However, as we’ll learn shortly, FADH2 ultimately ends up giving its electrons to another compound, CoQ, to make CoQH2. We also make a GTP (ATP), but other than that, there doesn’t appear to be any chemical energy coming out of this. What the frickity-frack?

Yeah, yeah, right, right. Electrons. Those are golden, remember. We’ll be using them later.

All right, that’s nice, but how is all of this accomplished? Well, dearest reader, it sounds like you’re begging for a step-by-step walkthrough! Awesome, because I’m more than happy to oblige!

Step 1: Condensation of Acetyl-CoA and Oxaloacetate

In the first step of the citric acid cycle, that acetyl-CoA that we made in the mitochondrion condenses with a molecule of oxaloacetate by an aldol condensation reaction. This generates a tricarboxylic acid (!!), citrate.

This reaction is carried out by an enzyme called Citrate Synthase, which is a transferase. The reaction itself is spontaneous enough under standard conditions (releasing 32 kJ/mol), but under cellular conditions, it’s even more favorable due to relative concentrations of reactants and products (-54 kJ/mol). Here’s a picture:

Step 1 cac

Step 2: Isomerization of Citrate to Isocitrate

After citrate is made, it’s isomerized into its isomer, the cleverly named “isocitrate.” This reaction is mediated by a S-Fe containing lyase (not an isomerase) called Aconitase. It’s a lyase because it uses a dehydration and hydration reaction to interconvert the two isomers. This step has a very small free energy change, and, thus, it is reversible. Observe:

step 2 cac

Step 3: Oxidation and Decarboxylation of Isocitrate

Once we make isocitrate, we start with our redox shenanigans. In the first redox step of the citric acid cycle proper, isocitrate is oxidized and decarboxylated into alpha-ketoglutarate. This generates our first molecule of CO2, as well as our first molecule of NADH.

This reaction is carried out by an enzyme called Isocitrate Dehydrogenase, which is an oxidoreductase. It’s dependent on divalent cations (such as Mg2+ or Mn2+) that can stabilize an intermediate in the mechanism. The free energy change for this reaction is about -18 kJ/mol under cellular conditions, which means it’s essentially irreversible. Here’s a picture:

step 3 cac

Step 4: Oxidation and Decarboxylation of alpha-Ketoglutarate

All right! So, we’ve oxidized our isocitrate to alpha-ketoglutarate and made NADH and CO2. Now what are we going to do? Well, we’re gonna do it again!

This time, alpha-ketoglutarate is being oxidized and decarboxylated to succinyl-CoA by an enzyme called alpha-Ketoglutarate Dehydrogenase (an oxidoreductase), producing another molecule of NADH and CO2. The free energy change for this reaction, under cellular conditions, is ridiculously negative: it releases 44 kJ/mol, to be exact. See?

step 4 cac

Step 5: Succinyl-CoA to Succinate and GTP

All right, now it’s time for us to make some energy directly. You’ll recognize that C-SCoA as being the really high-energy bond that was in Acetyl-CoA. Now we’re going to split that group off to make succinate, and we’ll use the energy we get to make GTP in the process. Because the energy in the thioester bond that’s broken and the energy in the phosphoanhydride bond that’s formed are roughly equivalent, this reaction is, surprisingly, reversible. In fact, the enzyme that does this reaction is named for the reverse reaction: it’s a ligase called Succinyl-CoA Ligase.

step 5 cac

Step 6: Oxidation of Succinate to Fumarate

All right, now, as you can tell from the net reaction, we aren’t going to lose any more carbons, but we are going to pull out some more electrons. This reaction is the first of the oxidation reactions that don’t also get rid of carbons. Here, we’re going to oxidize succinate to fumarate using an enzyme called Succinate Dehydrogenase (another oxidoreductase) in a reaction that’s reversible.

The first hydride (electron) receptor in this redox reaction is a lil’ thing called FAD. It picks up a hydride and a proton, becoming FADH2. However, FAD/FADH2 is covalently bound to Succinate Dehydrogenase. To properly transport its electrons, it gives them to a coenzyme called CoQ, which becomes CoQH2. This molecule is highly lipid soluble (hydrophobic, due to a freaking huge isoprene tail), and it goes to hang out in the inner mitochondrial membrane, where it can be useful.

step 6 cac

Step 7: Hydration of Fumarate

Now that we’ve got fumarate, we’re going to add water across its carbon-carbon double bond using an enzyme called Fumarase (a lyase). This reaction, perhaps predictably, is reversible, and it produces exclusively L-Malate (as opposed to a mix of L-Malate and D-Malate). Here’s the reaction:

step 7 cac

Step 8: Oxidation of L-Malate to Oxaloacetate

Finally, to make this a proper cycle, we’re going to regenerate our starting material. To do this, an enzyme called Malate Dehydrogenase (an oxidoreductase) oxidizes L-malate into oxaloacetate, making our final molecule of NADH. Now all we need is more Acetyl-CoA, and we’re ready to start over.

Interestingly, this reaction is the only one in the citric acid cycle that has an extremely positive free energy change under standard conditions (30 kJ/mol). However, because the cellular concentration of oxaloacetate is so much smaller than it would be at equilibrium (oxaloacetate is getting used in the citric acid cycle and gluconeogenesis, mind you!), this reaction proceeds in a reversible fashion under cellular conditions.

step 8 ca

A final note about these reactions: the three steps that are out of equilibrium (Step 1, 3 and 4), as well as the “pre-step” that converts pyruvate to acetyl-CoA, are dependent on two factors: concentrations of ATP and concentrations of NADH. If either is particularly high, those enzymes will slow down, and the citric acid cycle will slow down overall. That makes sense, if you think about it; why should we by trying to make tons of energy if we already have it?

Phew! Pretty complicated huh? Yep, yep it is. However, it’s a good thing we do this, because it really is a great source of energy. You can’t see that now, but just wait; when we talk about the ETC, it’s going to blow your mind.

Listening to “The Real Slim Shady” got me feeling the weird nostalgia feels. I mean, um, comment with anything fun or interesting~! Thanks!


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