The Krebs Cycle Made Easy

The Krebs cycle, named after 1953 Nobel Prize winner and physiologist Hans Krebs, is a series of metabolic reactions that take place in the mitochondria of eukaryotic cells. Put more simply, this means that bacteria do not have the cellular machinery for the Krebs cycle, so it limited to plants, animals and fungi.

Glucose is the molecule that is ultimately metabolized by living things to derive energy, in the form of adenosine triphosphate, or ATP. Glucose can be stored in the body in numerous forms; glycogen is little more than a long chain of glucose molecules that is stored in muscle and liver cells, while dietary carbohydrates, proteins and fats have components that can be metabolized to glucose as well. When a molecule of glucose enters a cell, it is broken down in the cytoplasm into pyruvate.

What happens next depends on whether the pyruvate enters the aerobic respiration path (the usual result) or the lactate fermentation path (used in bouts of high-intensity exercise or oxygen deprivation) before it ultimately allows for ATP production and the release of carbon dioxide (CO₂) and water (H₂O) as by-products.

The Krebs cycle – also called the citric acid cycle or the tricarboxylic acid (TCA) cycle – is the first step in the aerobic pathway, and it operates to continually synthesize enough of a substance called oxaloacetate to keep the cycle going, although, as you'll see, this is not really the cycle's "mission." The Krebs cycle supplies other benefits as well. Because it includes some eight reactions (and, correspondingly, nine enzymes) involving nine distinct molecules, it is helpful to develop tools to keep the important points of the cycle straight in your mind.

Glucose is a six-carbon (hexose) sugar that in nature is usually in the form of a ring. Like all monosaccharides (sugar monomers), it consists of carbon, hydrogen and oxygen in a 1-2-1 ratio, with a formula of C₆H₁₂O₆. It is one of the end products of protein, carbohydrate and fatty-acid metabolism and serves as fuel in every type of organism from single-celled bacteria to human beings and larger animals.

Glycolysis is anaerobic in the strict sense of "without oxygen." That is, the reactions proceed whether O₂ is present in cells or not. Be careful to distinguish this from "oxygen must not be present," although this is the case with some bacteria that are actually killed by oxygen and are known as obligate anaerobes.

In the reactions of glycolysis, the six-carbon glucose is initially phosphorylated – that is, it has a phosphate group appended to it. The resulting molecule is a phosphorylated form of fructose (fruit sugar). This molecule is then phosphorylated a second time. Each of these phosphorylations requires a molecule of ATP, both of which are converted to adenosine diphosphate, or ADP. The six-carbon molecule is then converted into two three-carbon molecules, which are quickly converted to pyruvate. Along the way, in the processing of both molecules, 4 ATP are produced with the help of two molecules of NAD+ (nicotinamide adenine dinucleotide) that are converted to two molecules of NADH. Thus for every glucose molecule that enters glycolysis, a net of two ATP, two pyruvate and two NADH are produced, while two NAD+ are consumed.

As noted previously, the fate of pyruvate depends on the metabolic demands and the environment of the organism in question. In prokaryotes, glycolysis plus fermentation provides almost all of the single cell's energy needs, although some of these organisms have evolved _electron transport chains_ that allow them to make use of oxygen to liberate ATP from metabolites (products) of glycolysis. In prokaryotes as well as in all eukaryotes but yeast, if there is no oxygen available or if the cell's energy needs cannot be fully met through aerobic respiration, pyruvate is converted to lactic acid via fermentation under the influence of the enzyme lactate dehydrogenase, or LDH.

Pyruvate destined for the Krebs cycle moves from the cytoplasm across the membrane of cell organelles (functional components in the cytoplasm) called mitochondria. Once in the mitochondrial matrix, which is a sort of cytoplasm for the mitochondria themselves, it is converted under the influence of the enzyme pyruvate dehydrogenase to a different three-carbon compound called acetyl coenzyme A or acetyl CoA. Many enzymes can be picked out from a chemical line-up because of the "-ase" suffix they share.

At this point you should avail yourself of a diagram detailing the Krebs cycle, as it is the only way to meaningfully follow along; see the Resources for an example.

The reason the Krebs cycle is named as such is that one of its main products, oxaloacetate, is also a reactant. That is, when the two-carbon acetyl CoA created from pyruvate enters the cycle from "upstream," it reacts with oxaloacetate, a four-carbon molecule, and forms citrate, a six-carbon molecule. Citrate, a symmetrical molecule, includes three carboxyl groups, which have the form (-COOH) in their protonated form and (-COO-) in their unprotonated form. It is this trio of carboxyl groups that lends the name "tricarboxylic acid" to this cycle. The synthesis is driven by the addition of a water molecule, making this a condensation reaction, and the loss of the coenzyme A portion of acetyl CoA.

Citrate is then rearranged into a molecule with the same atoms in a different arrangement, which is fittingly called isocitrate. This molecule then gives off a CO₂ to become the five-carbon compound α-ketoglutarate, and in the next step the same thing occurs, with α-ketoglutarate losing a CO₂ while regaining a coenzyme A to become succinyl CoA. This four-carbon molecule becomes succinate with the loss of CoA, and is subsequently rearranged into a procession of four-carbon deprotonated acids: fumarate, malate and finally oxaloacetate.

The central molecules of the Krebs cycle, then, in order, are

1. Acetyl CoA
2. Citrate
3. Isocitrate
4. α-ketoglutarate 
5. Succinyl CoA
6. Succinate
7. Fumarate
8. Malate
9. Oxaloacetate

This omits the names of the enzymes and a number of critical co-reactants, among them NAD+/NADH, the similar molecule pair FAD/FADH₂ (flavin adenine dinucleotide) and CO₂.

Note that the amount of carbon at the same point in any cycle remains the same. Oxaloacetate picks up two carbon atoms when it combines with acetyl CoA, but these two atoms are lost in the first half of the Krebs cycle as CO₂ in successive reactions in which NAD+ is also reduced to NADH. (In chemistry, to simplify somewhat, reduction reactions add protons while oxidation reactions remove them.) Looking at the process as a whole, and examining only these two-, four-, five- and six-carbon reactants and products, it is not immediately clear why cells would engage in something like resembles a biochemical Ferris wheel, with different riders from the same population being loaded on and off the wheel but nothing changing at the end of the day except for a great many turns of the wheel.

The purpose of the Krebs cycle is more obvious when you look at what happens to hydrogen ions in these reactions. At three different points, a NAD+ collects a proton, and at a different point, FAD collects two protons. Think of protons – because of their effect on positive and negative charges – as pairs of electrons. On this view, the point of the cycle is the accumulation of high-energy electron pairs from small carbon molecules.

You may notice that two critical molecules expected to be present in aerobic respiration are missing from the Krebs cycle: Oxygen (O₂) and ATP, the form of energy directly employed by cells and tissues to carry out work such as growth, repair and so on. Again, this is because the Krebs cycle is a table-setter for the electron transport chain reactions that occur nearby, in the mitochondrial membrane rather than in the mitochondrial matrix. The electrons harvested by nucleotides (NAD+ and FAD) in the cycle are used "downstream" when they are accepted by oxygen atoms in the transport chain. The Krebs cycle in effect strips away valuable material in a seemingly unremarkable circular conveyor belt and exports them to a nearby processing center where the real production team is at work.

Also note that the seemingly unnecessary reactions in the Krebs cycle (after all, why take eight steps to accomplish what might be done in perhaps three or four?) generate molecules that, though intermediates in the Krebs cycle, can serve as reactants in unrelated reactions.

For reference, NAD accepts a proton at Steps 3, 4 and 8, and in the first two of these CO₂ is shed; a molecule of guanosine triphosphate (GTP) is produced from GDP at Step 5; and FAD accepts two protons at Step 6. In step 1, CoA "leaves," but "returns" in Step 4. In fact, only Step 2, the rearrangement of citrate into isocitrate, is "silent" outside of the carbon molecules in the reaction.

Because of the importance of the Krebs cycle in biochemistry and human physiology, students, professors and others have come up with a number of mnemonics, or ways to remember names, to help with remembering the steps and reactants in the Krebs cycle. If one only wishes to remember the carbon reactants, intermediates and products, it is possible to work from the first letters of successive compounds as they appear (O, Ac, C, I, K, Sc, S, F, M; here, notice that "coenzyme A" is represented by a small "c"). You can create a pithy personalized phrase from these letters, with the first letters of the molecules serving as the first letters in the words of the phrase.

A more sophisticated way of going about this is to use a mnemonic that lets you keep track of the number of carbon atoms at every step, which may allow you to better internalize what is happening from a biochemical standpoint at all times. For example, if you let a six-letter word represent the six-carbon oxaloacetate, and correspondingly for smaller words and molecules, you can produce a scheme that is both useful as a memory device and information rich. One contributor to the "Journal of Chemical Education" proposed the following idea:

1. Single
2. Tingle
3. Tangle 
4. Mangle
5. Mange
6. Mane
7. Sane
8. Sang
9. Sing

Here, you see a six-letter word formed by a two-letter word (or group) and a four-letter word. Each of the next three steps includes a single letter substitution with no loss of letters (or "carbon"). The next two steps each involve the loss of a letter (or, again, "carbon"). The rest of the scheme preserves the four-letter word requirement in the same way the last steps of the Krebs cycle include different, closely related four-carbon molecules.

Apart from these specific devices, you may find it beneficial to draw yourself a complete cell or portion of a cell surrounding a mitochondrion, and sketch the reactions of glycolysis in as much detail as you like in the cytoplasm part and the Krebs cycle in the mitochondrial matrix part. You would, in this sketch, show pyruvate being shuttled into the interior of the mitochondria, but you could also draw an arrow leading to fermentation, which also occurs in the cytoplasm.