Glycolysis is the process through which a molecule of glucose is sequentially broken down into two molecules of pyruvate. This process is catalyzed by several enzymes, all of which have different properties. These enzymes and some of their properties will be discussed below, as well as the ultimate yield of glycolysis and what this means to the organism in question. We recommend that you concurrently open the image of the glycolytic pathway while reading this text. This can be done via the, "open in new window" command which appears in the list after right clicking on the link.


Hexokinase is listed as enzyme one on our flowchart. It adds a phosphate to glucose or other hexoses. (Remember, a hexose is a six carbon sugar, and a kinase is an enzyme which adds a phosphate; therefore hexokinase is an enzyme which adds a phosphate to a six carbon sugar).
The addition of this phosphate introduces a negative charge on the glucose molecule. This is important for two major reasons. First and foremost, it makes the new molecule, glucose-6-Phosphate, impermeable to the cell membrane. This is crucial for the cell, as glucose in it's native state can and does penetrate the cell easily. The cell, to survive, needs to accumulate high concentrations of glucose, usually concentrations that are much higher inside the cell than outside. If this phosphate were not added the glucose molecule would simply move towards equilibrium and leave the cell, in effect starving the cell of glucose. The second reason is that the phosphate group provides a strong charge to interact with future enzyme active sites. This allows higher specificity of reaction and more efficient turnover, all of which serve to help glycolysis go.
This enzyme has a large negative delta G (or Gibbs free energy) meaning that it's actions are for the most part irreversible. The reason the delta G is so negative is that ATP is used as the phosphate donor. The bond energy between the glucose and the phosphate is much less than that of the ADP and the phosphate. Therefore, a large amount of free energy is released, creating a negative delta G. Aside from ATP, this enzyme also requires Mg2+ as a cofactor. The product of the enzyme, Glu-6-P, is then used by the next enzyme in the pathway (as occurs in all pathways, the product of enzyme one is used by enzyme two is used by enzyme three... and so on).

As we have seen, a hexose is a six carbon sugar, so it stands to reason that a phosphohexose is a six carbon sugar with a phosphate attached. And it is. But what is an isomearse? An isomerase, as it turns out, is the name given to any of a class of enzymes which interconvert one molecule to another related molecule. So from the name of this enzyme we can guess that a phosphohexoisomerase is a molecule that converts phosphated hexoses into other phosphated hexoses. And this guess would be correct.
Specifically, this enzyme in this pathway converts Glu-6-P to Fructose-6-P. This is a reversible reaction, meaning that the delta G is very close to zero. Again this enzyme requires Mg2+, although no ATP are consumed.

Phosphofructokinase (I and II)
Phosphofructokinase (or PFK as it will be abbreviated from now on) is an enzyme that adds a phosphate to a phosphorylated fructose. But of course, from looking at the name you already knew that. What you may not have known is that this is done in a stereospecific manner; the phosphate is not just thrown on randomly. Fru-1,6-bP is the standard product of this reaction (where bP is bisphosphate).
Here again we see a phosphate being added. As in the case of the hexokinase, this phosphate may be from ATP, or it may be from somewhere different, from a pyrophosphate (PPi). Here again we see an irreversible reaction due to the large negative delta G, as we expect from our studies of hexokinase. Also like hexokinase, this enzyme requires Mg2+ as a cofactor. However, one thing we did not observe with hexokinase is seen here: PFK is regulated by ATP.
This enzyme is one of the focal points of glycolytic regulation. In the presence of a low concentration of ATP (hereon abbreviated low ATP) and a high concentration of ADP or AMP this enzyme is stimulated. When we think about this, it does make sense. This aspect of the enzyme will be discussed in the "regulation of glycolysis" section of this site.

FbP aldolase
With the enzyme the first thing you should note is that the name is much shorter due to the unannounced abbreviation. It should be relatively apparent that F stands here for Fructose, and bP for bisphosphate. But now we are on to something new; what is an aldolase? An adolase is an enzyme that mediates and aldol condensation reaction. In this case it produces two three carbon compounds from one six carbon one. The enzyme may require Zn2+ as a cofactor, although that is not strictly known for all species.
Of the specific 3C compounds (glyceraldehyde-3-P and Dihydroxyacetone-P), only one (dihydroxyacetone-P) moves on to become a substrate for the next enzyme. At that step it is converted into the other 3C compound produced, both of which are acted upon by the following enzyme. Please see the flow chart for clarification.

Triose Phosphate Isomerase
Here again we see an enzyme called an isomerase. As we learned before an isomerase is an enzyme responsible for the interconversion of molecules into different but related molecules. In this case this enzyme acts to convert hidydroxyacetone-phosphate into glyceraldehyde-3-P. After this is accomplished, please note that there are TWO glyceraldehyde-3-Ps moving on into the next part of the pathway, one as a result of this enzyme and one as a result of FbP aldolase.

By now you may, if you are astute enough, be scratching your head. "Wait a moment," you may be saying, "Isn't glycolysis designed to provide energy for cells? I'm quite sure I read that somewhere, and I'm quite sure I saw two ATP go into this process, and none come out." And if you are thinking this, you are correct.
Thus far glycolysis has consumed two ATP (the chemical energetic currency of the cell) and produced nothing other than further products. However, we can break glycolysis down into two phases which helps explain this. The first phase, through which we have just progressed, is the "preparatory phase." In this phase we are preparing the glucose, through a series of interactions, to provide energy or compounds that can later provide energy. The use of ATPs to this point is an example of priming the pump if you will. It is necessary to put energy into the system to get energy out of it. The next phase of glycolysis, the "payoff phase," is the beginning of the benefits. Both the costs and the benefits of these two phases will be discussed following the discussion of the enzymes. Now, back to the enzymes and the beginning of the payoff phase.

Glyceraldehyde-3-P dehydrogenase
Boy that's a long word, and I'm sure you're wondering what it means. The glyceraldehyde-3-P of course means that it has that molecule (which is, quite frankly, too long to type again) as its substrate. But the dehydrogenase? Don't recognize that word. But then, you don't have to recognize it, only think it through. Hydro, I'm sure you've seen that before. It means having to do with water. And "de" should be familiar also. Deemphasize, debug, decapitate, starting to get the picture? A dehydrogenase then is something that removes water from the picture. In this step the enzyme oxidizes and phosphorylates the Gly-3-Ps from the preparatory phase. It does this by adding inorganic free phosphates (which makes delta G more positive) and removing hydrogens to NAD+s (which become NADHs). Again, here it is good to look at the provided jpg. The end result of this enzyme are two molecules of 1,3-bisphosphoglycerate, which have high energy acyl phosphates...

Phosphoglycerate Kinase
This enzyme uses the acyl phospahtes to make ATP through substrate level phosphorylation, something you simply must read about in a later section. And yes, finally, we are making ATP. And in the last step we had two acyl phosphates (one per molecule) so now we get two ATP. This makes up for all that is "spent" in the preparatory phase. The product of this reaction are two molecules of 3-phosphoglycerate.

Phosphoglycerate mutase
What's this? A mutase? Don't know what it is? Guess what? It's a bit like an isomerase. But instead of creating entire new molecules, it simply rearranges them. In this case it converts 3-phosphoglycerate into 2-phosphoglycerate. It is nothing overly complex and there is not much for the beginning student to know about this enzyme, aside from it's dependence on Mg2+ as a cofactor and it's near-zero delta G.

Okay, I admit, this one is kind of hard to figure out from it's name. What in the heck is an enolase. Well, the ase is just a post script to tell you it's an enzyme. And an Enol is a tautomer of a ketone. So this must be something which makes an enol of some sort. And it does. But that isn't all it does. It removes H2O to raise the free energy of hydrolysis and produce phosphoenolpyruvate, or PEP. Remember now that there are two molecules here, as we began with two molecules of gly-3-P. But what happens to PEP? It's such a cute name I hope nothing happens to it...

Pyruvate kinase
That's right, another kinase hops into action, this time on our good friend PEP. This kinase again relies on substrate level phosphorylation, which we'll explain in just a tad. And just like the last example of substrate level phosphorylation that we saw, this one produces ATP from ADP. It utilizes the breaking of a high energy phosphate bond to provide the delta G necessary to charge an ADP to an ATP. An enol is formed, which then rapidily converts to the favored keto position. A whole host of cofactors are needed here, essential K+ and Mg2+ or Mn2+. Regulation also occurs at this step, again through ATP (just like with PFK). Low ATP stimulates it (like PFK) and high ADP and AMP inhibit pyruvate kinase (like PFK). We'll learn more about this when we focus on regulation.