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As stated previously, glycolysis is essential for converting carbohydrates to ATP, the energy currency of the cell. A previous post discussed the overall process. Some people want more information on how this process occurs. This article is for you!

Step 1 in glycolysis is the phosphorylation of glucose to glucose-6-phosphate by the enzyme hexokinase. The high-energy phosphate comes from the conversion of one molecule of ATP to ADP. In other words, one molecule of ATP is ‘spent’ to ‘prime the pump’ for glycolysis. This step is critical in locking glucose into the cell. It also enables the glucose to go on to step 2 of glycolysis. There is a control mechanism (as indicated by the black box on the arrows in the diagram) at this step. Glucose-6-Phosphate inhibits the action of hexokinase and reduces its ability to phosphorylate glucose. Therefore, when the levels of glucose-6-phosphate increase, the activity of hexokinase decreases and less glucose is phosphorylated. This type of control (where the product inhibits the enzyme) is known as product inhibition. There is another enzyme that can catalyze this reaction, glucokinase, but it is only active when glucose levels are very high. It is specific to the glucose molecule while hexokinase is active on any hexose (6-carbon sugar).

Step 2 is simply the conversion of glucose-6-phosphate to fructose-6-phosphate by the enzyme isomerase (also known as phosphoglucoisomerase).

Step 3 is another step that consumes an ATP. Fructose-6-phosphate is converted to Fructose-1,6-bisphosphate (also known as Diphosphate). As you can see, another ATP gives up its terminal high-energy phosphate and donates it. Now, our molecule has 2 high-energy phosphates. This is the most regulated step in glycolysis. It is catalyzed by phosphofructokinase 1 or PFK 1. This step is activated by low cellular energy such as when the cell is high in AMP, ADP, Fructose-1,6-bisphosphate, and Fructose-2,6-bisphosphate. It is inhibited by high cellular energy such as when the cell is high in ATP, NADH, and citrate. In this way, the cell responds to high energy demands (such as exercise) by increasing energy production and decreasing production when the demand decreases.

Step 4 involves the splitting of the ring into two 3-carbon structures by the enzyme Aldolase. One of them is dihydroxyacetone-phosphate. The other is glyceraldehyde-3-phosphate. The two are interchangeable via the enzyme Triose Phosphate Isomerase. Only glyceraldehyde-3-phosphate is able to complete glycolysis. However, since equilibrium dictates that as soon as the product is used up (or moves on) the reaction shifts toward the left, dihydroxyacetone-phosphate is converted to glyceraldehyde-3-phosphate. Therefore, for every molecule of glucose that enters glycolysis, two molecules of glyceraldehyde-3-phosphate are produced. From this point on, all reactions are doubled. The second picture (on the overview) should be doubled. In other words, for every molecule of ATP or NADH produced in the diagram, double it.

Step 5 The two molecules of glyceraldehyde-3-phosphate are converted to 1,3-bisphosphoglycerate by the enzyme glyceraldehyde-3-phosphate dehydrogenase (dehydrogenase is often abbreviated DH). This step causes the reduction of NAD+ to NADH + H+. I will describe a useful analogy to help conceptualize the whole process with NADH + H+ later. For now, just understand the process. NADH cannot cross the mitochondrial membrane so it must use a transport mechanism. Click here to see it. Therefore, our net yield so far is:

-2ATP + 2 (NADH + H+)

We haven’t really yielded any return on our investment! What’s the deal?

Our investment was putting 2 ATP into the system in the hopes of getting more out.

Step 6 Finally, we are getting a return that we can use immediately! This step uses phosphoglycerate kinase to convert 1,3-diphosphoglycerate to 3-phosphoglycerate. The phosphate on the number one carbon was transferred (along with its energy) to ADP to form ATP. We have 2 molecules of the 1,3-diphosphoglycerate so we get 2 molecules of ATP (remember, this is per one molecule of glucose). Reaction thus far:

-2 ATP + 2(NADH + H+) + 2 ATP

Hey, we finally broke even! We also have that NADH + H+ stuff but we can’t use it… yet!

Step 7 All we do in this step is use phosphoglyceromutase to change 3-phosphoglycerate to 2-phosphoglycerate. Not much happens here. No energy transfer, no energy production. The net reaction is still the same as in step 6.

Step 8 2-phosphoglycerate is converted to phosphoenolpyruvate via the enzyme enolase. We do gain a water out of it though. In fact, this is part of where the water comes from when they talk about water intake vs. output and mention metabolic water production. Overall reaction is the same just add two water molecules to it.

Step 9 This is the final step in glycolysis. Here, phosphoenolpyruvate loses its last high-energy phosphate. Pyruvate is produced from phosphoenolpyruvate by pyruvate kinase. This step is virtually irreversible. ADP is converted to ATP. Therefore, the final overall reaction is as follows:

-2 ATP + 2(NADH + H+) + 2 ATP + 2 H2O + 2 ATP + 2 Pyruvate

Two of the ATP’s cancel and we get:

2 NADH + H+ + 2 ATP + 2 Pyruvate

I didn’t mention the water because it really isn’t significant for understanding the process. The 2 pyruvate will either enter the Kreb’s cycle (TCA cycle or citric acid cycle) or be converted to lactic acid (we’ll discuss that shortly). But it isn’t available for immediate energy. The 2 NADH must go into the mitochondria and enter the ETS to get energy so it can’t be of use to use immediately either. The only thing we can use immediately are the 2 ATP. When we need energy right away, we must rely on ATP/CP stores or glycolysis to provide it. The other energy sources (Kreb’s and ETS) simply aren’t fast enough to provide the energy for things such as a sprint.

As stated previously, pyruvate can serve as an acceptor of the hydrogens (electrons) from NADH + H+. The addition of these two electrons oxidizes pyruvate to lactate. This process in catalyzed by the enzyme Lactate Dyhydrogenase (LDH). Once lactic acid is formed, it easily diffuses into the blood and is carried away from the site. Therefore, anaerobic glycolysis can continue without a localized buildup of lactic acid. However, as lactic acid builds up, pH decreases. If you’ll recall, most enzymes are less active at lower pH levels. This reduces the ability of the body to regenerate ATP adequately and fatigue ensues.

If you didn’t know, the acid form (pyruvic acid or lactic acid) is not the ionized form. It has all the hydrogens it can have. It serves as the Bronstead-Lowry acid. The other form (pyruvate or lactate) is the ionized form. It has lost a hydrogen and has a negative charge.

The lactic acid must be dealt with. It may go to the liver to enter the Cori cycle, reconverted to pyruvate in the muscle when NADis freed up, or transported to other tissue and converted back to pyruvate.