Glycolysis

This blog covers:
a) The chemcical interconversions of glycolysis at the molecular level.
b) The enzymes involved in the interconversions.
c)The envergitics of these interconversions.
d)The mechanisms of flux control and regulation for the maintainance of steady state.

(Refer to this blogpost for an introduction on flux and steady state.)

General outline: What does glycolysis hope to achieve?

Glucose, the end product of carbohydrate digestion is a six carbon molecule. Our body breaks glucose down into two three carbon molecules of lower energy called pyruvate. During this process, energy is liberated a little at a time so that the body can store it as chemical energy (in the terminal bond of $ATP$) and utilize it when required. Phosphorylation is the addition of an inorganic phosphoryl group to a molecule. Phosphorylations take place in the initial reactions of glycolysis. These phosphorylated intermediates are then converted into compounds with high group transfer potential which undergo hydrolysis to release free energy. This release of free energy is coupled with $ATP$ synthesis. (Refer to this blogpost for an explanation of group transfer potentials.)

Two such high energy phosphorylated intermediates are phosphoenolpyruvate and 1,3Bisphosphoglycerate. In conclusion, the pathway of glycolysis aims to form both of these compounds and extract free energy by hydrolyzing them.

Steps in glycolysis

The free energy in glycolysis comes from the hydrolysis of 2 high energy intermediates: 1,3 Bisphosphoglycerate and Phosphoenolpyruvate. Both of these compounds are 3 carbon molecules. Thus, the first half of glycolysis prepares glucose (a 6 carbon molecule) for cleavage into two 3 carbon molecules. After cleavage, formation of 1,3 Bisphosphoglycerate becomes the primary goal. Liberation of free energy from this compound is followed by the formation of phosphoenolpyruvate. Hydrolysis of the phosphoryl group of phosphoenolpyruvate marks the end of glycolysis.

Keep the structures of the molecules in mind while reading the following sections. It greatly enhances the glycolysis experience.

Preparing glucose for cleavage:

1. Glucose is converted to Glucose-6-Phosphate
Enzyme: Hexokinase
Depending on the cell type, glucose is either transported into it by facilitated diffusion or through active transport. Phosphorylating glucose reduces the concentration of glucose within the cell. This facilitates the inward movement of glucose. Secondly, phosphorylation of glucose adds a negative charge to the previously neutral molecule. This prevents it from diffusing out.

Phosphorylation of glucose effectively traps it within the cell.

2. Glucose-6-Phosphate is isomerized to Fructose-6-Phosphate
Enzyme: Phosphoglucose Isomerase (PGI)
The isomerization of glucose fructose is important for the step of cleavage. This will be further explained in step 5.
3. Fructose-6-Phosphate is converted to Fructose 1,6 Bisphosphate.
Enzyme: Phosphofructokinase (PFK)
This step is irreversible. It commits the substrate to the pathway and hence is essential. Many complex regulation mechanisms are associated with this step. This second phosphorylation results in two similar compounds (both phosphorylated) being formed after cleavage of the six carbon molecule.

The cleavage of the 6 carbon molecule into 2 three carbon molecules:

4. Fructose 1,6 Bisphosphate is broken down into Glyceraldehyde 3 Phosphate (G3P) and Dihydroxy Acetone Phosphate (DHAP).
Enzyme: Aldolase
This step is a reverse aldol condensation reaction. Aldol cleavage to form 2 three carbon products requires a carbonyl carbon at the C2 carbon and a hydroxyl group at the C4 carbon. Thus, the isomerization of glucose to fructose earlier was necessary. The aldol cleavage
of Glucose 6 Phosphate would have resulted in products of unequal carbon chain length while that of Fructose 6 Phosphate results in two interconvertible three carbon compounds.

Formation of 1,3 Bisphosphoglycerate:

5. DHAP is converted to G3P
Enzyme: Triose Phosphate Isomerase (TPI)
Converting DHAP to G3P means that both the molecules can enter a common degenerative pathway. The number of substrate molecules has now doubled. Each of the reactions that follow take in twice the amount of substrate and yield twice the amount of product molecules. Thus, in the substrate level phosphorylations that follow, two $ATP$ molecules are synthesized per molecule of glucose.

6. G3P is oxidized to 1,3 Bisphosphoglycerate
Enzyme: Glyceraldehyde 3 Phosphate Dehydrogenase (G3PDH)
This step utilizes the cofactor $NAD^+$ along with an inorganic phosphate ($P_i$). To properly understand this reaction, lets break it down into parts:

• The active site of the enzyme G3PDH has a sulfhydryl group ($S-H$).
• The Sulphur atom of this group attacks the carbonyl carbon of G3P forming a thiohemiacetal.
• The oxidation step that follows involves hydride ($H^-$) transfer from this thiohemiacetal to $NAD^+$. Thus, the energy liberated during the oxidation of the aldehyde is now stored in $NADH$ and the acyl thioester intermediate.
• Finally, the $P_i$ group attacks to form an acyl phosphate, a high energy compound.
• Note that this attacking $P_i$ group has a proton attached to one of its oxygens. This proton is released into the solution. Thus, $NAD^+$ reduction makes the surrounding environment acidic (lowers its pH).

Phosphorylation of $ADP$ to $ATP$: A Substrate Level Phosphorylation

7. 1,3 Bisphosphoglycerate is converted to 3 Phosphoglycerate
Enzyme: Phosphoglycerate Kinase
This reaction results in the formation of the first $ATP$. The hydrolysis of the high energy phosphoryl bond formed in the previous step is coupled with $ATP$ synthesis.

Formation of Phosphoenolpyruvate:

8. 3 Phosphoglycerate is converted to 2 Phosphoglycerate
Enzyme: Phosphoglycerate Mutase (PGM)
A mutase catalyzes the transfer of a functional group from one position in the molecule to another. In accordance with the structure of phosphoenolpyruvate, Phosphoglycerate Mutase adds its phosphoryl ($PO^{2-}_3$) group to the second carbon and removes the $PO^{2-}_3$ group from the third carbon. A bisphosphate intermediate 2,3 bisphosphoglycerate (2,3BPG) is formed during this reaction. Sometimes, this intermediate dissociates from the enzyme reversibly. This renders the enzyme inactive. A pool of 2,3 bisphosphoglycerate is present in cells to perform the reverse reaction and reactivate the enzyme. Read about 2,3 BPG’s affect on hemoglobin’s oxygen affinity here.

9. 2 Phosphoglycerate is dehydrated to Phosphoenolpyruvate
Enzyme: Enolase
In this step, the substrate is dehydrated to form the high energy compound phosphoenolpyruvate. The enzyme enolase forms a complex with divalent cations such as $Mg^{2+}$ before the substrate is bound. In the presence of fluoride ($F^-$) and inorganic phosphate ($P_i$), the divalent cation forms a complex with $F^-$ and $P_i$ at the enzyme’s active site. The substrate can no longer bind to this site. This results in loss of catalytic activity and therefore, inhibition of glycolysis. In such a condition, build up of 2 phosphoglycerate (the substrate) and 3 phosphoglycerate (that is in equilibrium with the substrate) is seen.

Phosphorylation of $ADP$ to $ATP$: A Substrate Level Phosphorylation

Phosphoenolpyruvate is converted to Pyruvate (End product of glycolysis)
Enzyme: Pyruvate Kinase (PK)
In the final reaction of glycolysis, pyruvate kinase couples the free energy of phosphoenolpyruvate hydrolysis to the
synthesis of $ATP$ to form pyruvate.

The fate of pyruvate

The concentration of $NAD^+$ in a cell is limited. Thus, in order for glycolysis to continue, a constant replenishment of $NAD^+$ in the cell is necessary (for step 6). In this section, we will go over the mechanisms cells employ to oxidize the formed $NADH$ back to $NAD^+$ so as to continue glycolysis. These mechanisms differ considerably in the presence (aerobic) and absence (anaerobic) of oxygen. The amount of $ATP$ synthesized from the aerobic mechanism is eight times more than that synthesized in the anaerobic mechanism.

Homolactic Fermentation:

The reaction
Pyruvate formed at the end of glycolysis is reduced to lactic acid so that $NADH$ and be oxidized back to $NAD^+$. This reaction absorbs a proton from the solution. The formation and accumulation of lactic acid that occurs in muscle cells under anaerobic conditions leads to muscle fatigue. Note that it is not lactate but lactic acid alone that causes fatigue. If the pH were somehow maintained during glycolysis such that there was no acid formation, the muscle would not fatigue.

Enzyme: Lactate Dehydrogenase (LDH)
The enzyme lactate dehydrogenase, has two types of subunits: the $M$ type and the $H$ type that join together to form five types of isoenzymes. They are: $M_4$, $M_3H_1$, $M_2H_2$, $M_1H_3$ and $H_4$. The $H_4$ isoenzyme of LDH is primarily found in the heart and the $M_4$ type in skeletal muscle cells. The $H_4$ isoenzyme is allosterically inhibited by high quantities of pyruvate while $M_4$ is not. The $K_m$ value for pyruvate is high for the $M_4$ isoenzyme and low for the $H_4$ isoenzyme. This means the $H_4$ isoenzyme is quickly saturated, even in low concentrations of pyruvate. This way, the rate of conversion of pyruvate to lactate is by the $H_4$ isoenzyme is independent of pyruvate concentration. Thus, $M_4$ is better suited for the conversion of pyruvate to lactate while $H_4$ is better suited for the reverse reaction.

Understanding Efficiency
Although it does not synthesize as much $ATP$, anaerobic glycolysis is up to 100 times faster than aerobic glycolysis. Thus, in muscle tissue, when there is rapid consumption of $ATP$, anaerobic glycolysis must take place.

Although only 33% efficient, anaerobic glycolysis is much faster than aerobic glycolysis and is employed in times of rapid $ATP$ consumption.

An alternative process alcoholic fermentation is seen in yeast. This reaction is not visited here.

Under aerobic conditions

Under aerobic conditions, pyruvate enters the citric acid cycle. Here, it is completely oxidized to three molecules of $CO_2$. The reducing equivalents formed during the cycle are re-oxidized through the electron transport chain. Here, their electrons are fed to molecular oxygen ($O_2$) one at a time until it is reduced to water. This process is coupled with $ATP$ synthesis.

Metabolic control and regulation of glycolysis

Glycolysis is the energy supply block while the cells requirement for energy is the demand block. In this case, the demand block controls flux and the supply block regulates the flux. The substrate cycle involving phosphofructokinase plays an important role in regulation. It however, has no role in flux control. Increase in its concentration does not lead to an increased rate of glycolysis. The flux control of glycolysis is external and lies in the demand block. For a better understanding of flux control and regulation, refer to this blogpost.

FAQs

Bisphosphate, Diphosphate, Biphosphate: What’s the difference?
Bisphosphates are compounds to which two phosphoryl groups are attached to the compound individually.

A diphosphate is a compound in which two phosphate groups are attached to each other this whole structure is attached to the rest of the compound. This can be seen in the structure of adenosine diphosphate.

Biphosphate is used to name any salt of phosphoric acid in which only one of the hydrogen atoms has been replaced by a metal ion. It deals with ionic bonds in inorganic chemistry. For more insight on the prefix ‘bi’ as in bicarbonate, bisulphate, biphosphate etc. refer to this stack exchange page .

What is a kinase enzyme?
A kinase is an enzyme that transfers phosphoryl groups between $ATP$ and a metabolite. The metal ion $Mg^{2+}$ is essential for kinase activity. Nothing is implied about the direction of phosphoryl transfer. The word kinase comes from the Greek word kinein which means to move.

Hexokinase makes my heart soar

In this blog post, the mechanisms of actions of the enzymes of glycolysis are barely touched upon. However, while reading about them one particular enzyme fascinated me. Hexokinase catalyzes the first step in glycolysis. It follows the induced fit mechanism of action. When the substrate binds to an enzyme of this nature, the enzyme undergoes a conformational change so as to fit the substrate and exclude other substances (such as water) from interfering with the reaction. Thus, although thermodynamically more feasible, the phosphate group of $ATP$ is not transferred to water but to the hexose sugar (the substrate).

Note the structure of hexokinase is not realistic. This diagram simply shows the functional aspects of the induced fit mechanism.

Read More

• For an animation of the hexokinase reaction refer to this YouTube video by Molecular Memory.
• For a deeper look into the energetics of glycolysis refer to this stack exchange answer.