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The molecule Glucose is a hydrated 6-membered ring classified as a carbohydrate. This makes sense, after examining the empirical formula for glucose, C6(H20)6, which suggests that glucose is a hydrated hydrocarbon ring. Glucose and other polysaccharides are the cell's major source of ATP, in addition to lipids and proteins.  Glucose can not be synthesized within the human body, therefore we must injest it through eating plants, which are rich with glucose.  Glucose from the bloodstream is then taken up by individual cells through active transport.  While the major production of ATP occurs in later reactions during the electron transport chain, glycolysis generates two molecules of ATP in the conversion of glucose to pyruvate. Pyruvate is a very important molecule because, depending on the amount of oxygen available, it will continue on to the citric acid cycle (Krebs), or be converted into lactate (in animals), or into ethanol (plants and microorganisms). Furthermore, glycolysis is a useful pathway because it produces ATP without oxygen in a highly exergonic oxidation reaction.  Let's now take a closer look at the chemistry of glucose.

Glucose has several different classifications, some of which are:

  • Aldose- because Glucose' open chain form has an aldehyde
  • Hexose- a 6 Carbon sugar
  • Pyranose- an oxacyle 6 membered ring (6 membered ring with one of the C replaced by an O)
  • Monosaccharide- 1 sugar unit. Single sugar units take on the form (C6H12O6)n. C=O bonds make sugars reactive
  • Hemiacetal- has the form RO-C-OH 

Conformations of Glucose

Glucose itself can be found in three different conformations. Beta-Glucose, open chain glucose, and Alpha-Glucose, which are shown in the figure below.

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Take a look at these three structures:  the structural difference between Beta-Glucose and Alpha-Glucose is merely the position of the hydroxyl group on the hemiketal carbon, which means they are anomers. For that reason, the hemiketal carbon is known as the anomeric carbon. The anomeric carbon in Beta-Glucose has a hydroxyl group in the "up" position (equatorial) and the anomeric carbon in Alpha-Glucose has a hydroxyl group in the "down" position (axial).  The Beta-Glucose is favored because the axial position of the electron-rich and very large hydroxyl group in the Alpha-Glucose leads to one additional gauche interaction over the Beta form, at a cost of 1kcal/mol. Now, a knowledge of steric interactions and a little common sense should tell us that due to this additional energy cost, Beta and Alpha Glucose should exist in a ratio of 10:1 in the human body. However, experimental results show otherwise: The ratio is about 66% in Beta  form, 33% in Alpha form, and 1% in the open chain form.

This may come as a bit of a shock, and there must be a certain stabilizing factor possessed by the Alpha form that slightly lessens the pain caused by the extra gauche interaction: the Anomeric Effect.

The Anomeric Effect

As previously stated glucose has two conformations: beta and alpha. In the beta form, the hydroxyl group on the anomeric carbon is in the equatorial position. Because of the more favorable sterics of the beta glucose conformation, we would expect to find a 10:1 ratio of beta glucose to alpha glucose. However, what we find is a 2:1 ratio. What causes this unexpected ratio? The answer to this is the anomeric effect.

 The anomeric effect is a special type of hyperconjugation. In the anomeric effect a lone pair from oxygen fills the sigma star bonding orbital of the adjacent C-OH bond. In the alpha glucose on the left, we see oxygen is able to send its electron density down into the sigma star of the C-O bond because the axial position of the bond aligns the orbital properly with the oxygen’s lone pair. In the beta glucose on the right, oxygen is unable to send its electron density down into the sigma star orbital because in the equatorial position the sigma star orbital does not align with the oxygen’s lone pair. The stabilizing hyperconjugation of the anomeirc effect found in alpha glucose is what causes more glucose to be found in alpha form and gives us the unexpected ratio of beta to alpha glucose. 

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Note also that hyperconjugation still occurs in the beta glucose with the sigma star of the -OH bond interacting with a Hydrogen; this of course is not as stabilizing as interacting with a juicy lone pair as the alpha glucose does. Also the lone pair in the beta glucose still has a small interaction with the sigma star of another Hydrogen, but the hydrogen is not as big a polarizable as the oxygen, and so this interaction is also much weaker.

Phosphorylation of Glucose

Mechanism

So, earlier we established that Glucose is important to the body because it is broken up into two useful molecules: ATP and Pyruvate. This is a long process that involves 9 different enzymes, but don't be intimidated. It may help to look at the names of the enzymes to remember what to do.  For example, kinases usually add phosphates by transferring them from a high energy molecule like ATP.  Another useful tool is to follow the "pink" carbon (think of the little ball that used to bounce on the words in those cheesy Disney sing-a-longs we watched as children) and you should be good to go. Now, let's get started!

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All right, this step may seem kind of dumb. First of all, why are wasting valuable ATP when it is one of the products we are generating, and also, why are we even phosphorylating the glucose in the first place? To answer the first question; the amount of ATP used is relatively small, and will lead to a big payoff later in the mechanism.  As for the second question, if you take a good look at Glucose, you will realize that it does not particularly stick to enzyme receptors because it does not have any good recognition elements. In fact, it's kind of like a big greasy hydroxyl ball. So, by adding the phosphate group we add a valuable recognition element that will facilitate further reactions, we are essentially "activating" the glucose molecule. Also, note that the Magnesium serves to stabilize the charged oxygens, which makes them feel less charge, and the carbon becomes more partially positive, which makes it easier to attack-a good situation for everyone involved.

Isomerization of Glucose-6 Phosphate

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This reaction uses phosphohexose isomerase to convert the Glucose-6-Phosphate, an aldopyranose ring, to fructose-6-Phosphate, a Ketofuranose, or 5-membered sugar whose open form contains a ketone. This is a reversible step and it is helpful to remember that an isomerase will isomerize a molecule, in both directions of course. Note that one important component of this reaction is that the southwest hydroxyl group gains a specific stereochemistry that it lacked in Glucose-6-Phosphate. This reaction is a bit beyond us now, but the next lecture will cover this type of reaction. 

Phosphorylation of Fructose-6-Phosphate

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Yeah, this step is pretty repetitive. A kinase uses a small amount of ATP, like the first step, to phosphorylate the F6P molecule. This is the rate limiting step in eukaryotes.

To simplify things a bit, you may want to think of glycolysis as being broken down into two major phases. Phase 1 is sort of like the priming phase, where there are two phosphorylation reactions requiring 2 equivalents of ATP.  The step above is the last of this phosphorylation phase of glycolysis, and there will be no more ATP used after this step. Phase 2 is more complex and will break this six carbon molecule into two three carbon molecules, it will also create 4 equivalents ATP and 2 equivalents of NADH, as well as eventually forming pyruvate. Phase 2 starts below.

Retro-Aldol of Fructose-1,6-Diphosphate

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All right, this step is the biggie, the step where we form DHAP and GAP.  GAP will continue on to be converted into pyruvate, and DHAP is a "dead end" molecule (much like lactate). But wait, how would Glycolysis be an effective process if half of these products were a metabolic dead end? Wouldn't ATP, the currency of metabolism, be completely wasted? Nature of course has a solution for this, an extremely efficient enzyme called TIM will convert DHAP into GAP (this is the topic of the next lecture).  First, an enzymatic base is going to deprotonate the southeast hydroxyl group, which will bust open the ring. For the next step, think back to Chem 120....Remember the Aldol reaction? This next part is a Retro-Aldol reaction, which means that we'll be doing the absolute of the aldol reaction. For this to be possible, we have the Lysine side chain eliminate our southeast ketone, much like in a PLP reaction. The lysine bound to the ring forms an electron sink which will enable the retro aldol reaction to occur. When the ring snaps, the addition of water to displace the Lysine forms DHAP, along with the desired product GAP.

Oxidative Phosphorylation of GAP

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In this step we're trying to replace the aldehyde's H with a Phopsphate group. However, we can't just kick this H off--instead, we need to trick the H into leaving by attacking the aldehyde with a cysteine side chain. The cysteine's nucleophilic sulfur will attack, forming an acid anhydride. Once the anhydride is formed, the Oxygen's electrons are able to fold down and kick off the H as a hydride to the NAD+. Then, the phosphate is able to attack and kick off the cysteine, forming the desired product. In this step, we go through an enzyme intermediate. Does this remind you of anything? Yes, in this step the cysteine is acting very much like a cysteine protease.

Note that while this is a phosphorylation step, the enzyme here is not a kinase because it is not driven by ATP. The phosphate is not just added for recognition, but to  also provide a good leaving group in the next step to make a carboxylate. NADH is generated in this step.  NADH and FADH2 are used in the electron transport chain to transfer electrons down a complex so that protons can be pumped into the intermembrane space of the mitochondria.  Then once enough has been pumped through, the protons can flow down their concentration gradient through an ATP pump into the matrix to make ATP.

Dephosphorylation of 3-PGP

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3-PGP is dephosphorylated allowing ADP to be phosphorylated, so here we make the first molecule of ATP.

Isomerization of 3-PGA

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This step is one of the most infuriating and also most interesting steps of the whole process. A serine protease-like mechanism is used as the histidines ping-pong hydrogens and phosphates back and forth. Don't be alarmed by this game of molecular ping-pong. We understand that things in the enzyme are generally held in place for a reason, but remember that histidines are built to swivel, so they are able to swing around and catalyze reactions on different parts of the molecule. Basically, the histidines ping-pong the phosphate back in forth, forming an isomer of the starting molecule.

Dehydration of 2-PGA to PEP

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In this step, the Lysine side chain and Mg 2+ salt bridge and hydrogen bond with the carboxylate to make it feel less electron rich, and therefore more open to attack, as it is very difficult to enolize a carboxylic acid. However, this "juicing up" of the carboxylate allows an enzymatic base to pull a hydrogen alpha to the carboxylate, and the electrons come down and kick off the hydroxyl group as water. This last reaction is a conjugate elimination, which you have seen in PLP reactions. Get to know it well.

Dephosphorylative Tautomerization of PEP

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The final step is relatively simple--ADP attacks the phosphate group, leaving an unstable enol form of pyruvate with a negatively charged Oxygen. This will spontaneously tautomerize to a stable keto form when the oxygen's electrons come down and prompt the double bond to pick up a hydrogen. This step is extremely exergonic and irreversible, and produces more ATP. Remember we broke down two ATP's to make GAP and DHAP then generated another two per molecule of GAP (DHAP will be converted to GAP by TIM as explained by next lecture).  Therefore 2 molecules were consumed and 4 made, for a net of 2 molecules of ATP through glycolysis. Finally, after these nine enzymes have done their work, we have pyruvate containing the original "pink" carbon.

Drill Problems

D18-1. Starting with the isotopically-labeled analog of DHAP, show the products of each of the enzymatic reactions in the spaces provided.  No mechanisms are necessary.

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D18-3. The alpha and beta forms of glucose are in rapid equilibrium in water that contains a trace of HCl.  Provide a mechanism for this interconversion.  

D18-5. Draw the all the intermediates that connect glucose to pyruvate along the glycoloysis pathway.  Now, circle the anomeric carbon of glucose, and follow that carbon through the pathway, circling all corresponding carbon(s) in each subsequent intermediate.

D18-2. The enzyme glucose phosphate isomerase catalyzes the reaction shown below.  Its mechanism of action is strikingly similar to that of TIM. Predict the products of the following reactions or sequences of reactions.  Be sure to describe the stereochemistry of your predicted products.  Imagine that the enzyme active site is identical to that of TIM with respect to both mechanism and stereochemistry.

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D18-4. Provide a mechanism for the final hydrolysis step of the reaction catalyzed by fructose-1,6-diphosphate aldolase.

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